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Objectives: To observe the reparative effects of human umbilical cord mesenchymal stem cell (hUC-MSC) transplantation on white matter injury (WMI) in neonatal rats and explore its mechanism through the nuclear factor-kappa B (NF-κB) signaling pathway mediated by microglial cells., Methods: Sprague-Dawley rats, aged 2 days, were randomly divided into three groups: sham-operation,WMI, and hUC-MSC ( n =18 each). Fourteen days after modeling, hematoxylin-eosin staining was used to observe pathological changes in the white matter, and immunofluorescence staining was used to measure the expression level of ionized calcium-binding adapter molecule 1 (Iba1). Western blotting was used to measure the protein expression levels of inhibitory subunit of nuclear factor-kappa B alpha (IκBα), phosphorylated IκBα (p-IκBα), phosphorylated NF-κB p65 (p-NF-κB p65), myelin basic protein (MBP), and neuron-specific nuclear protein (NeuN). Quantitative real-time PCR was used to assess the mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), MBP, and NeuN. Immunohistochemistry was used to measure the protein expression levels of MBP and NeuN. On day 28, the Morris water maze test was used to evaluate spatial cognitive ability., Results: Fourteen days after modeling, the sham-operation group exhibited intact white matter structure with normal cell morphology and orderly nerve fiber arrangement. In the WMI group, large-scale cell degeneration and necrosis were observed, and nerve fiber arrangement was disordered. The hUC-MSC group showed relatively normal cell morphology and more orderly nerve fibers. Compared with the sham-operation group, the WMI group had significantly higher proportions of Iba1-positive cells, increased protein levels of p-IκBα and p-NF-κB p65, and higher mRNA levels of TNF-α and IL-1β. The protein expression of IκBα and the positive expression of MBP and NeuN, as well as their protein and mRNA levels, were significantly reduced in the WMI group ( P <0.05). Compared with the WMI group, the hUC-MSC group showed reduced proportions of Iba1-positive cells, decreased protein levels of p-IκBα and p-NF-κB p65, and lower mRNA levels of TNF-α and IL-1β. Furthermore, IκBα protein expression and MBP and NeuN expression (both at the protein and mRNA levels) were significantly increased in the hUC-MSC group ( P <0.05). On day 28, the Morris water maze results showed that compared with the sham-operation group, the WMI group had significantly longer escape latency and fewer platform crossings ( P <0.05). In contrast, the hUC-MSC group had significantly shorter escape latency and more platform crossings than the WMI group ( P <0.05)., Conclusions: hUC-MSC transplantation can repair WMI in neonatal rats, promote the maturation of oligodendrocytes, and support neuronal survival, likely by inhibiting activation of the NF-κB signaling pathway mediated by microglial cells.

Objective To explore the mechanism of butylphthalide (NBP) in regulating microglia activation and inflammatory cytokine expression in the hippocampus of the mouse model of delayed encephalopathy after carbon monoxide poisoning (DEACMP). Methods Wild-type C57 adult mice with normal cognitive function were selected,and DEACMP was modeled by static inhalation of carbon monoxide.The mice were randomized into three groups:DEACMP,control,and NBP.The NBP group was administrated with NBP suspension at 6 mg/kg by gavage for 21 days,and the DEACMP and control groups were administrated with the same amount of vegetable oil by gavage.The hippocampal injury was observed by HE staining.The protein level of ionized calcium-binding adapter molecule 1 (IBA1) was determined by Western blotting,and the levels of downstream inflammatory cytokines were measured by ELISA. Results Compared with the control group,the DEACMP and NBP groups showed prolonged escape latency ( P =0.001, P =0.029),reduced nerve cells ( P =0.001, P =0.035),up-regulated expression of IBA1 ( P =0.001, P =0.042),increased mean fluorescence intensity of IBA1 ( P =0.001, P =0.021),and elevated levels of tumor necrosis factor-α (TNF-α) ( P =0.002, P =0.024),interleukin (IL)-6 ( P =0.001, P =0.015),and IL-1β ( P =0.001, P =0.023).Compared with the DEACMP group,the NBP group showed shortened escape latency ( P =0.025),increased nerve cells ( P =0.039),down-regulated expression of IBA1 ( P =0.035),decreased average fluorescence intensity of IBA1 ( P =0.031),and lowered levels of TNF-α ( P =0.028),IL-6 ( P =0.037),and IL-1β ( P =0.034). Conclusion NBP can inhibit the activation of microglia and reduce the expression of inflammatory factors,thereby alleviating cognitive dysfunction and brain tissue damage caused by DEACMP.

Objectives: To investigate the effect of osteoblast-derived extracellular vesicles (OB-EVs) on the proliferation and differentiation of osteoclasts, and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts., Methods: Primary osteoblasts were isolated from newborn mouse calvarial bone and induced by β-glycero phosphate, ascorbic acid and dexamethasone. Osteogenic feature was tested by alkaline phosphatase (ALP) and alizarin red S staining. Extracellular vesicles were isolated by ultracentrifugation from the cell culture supernatant. Vesicle morphology was observed by transmission electron microscopy, and the characteristic markers of tumor susceptibility gene 101 (TSG101), ALG-2 interacting protein X (Alix) and cluster of differentiation 9 (CD9) on the surface of extracellular vesicles were identified by Western blotting. Cell counting kit 8 (CCK-8) assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells. Furthermore, the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and receptor activator for nuclear factor κB ligand (RANKL). The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages (BMMs) by tartrate-resistant acid phosphatase (TRAP) staining, and the effect of OB-EVs on osteoclast differentiation was determined., Results: The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm, and TSG101, Alix and CD9 were expressed. RAW264.7 cells were stimulated with OB-EVs, and the results of CCK-8 assay showed that high concentration of OB-EVs (more than 20 μg/mL) inhibited cell proliferation ( P <0.05). Western blotting analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos, activated T cell nuclear factor (NFATc1) and c-Jun N-terminal kinase (JNK) in RAW264.7 cells were significantly increased, and the promoting effect was enhanced with increasing of OB-EVs concentration ( P <0.05). In addition, the combination of OB-EVs and RANKL on BMMs showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone ( P <0.05)., Conclusions: OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts, but high concentration of OB-EVs can inhibit proliferation of RAW264.7 cells.

Nesfatin-1 is a neuropeptide hormone known for its biological functions, including inhibiting food intake, regulating glucose and lipid metabolism, promoting apoptosis, and providing anti-inflammatory and anti-tumor effects. Glucose metabolism is a crucial pathway for the body's energy supply. Current research has demonstrated that Nesfatin-1 can affect glucose metabolism through various mechanisms, such as inhibiting food intake, regulating enzyme activity, and improving insulin resistance, though the findings are not entirely consistent. Investigating the relationship between Nesfatin-1 and glucose metabolism may offer new insights into the diagnosis and treatment of diseases related to glucose metabolism disorders.

Objective: To establish patient-derived organoid models of pleomorphic adenomas (PA) of the parotid gland and preliminarily characterize their histology, related biomarkers and functions. Methods: Fresh tumor tissue specimens were collected from surgical procedures of Oral and Maxillofacial Department. The harvested tissues were processed and cultured in a head and neck tumor organoid culture system to establish organoid models from parotid gland pleomorphic adenomas. The in vitro growth of PA organoids was recorded by light microscopy. The successfully established organoids were passaged and cryopreserved, and the cryopreserved PA organoids were revived and re-cultured to observe their viability and organoid regeneration ability. Histological characterization, as well as characterization and detection of related markers and functional proteins, were performed on the organoids, comparing them with the patient-derived tissues. Results: The constructed organoid model of pleomorphic adenoma exhibited a dense and compact three-dimensional spherical structure. Hematoxylin and eosin staining indicated morphological similarities between the organoid and its tissue of origin. Immunohistochemistry showed positive cytoplasmic staining for Calponin, cytokeratin 7, and epithelial membrane antigen in both the organoid and the source tumor tissue, suggesting consistent histopathological characteristics between the organoid and its tissue of origin. Periodic acid-Schiff staining of the organoid showed positive staining for glycogen, with positive staining located in the interior and periphery of the organoid, indicating that the organoid possessed secretory functions like the salivary gland. Conclusions: This study successfully constructed organoids of pleomorphic adenoma derived from patient samples. This model faithfully replicates the tissue morphology and biomarkers of the source tissue and exhibits biological functions associated with mucus secretion. It serves as a valuable in vitro model for studying the development and progression of salivary gland tumors.

Objective: To explore the effect of the absent in melanoma 2 (AIM2) -mediated neuroinflammation in noise-induced cognitive dysfunction in rats. Methods: In April 2023, sixteen male Wistar rats were randomly divided into control group and noise group, with 8 rats in each group. The rats in the noise group were placed in 50 cm×50 cm×40 cm transparent boxes and exposed to 100 dB (A) white noise with a sound pressure level of 100 dB (A) (4 h/d for 30 d) . At the same time, rats in the control group were kept in similar boxes with environmental noise less than 60 dB (A) . After 30 days of noise exposure, the Morris water maze experiment was applied to test the learning and memory abilities of the rats; the pathological morphology of hippocampal tissues was observed by Hematoxylin-Eosin (HE) staining. Western blot was used to detect the protein expression levels of AIM2, cysteinyl aspartate specific proteinase-1 (caspase-1) , apoptosis-associated speck-like protein (ASC) , interleukin-1β (IL-1β) , IL-18, ionic calcium-binding articulation molecule-1 (Iba-1) , and glial fibrillary acidic protein (GFAP) . The expression of both Iba-1 and GFAP in hippocampal tissue was assessed by immunohistochemical staining. The co-localization of AIM2 with Iba-1 or GFAP was determined by immunofluorescence double staining. Results: Compared with the control group, the escape latency of rats in the noise group was increased by 16.29 s, 17.71 s, and 20.26 s on days 3, 4, and 5, respectively. On day 6, the noise-exposed rats spent shorter time in the target quadrant and had fewer times in crossing the platform[ (7.25±2.27) s and (1.13±0.64) times] than the control group[ (15.64±3.99) s and (4.25±2.12) times] ( P <0.05) . After noise exposure, hippocampal neurons of rats displayed marked nuclear hyperchromatic and pyknosis phenomenon. The noise-exposed rats had higher numbers of both microglia and astrocytes (27.00±2.65 and 43.33±5.51) in the DG area of the hippocampus relative to the control group (14.67±3.06 and 20.00±4.58) ( P <0.05) . Moreover, the glial cells in the noise group had larger cell cytosol with more and thicker branches. The protein expression levels of inflammatory cytokines Cleaved-IL-1β and Cleaved-IL-18 in the hippocampus of rats in the noise group (1.55±0.19 and 1.74±0.12) were significantly higher than the control group (1.00±0.11 and 1.00±0.13) ( P <0.05) . After noise exposure, the protein expression levels of AIM2, Cleaved-Caspase-1 and ASC (1.19±0.09, 1.34±0.07 and 1.14±0.01) were higher than the control group (1.00±0.07, 1.00±0.14 and 1.00±0.06) and differences between the two groups were statistically significant ( P <0.05) . A significant increase in the number of cells co-localizing AIM2 with Iba-1 or GFAP in the noise group (28.67±4.04 and 40.67±5.13) compared with the control group (15.67±4.04 and 17.67±3.79) , and statistically significant differences were observed between the two groups ( P <0.05) . Conclusion: Noise exposure may activate the AIM2 inflammasome in hippocampal glial cells of rats, releasing excessive inflammatory cytokines and causing neuroinflammation that damages neurons.

Objective: Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation., Methods: HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1 -/- cells), and SW620-nkd1 -/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells., Results: In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells ( P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter ( P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells ( P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells ( P < 0.05)., Conclusion: NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.

Objective: To explore the clinical value of the inflammasomes NLRP1 and NLRC4 in the diagnosis and treatment of PCa., Methods: Using immunohistochemical staining, we detected the expressions of the inflammasomes NLRP1 and NLRC4 and the inflammatory cytokines IL-18 and IL-1β in 54 cases of BPH and 58 cases of PCa treated in Pinghu First People's Hospital from January 2022 to May 2022. We analyzed the characteristics of their expressions in the two groups of patients and the correlation of NLRP1 and NLRC4 expressions with tPSA, fPSA and Gleason scores in the PCa patients. Based on the Cancer Genome Atlas dataset, we compared the expressions of NLRP1 and NLRC4 in different stages of PCa., Results: The NLRP1 and NLRC4 expressions were significantly increased in the PCa patients (P < 0.001). The expression of NLRP1 was linearly correlated with those of IL-1β and IL-18 (P < 0.05), and so was the expression of NLRC4 with that of IL-18 (P < 0.05). The expressions of NLRP1 and NLRC4 were positively correlated with the Gleason scores of the PCa patients (P < 0.05), the former remarkably higher in T3 and T4 than in T1 (P > 0.05), and the latter markedly higher in T2, T3 and T4 than in T1 (P < 0.05)., Conclusion: The inflammasomes NLRP1 and NLRC4 are highly expressed in PCa and facilitate tumorgenesis by promoting the maturation and release of the inflammatory cytokines IL-1β and IL-18, which indicates their important role in the progression of tumors and clinical value in the risk assessment and prognosis of PCa.

Objective: To examine the correlation of CCBE1 expression in adjacent tissues of tongue squamous cell carcinoma (TSCC) with pericancerous lymphatic vessel proliferation, cervical lymph node metastasis and survival outcomes of the patients., Methods: Lymphatic vessel density was quantified in pericancerous tissue sections of 44 cases of cT 1-2 N 0 TSCC using D2-40 as the lymphatic vessel endothelial marker for calibration and counting of the lymphatic vessels. Of these 44 cases, 22 showed a relatively low lymphatic vessel density (group A) and the other 22 had a high lymphatic vessel density (group B), and the expression levels of CCBE1 in the adjacent tissues determined using immunohistochemistry, immunofluorescence assay and Western blotting were compared between the two groups. The expression level of CCBE1 was also measured in another 90 patients with TSCC using immunohistochemistry, and all the patients were followed up for their survival outcomes., Results: Immunohistochemistry and Western blotting showed a significantly lower rate of high CCBE1 expression in group A than in group B ( P < 0.05). Immunofluorescence assay showed co-localization of CCBE1 and D2-40 in the adjacent tissues of TSCC. In the 90 TSCC patients with complete follow-up data, a high expression of CCBE1 was found to correlate with lymph node metastasis and a poor 5-year survival outcomes of the patients ( P < 0.05)., Conclusion: A high expression of CCBE1 in the adjacent tissues of TSCC is closely related with pericancerous lymphatic vessel proliferation, cervical lymph node metastasis and a poor 5-year survival of the patients, suggesting the value of CCBE1 as a potential prognostic predictor for TSCC.

Objective: To identify rare variants in exon and exon-intron boundary of containing NLR family CARD domain protein 4 (NLRC4) in type 1 diabetes (T1DM) patients, and to explore their effects on gene function. Methods: A total of 508 T1DM patients and 527 healthy controls in the Department of Metabolic Endocrinology, Second Xiangya Hospital of Central South University from August 2017 to September 2020 were selected. The case group included 264 males and 244 females, and the age [ M ( Q 1 , Q 3 )] was [27 (11, 43)] years. The control group included 290 males and 237 females, and their age［ M （ Q 1 ， Q 3 ）］was [47 (36, 60)] years old. Identification of rare variants in exons of NLRC4 gene in T1DM patients and healthy controls was performed and verified by next-generation sequencing and sanger sequencing. The NLRC4 gene wild-type and mutant plasmids were constructed and transfected into 293T cells. Western blot (WB) was used to detect the expression of NLRC4 protein and cleavage products of pro-cysteinyl aspartate specific proteinase(procaspase-1). Cycloheximide (CHX) was added to 293T cells transfected with wild-type or mutant NLRC4 plasmid to detect the degradation of NLRC4 protein. The localization of NLRC4 protein was detected by immunofluorescence, and the concentration of IL-1β in the cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Results: The sequencing results showed that 4 patients and 2 healthy controls had a heterozygous variant c.208C>T in exon 3 of the NLRC4 gene. Two patient had a heterozygous variant c.1564T>C in exon 4, and 1 patients had c.1219G>C in exon 4. These three variants might be pathogenic variants in T1DM. In 293T cells transfected with NLRC4 wild-type and c.208C>T、c.1564T>Cc.1219G>C mutant plasmids, the expression level, degradation rate, localization of NLRC4 protein and the content of cleavage products of procaspase-1 did not change significantly. However, the concentration of IL-1β secreted by 293T cells transfected with c.1219G>C and c.208C>T plasmid [ M ( Q 1 , Q 3 )] was 15.25 (12.98, 17.52) and 15.44 (13.81, 17.07) ng/L, respectively, which was lower than 18.70 (16.59, 20.81) ng/L of 293T cells transfected wild-type plasmid ( P =0.020, 0.010). Conclusions: NLRC4 gene rare variants c.208C>T, c.1564T>C and c.1219G>C may not change the protein expression, degradation and localization, but c.208C>T and c.1219G>C may inhibit the secretion of IL-1β. This result suggests that NLRC4 rare variants may have an impact on gene function.

Objectives: To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism., Methods: FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-In TM T-REx TM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation., Results: After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine., Conclusions: This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.

Objective: To investigate the change in expression of anti-senescence marker protein calmodulin (RGN) in liver tissues of rats with immune hepatic fibrosis, and to observe the effect of compound glutathione inosine injection (CGII) on it. Methods: Rat liver fibrosis model was induced by intraperitoneal injection of porcine serum, and CGII intervention was administered at the appropriate time. Rat liver tissues were stained with HE and Masson. RGN and protein expression at mRNA in liver tissues was detected by fluorescence quantitative PCR and immunohistochemistry. One-way Anova was used for measurement data. LDS test was used for two-way comparison, and pathological semi-quantitative results were analyzed by rank-sum test. Results: The relative expression of RGN mRNA and protein in liver tissue of fibrotic rats was 82.23 ± 15.21 and 12.52 ± 3.23, respectively, which were significantly lower than that of normal rats 176.39 ± 11.35 and 59.23 ± 9.13 ( P < 0.01). The degree of liver fibrosis in fibrotic rats after CGII intervention was significantly lower than fibrotic rats. The relative expression of RGN mRNA and protein in the intervention group was 168.78 ± 21.31 and 46.42 ± 4.71, respectively, which were significantly higher than fibrosis and spontaneous recovery group. The difference was statistically significant ( P < 0.01). The relative expression of RGN mRNA and protein in the spontaneous recovery group was 86.23 ± 17.16 and 14.34 ± 5.16, which was higher than model group. The difference was not statistically significant ( P > 0.05). Conclusion: The expression of RGN in liver tissue of rats with hepatic fibrosis induced by porcine serum is decreased, while the expression of RGN increases with the decrease of fibrosis after CGII intervention, suggesting that the protein may play an important role in the development of liver fibrosis.

Objective: To investigate the role of miR-144-3p in regulating osteogenic differentiation of bone marrow mesenchymal stem cells and predict its target genes., Methods: Rat bone marrow mesenchymal stem cells (BMSCs) with induced osteogenic differentiation were examined for the expressions of Runx2, OCN and miR-144-3p. The effects of transfection with a miR-144-3p mimic or a miR-144-3p inhibitor were tested on the osteogenic differentiation of the BMSCs. The changes in the expressions of the predicted target of miR-144-3p in the BMSCs during induced osteogenic differentiation were examined using Western blotting and qRT-PCR., Results: Rat BMSCs with induced differentiation into osteoblasts exhibited a progressive increase in the expressions of Runx2 and OCN (two markers of osteogenic differentiation), while the expression of miR-144-3p gradually decreased during the differentiation till reaching the lowest level at 21 days of induction. In rat BMSCs, transfection with the miR-144-3p mimic significantly decreased ALP activity ( P &lt; 0.05) wile transfection with the miR-144-3p inhibitor significantly increased ALP activity ( P &lt; 0.05) in rat BMSCs. Analysis based on miRanda, microRNA.org database and TargetScan suggested that Smad4 was the most likely target gene of miR-144-3p. The results of qRT-PCR showed no significant differences in expression levels of Smad4 among the cells with different treatments ( P &gt; 0.05), while Western blotting revealed a significantly decreased expression of Smad4 in the cells transfected with miR-144-3p mimics and an increased Smad4 expression in the cells transfected with the miR-144-3p inhibitor as compared with the control cells ( P &lt; 0.05)., Conclusions: miR-144-3p participates in the regulation of osteogenic differentiation of rat BMSCs, and its inhibitory effect on osteogenic differentiation is achieved probably by decreasing the expression level of Smad4.

Objective: To investigate the clinicopathologic features, immunohistochemical phenotypes and biological behavior of pseudosarcomatous myofibroblastic proliferation (PMP) of the urinary bladder which may be misdiagnosed as a malignant neoplasm and undergo extensive treatment. Methods: Six cases of PMP of the urinary bladder were collected from 2001 to 2016 at Beijing Tiantan Hospital, Capital Medical University. The clinicopathologic features and immunophenotypic profile were studied by histopathological and immunohistochemical investigations with clinical follow-up. At the same time, the translocation of ALK gene was detected by fluorescence in situ hybridization (FISH). Immunohistochemistry was carried out using EnVision method for the expression of AE1/AE3, vimentin, EMA, SMA, Caldesmon, Calponin, desmin, ALK, Ki-67, MyoD1, myoglobin, CD34, S-100, CD117, CK7, CK20, GATA3, p63 and CK5/6. The related literature was reviewed. Results: There were two male and four female patients, significantly more common in women. The age of the patients was 27 to 53 years, and the median age was 35 years. The main clinical symptom was painless gross hematuria, one case with dysuria, and one case showed recurrent cystitis. There was no history of surgery and trauma. Follow-up ranged from 4 months to 13 years and showed five cases without recurrence and one case with recurrence. Microscopy showed submucosal lesion with inflammatory exudate and bleeding on the surface, in some cases extending to the superficial muscles of the bladder wall. The lesion was characterized by the proliferation of plump spindle cells, which were loose or dense in arrangement. There were varying degrees of acute and chronic inflammatory cells infiltration in the myxoid matrix. Spindle cells arranged in disorder, or a dense stranding, especially abundant in the cell region. The median mitotic rate was <2/10 HPF cells, but there were no pathological mitotic figures and without nuclear atypia in most spindle cells. Spindle cells with eosinophilic cytoplasm showed long tapering cytoplasmic projections. Oval or short spindle nuclei had vacuolization with prominent nucleoli, looking like ganglionic cells. There were scarce collagen fibers, and a few spindle cells degenerated with chromatin blurred. Some areas showed a granuloma-like pattern and neutrophils within vascular cavity. Immunohistochemically, the spindle cells were diffusely positive for vimentin, SMA and caldesmon. CKpan was strongly and diffusely positive. Desmin and calponin expression was varying. Ki-67 positive cells were about 35% to 55%, but the spindle cells were negative for myoglobin, S-100, CD117, CD34, p63 and CK5/6. FISH test showed that there was no ALK isolated signal in 6 cases of PMP, and so no positive cases were found. Conclusions: PMP of the urinary bladder is a benign non-neoplastic myofibroblastic proliferative lesion. Morphology is extremely easy to be misdiagnosed as malignant tumors, and therefore more attention should be paid to avoid this misdiagnosis.

Objective: To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemia-reperfusion injury and the underlying mechanisms.
 Methods: Forty Sprague-Dawley rats were divided into 4 groups randomly: a control group, a KB-R7943 group, an ischemia-reperfusion group (IR group), and an IR plus KB-R7943 group (KB-R7943+IR group). Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion. The ratio of left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), the infarct size of myocardium, and the lactate dehydrogenase (LDH) activity in the coronary flow was determined. HE staining was used to assess the change of myocardial morphology. Western blot was used to determine the levels of cleaved caspase-3, cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the Thr17 site of phospholamban.
 Results: Compared with the control group, IR group significantly induced an enlarged infarct size, reduction of the ratio of LVDP, up-regulation of cytochrome c, cleaved caspase-3, p-CaMKII and p-phospholamban, and increased in the activity of LDH, the level of LVEDP (P<0.01) and the disordered myocardial morphology. These effects were significantly attenuated in the presence of KB-R7943 treatment (10 μmol/L).
 Conclusion: NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKII.

Objective: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS)., Methods: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected., Results: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression ( P <0.01), suppressed ERS mRNA levels ( P <0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression ( P <0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS ( P <0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels ( P <0.01)., Conclusions: Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation via suppressing ERS, which might be regulated by mTOR-P70S6K signaling.

Objective: This study was to observe the effect of SMP-30 on ultraviolet B (UVB)-induced apoptosis of human lens epithelial cells(HLE-B3) in vitro . Methods: Experimental study. The SMP-30 cDNA was amplified by PCR and inserted into the pRFP-N1 expressing vector which had been double digested by XhoI/HindIII. HLE-B3 cells were cultured and divided into three groups: normal group, pRFP-N1 vector plasmid group and pRFP-N1-SMP-30 plasmid group (SMP-30). Then cells were exposed to UVB and the survival rate of cells was detected by MTT assay. The effects of SMP-30 on UVB-induced HLE-B3 apoptosis were measured by the Cell Death Detection ELISA kit. Meanwhile, the influence of SMP-30 on UVB-induced apoptosis-relative protein expression in HLE-B3 cells was tested by Western blots. Moreover, 2', 7'-Dichlorofluorescin diacetate staining was performed to monitor the protective effects of SMP-30 on UVB-induced HLE-B3 reactive oxygen species(ROS). One-way analysis of variance combined with Dunnett's statistical method were performed to analyze the data. Results: The full length of PSF cDNA fragment was correctly inserted into the pRFP-N1 vector, which was confirmed by DNA sequencing. The SMP-30 fragment was inserted to the plasmid pRFP-N1 correctly, which was also confirmed by DNA sequencing. The PRFP-N1-SMP-30 plasmid was transfected into HLE-B3 successfully. SMP-30 expression was up-regulated in the transfection group, compared with the control group. Data showed that the survival rate of HLE-B3 after the pRFP-N1-SMP-30 plasmid transfection was 0.90±0.14, while the apoptosis rate was 0.43±0.06 and the ROS production was 0.52±0.02, showing significant difference in comparison with the vector plasmid group and the normal group( t= 5.830, 9.934, 12.19, P< 0.05). In the meantime, SMP-30 overexpression down-regulated the levels of Bax and cleav-caspase-3, but up-regulated the Bcl-2 and Pro-caspase-3 expression levels under UVB irradiation. Conclusion: SMP-30 overexpression plays a protective role in UVB-induced apoptosis via regulating the expression of apoptosis-related proteins and inhibiting the production of ROS in HLE-B3 cells. (Chin J Ophthalmol, 2017, 53: 835-841) .

Objective: To assess the effect of choline in ameliorating lipopolysaccharide (LPS)-induced central nervous system inflammation and cognitive deficits in mice and explore the underlying mechanism., Methods: Seventy-two mice were randomized into saline control group, LPS group, choline intervention group and choline control group. In the latter two groups, the mice received pretreatment with intraperitoneal injections of choline (40 mg/kg, 3 times daily for 3 consecutive days) prior to microinjection of LPS into the lateral cerebral ventricle to induce central nervous system inflammation; in saline and LPS groups, the mice were pretreated with saline in the same manner before intraventicular injection of artificial cerebrospinal fluid. Choline treatment was administered in the mice till the end of the experiment. The locomotor activity and spatial learning and memory capacity of the mice were examined. The expressions of Iba1 protein and proinflammatory cytokines (TNF-α and IL-β) I the hippocampal dentate gyrus, and the expressions of α 7nAchR, p38 MAPK and phosphorylated p38 MAPK in the hippocampus of the mice were detected., Results: Water maze test showed that compared with the saline control group, the mice in LPS group exhibited significantly reduced platform crossings (P<0.05), which was significantly increased by choline pretreatment (P<0.05). The mice pretreated with LPS expressed obviously increased levels of IBA-1 protein, TNF-α, and IL-1β in the hippocampus (P<0.01), and choline pretreatment significantly lowered the expressions of IBA-1 protein and IL-1β (P<0.05). The phosphorylation level of p38 MAPK increased significantly after LPS pretreatment (P<0.05), and was reduced by choline pretreatment (P<0.05); α 7nAchR expression increased significantly in choline intervention group as compared with that in the other 3 groups (P<0.05)., Conclusion: Choline can probably antagonize LPS-induced hippocampal p38 MAPK phosphorylation in mice via the α 7nAchR signaling pathway to protective against LPS-induced neuroinflammation and cognitive impairment in mice.

Objective: To investigate the effects of silencing miRNA378 * on apoptosis, endoplasmic reticulum stress and calumenin of cardiomyocyte with coxsackie virus B3 (CVB3) infection., Methods: Primary cultured suckling mouse myocardium were divided into control group (normal cell), coxsackie virus infection group (normal cell and coxsackie virus B3), miRNA378 * control group (normal cell +coxsackie virus B3+miRNA378 * empty plasmid), miRNA378 * silencing plasmid group(normal cells + coxsackie virus B3 + miRNA378 * silencing plasmid). Four groups of cells were transfected, infected and treated in CO 2 incubator at 37℃. The α-SMA protein, cell apoptosis rate, calumenin, glucose regulated protein 78 (GRP78), activation transcription factor 6(ATF6) and transcription factors c/ebp homologue protein (CHOP) in endoplasmic reticulum were analyzed., Results: By detecting α-SMA protein, the isolated suckling mouse ventricular myocardium were confirmed. TUNEL detection of different groups of ventricular cell apoptosis found that coxsackie virus group of ventricular myocytes apoptosis was significant. Compared with the coxsackie virus infection group of myocardial cells, miRNA378 * silencing plasmid expression of cardiomyocyte apoptosis cells significantly reduced( P <0.01). The expressions of GRP78, ATF6 and CHOP were increased compared with those infected by Coxsackie virus infection ( P <0.01), while the expressions of calumenin were decreased ( P <0.01)., Conclusions: CVB3 infected myocardial cells effected miRNA378 * expression. It can trigger endoplasmic reticulum stress and activates signaling pathway factor and increase myocardial cell apoptosis.>.

Objective: To compare expression levels of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP-2) in Randall's plaque of renal papillary tissues in patients with calcium oxalate kidney stones and the underlying mechanism for stone formation.
 Methods: A total of 30 samples of Randall's plaque in renal papillary tissues from patients with calcium oxalate kidney stones were collected from the Department of Urology of Xiangya Hospital of Central South University from April, 2015 to December, 2015 and served as an experimental group. Ten samples of renal papillary tissues in patients undergone renal tumor nephrectomy were collected from the same hospital and served as a control group. The expressions of MGP and BMP-2 mRNA and protein were detected by quantitative real-time PCR and Western blot.Meanwhile, immunohistochemical technique was used to observe the expressions of MGP and BMP-2 in different parts of renal papillary tissues in the 2 groups.
 Results: 1) The mRNA expression levels of MGP in the experimental group and the control group were 0.760±0.804 and 1.365±0.348, respectively, with significant difference between them (P<0.05). Them RNA levels of BMP-2 in the experimental group and the control group were 2.500±0.725 and 1.485±0.870, respectively, with significant difference between them (P<0.05). The expression levels of MGP protein in the experimental group and the control group were 0.130±0.424 and 0.202±0.704, respectively, with no significant difference between them (P>0.05). The expression levels of BMP-2 protein in the experimental group and the control group were 0.885±0.220 and 0.682±0.272, respectively, with significant difference between them (P<0.05). The immunohistochemistry showed that the protein expression of MGP in the experimental group was lower than that in the control group, while the protein expression of BMP-2 in the experimental group was higher than that in the control group (both P<0.05).
 Conclusion: The BMP-2 expression is increased while MGP expression is decreased in renal papillary tissues in patients with calcium oxalate kidney stones, and the formation of calcium oxalate kidney stone might be a kind of osteogenetic reaction or ectopic calcification.

Objective: To study the effects of Caspase broad spectrum inhibitor Z-VAD-FMK on the expressions of calumenin,caspase-3, GRP78 and GRP94 in adriamycin-injured cardiomyocytes and to discuss whether there is a regulation relationship between calumenin and endo-plasmic reticulum stress and myocardial apoptosis., Methods: The primary cultured suckling mouse myocardium were randomly divided into control group (cardiomyocyte), adriamycin group (3 mg/L adriamycin + cardiomyocyte) and z-VAD-fm group (3 mg/L adriamycin + 0.1 μ mol/L Z-VAD-fmk + cardiomyocyte), each group of cardiomyocytes were cultured in CO 2 incubator at 37℃ for 24 h ( n =3). The expres-sion ofα -SMA protein in ventricular myocytes was detected by immunohistochemical method. The expressions of calumenin, GRP78, GRP94 and Caspase-3 in the myocardial cells were detected by Western blot., Results: Compared with the control group, the expression of calumenin in adriamycin induced myocardial cells was significantly decreased ( P < 0.01), while the expressions of GRP78, GRP94 and Caspase-3 ex-pression were increased ( P < 0.01). Compared with adriamycin group, the expression of calumenin in z-VAD-fm group was increased ( P < 0.01), while the expressions of GRP78, GRP94 and caspase-3 were decreased ( P < 0.01)., Conclusions: The caspase inhibitor z-VAD-fmk inhibited the expression of caspase and increased the expression of calumenin in adriamycin induced myocardial cells, and thus alleviated the endoplasmic reticulum stress.

Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-β2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-β2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-β and Notch signaling pathway.

Objective: To extract and identify semen-derived exosome using PEG6000., Methods: Exosomes were extracted from semen specimens from 6 healthy volunteers with step-by-step centrifugations and ultracentrifugation prior to 8% PEG6000 enrichment. The extracted exosomes were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting., Results: The pellets obtained were round or elliptic membrane vesicles 30 to 150 nm in diameter with intact double membranes and contained low electron density material. The pellets expressed CD63, ALIX and TSG101 molecules but not calnexin that was expressed in sperm cells., Conclusion: The PEG6000-based method for extraction of exosomes from semen samples facilitates future studies of seminal exosomes.

Objective: To evaluate the effect of ulinastatin on hypoxia-induced phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) and explore the underlying mechanism., Methods: Cultured PASMCs from SD rats were exposed to normoxic condition, normoxia with ulinastatin treatment, hypoxia, or hypoxia with ulinastatin treatment. After 24 h of exposures, the cells were examined for SM-α-actin and caplonin expressions with immunofluorescence assay and for cell migration with CCK-8 and 3 H-TdR assays. Western blotting was used for detecting the expressions of PPAR-γ in the cells, and PPAR-γ-responsive firefly luciferase reporter was employed for measuring the transcriptional activity of PPAR-γ. The PPAR-γ inhibitor GW9662 was used to explore the mechanism of the inhibitory effect of ulinastatin on hypoxia induced-phenotype modulation of PASMCs by measuring the changes in cell proliferation and migration., Results: Ulinastatin obviously enhanced the expressions of SM-α-actin and calponin (P<0.05), inhibited the proliferation and migration (P<0.05), and up-regulated the expression of PPAR-γ in PASMCs exposed to hypoxia (P<0.05). Pretreatment of the cells with GW9662 abolished the effect of ulinastatin on hypoxia-induced phenotype modulation of PASMCs and enhanced the cell proliferation and migration (P<0.05)., Conclusion: Ulinastatin inhibits hypoxia-induced phenotype modulation of PASMCs from rats possibly by up-regulating the expression of PPAR-γ.

Objective: To observe the effect of parathyroid hormone (PTH)(1-34) on the expression of matrix Gla protein (MGP) and Wnt/β-catenin signaling pathway and elucidate the possible molecular mechanism of PTH (1-34) in the prevention and treatment of osteoporosis., Methods: MG63 cells treated with PTH (1-34) at 10(-9), 10(-8), and 10(-7) mol/L, alone or in combination with Wnt/β-catenin signaling pathway inhibitors DKK-1 (200 ng/ml) were examined for mRNA and protein expressions related with Wnt/β-catenin signaling with real-time PCR and Western blotting. The cell differentiation after the treatment was assessed with alkaline phosphatase (ALP) staining and cell viability assay., Results: PTH (1-34) significantly increased the expression of MGP in a dose-dependent manner in MG63 cells (P<0.05 or P<0.01). PTH treatment obviously enhanced ALP activity in the cells, and this effect was suppressed by DKK-1. Combined treatment with DKK-1 partially blocked PTH-induced enhancement of ALP activity (P<0.05). PTH promoted the expression of MGP and enhanced LRP5, β-catenin, and Runx2 expressions in Wnt/β-catenin signaling pathway at both protein and mRNA levels (P<0.05 or P<0.01). DKK-1 partially blocked the effect of PTH (1-34) on Wnt/β-catenin signaling pathway (P<0.05) without affecting MGP expression., Conclusion: PTH (1-34) significantly increases the expressions of MGP and proteins in the Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling pathway and MGP mediate the regulation of osteogenosis by PTH.

Objective: To investigate the relationship between miRNA378, calumenin and endoplasmic reticulum stress in suckling mouse myocardium with myocarditis caused by adriamycin., Methods: The suckling mouse myocardium of primary culture were randomly divided into control group, adriamycin group, lentivirus infection miRNA378 over expression control group, lentivirus infection miRNA378 over expression group, lentivirus infection miRNA378 silence control group and lentivirus infection miRNA378 silence group. Firstly to identify the suckling mouse myocardium, α-SMA was monitored by immunohistochemical method, and secondly the ventricular myocytes were transfered by lentivirus plasmids. Then the expression change of miRNA378, calumenin and glucose regulated protein 78(GRP78) mRNA were detected by Quantitative Real-time PCR., Results: Compared with the adriamycin infection group, the expression of calumenin mRNA in lentivirus infection miRNA378 over expression group was increased( P <0.01), while the expression of GRP78 mRNA was reduced( P <0.01); Compared with the adriamycin infection group, the expressions of calumenin and GRP78 mRNA in lentivirus infection miRNA378 silence group did not change insignificantly., Conclusions: Adriamycin injection may cause expression of calumenin in suckling mouse myocardium with myocarditis reduced, which may lead to the endoplasmic reticulum stress. This effect is related with miRNA378.

Objective: To explore the expression of S100A14 in breast cancer tissue, and the EGF and S100A14 feedback regulatory mechanism., Methods: S100A14 mRNA level in 52 cases of of breast cancer and adjacent normal tissue was detected by quantitative real-time PCR. S100A14 protein in 21 cases of breast cancer and adjacent normal tissue was detected by Western blot. S100A14 mRNA after EGF treatment was detected by RT-PCR and real-time PCR. The levels of S100A14, p-ERK and t-ERK were detected by Western blot. Knocking down S100A14 expression was performed by siRNA technology., Results: The levels of S100A14 mRNA and protein were significantly increased in breast cancer tissues (P<0.05 for both). The high expression of S100A14 was related with the recurrence of breast cancer patients (P= 0.038). S100A14 mRNA level was significantly up-regulated in the MDA-MB-453 cells (1.50±0.11) and MCF-7 cells (1.40±0.03) after 1 ng/mL EGF treatment, and 1.66±0.08 and 1.71±0.17 in the MDA-MB-453 cells after 10 ng/mL EGF treatment, significantly higher than that of the control group (1.00±0.09 and 1.00±0.03) (P<0.05 for both). In the TD47 cells, the S100A14 mRNA levels in the control, 1 ng/ml EGF and 10 ng/ml EGF + U0126 treatment groups were 1.00±0.04, 1.56±0.04 and 1.00±0.10, respectively (P<0.05)., Conclusions: The expression of S100A14 mRNA and protein is promoted by EGF through p-ERK signaling pathway in breast cancer cells. There may be a feedback loop between EGF and S100A14.

Objective: To investigate the effect of total flavonoids of astragalus on the expression of endoplasmic reticulum chaperone, calumenin and connecxin 43 (CX43) in suckling mouse myocardium with myocarditis caused by coxsackievirus B3 (CVB3)., Methods: The primary culture of suckling mouse myocardium cells were randomly divided into control group, CVB3 infected group and total flavonoids of astragalus group. Firstly, to confirm the identity of the suckling mouse myocardium, α-SMA was monitored by immunohistochemistry method. Then the protein expression changes of endoplasmic reticulum chaperone-glucose regulatory protein 78 ( GRP78), calumenin and CX43 were detected by Western blot., Results: (1) Compared with that of the control group, the GRP78 expression level in CVB3 infected group was improved, the expression levels of calumenin and CX43 were all reduced. (2) Compared with that of CVB3 infected group, GRP78 expression level was decreased, and the expression levels of calumenin and CX43 were increased in total flavonoids of astragalus group., Conclusion: CVB3 infection may cause endoplasmic reticulum stress of rat myocardium cells by increasing the expression of GRP78 and decreasing the expression of calumenin and CX43. On the other hand, total flavonoids of astragalus can reduce the expression of GRP78 and increase the expression of calumenin and CX43.The results of this experiment may be closely related to the effects of anti-arrhythmia with viral myocarditis caused by CVB3.

Objective: To investigate the effects of triptolide on Notch receptor and ligand expressions in rats with adjuvant-induced arthritis (AA)., Methods: Forty rats were randomly divided into normal control (NC) group, model (MC) group, methotrexate group and triptolide groups. Rat models of AA were established by an intradermal injection of 0.1 mL Freund's complete adjuvant into the right paw. Twelve days after the injection, the rats were treated with corresponding drugs for 30 days; the rats in NC group and MC group were given saline only. Paw edema volume (E), arthritis index (AI), pulmonary function, histomorphologies, and Notch receptor/ ligand expression in the lung tissue were analyzed after the treatments., Results: Compared with the NC group, E, AI, Notch3, Notch4, and Delta1 expressions in the lung tissues significantly increased while pulmonary function and pulmonary expressions of Notch1, Jagged1, and Jagged2 significantly decreased the model rats (P<0.01). Compared with the MC group, triptolide-treated rats showed significantly improved pulmonary functions, increased expressions of Notch1, Jagged1, and Jagged2 and decreased expressions of Notch3, Notch4, and Delta1 in the lungs (P<0.05, P<0.01); the therapeutic effect of triptolide was better than that of methotrexate., Conclusion: Triptolide can reduce inflammatory reaction and immune complex deposition to improve joint and pulmonary symptoms in rats with AA possibly by up-regulating the expressions of Notch3, Notch4, and Delta1 and down-regulating the expressions of Jagged1, Jagged2, and Notch1.

Objective: To evaluate the effect of the platelet-derived growth factor-BB (PDGF-BB) on the phenotypic transformation of corpus cavernosum smooth muscle cells (CCSMC) in SD rats., Methods: CCSMCs were primarily cultured in the modified tissue sticking medium and subjected to immunofluorescence assay. The cells were divided into a blank control and four PDGF-BB groups, the latter exposed to 5, 10, 20, and 40 ng/ml of PDGF-BB, respectively, for 24 hours, and the cells in the 20 ng/ml PDGF-BB group treated for 24, 48, and 72 hours. The the relative expressions of α-SMA, SMMHC, calponin, and OPN mRNA were determined by real-time fluorescence quantitative RT-PCR (qRT-PCR)., Results: The α-SMA positive rate of the CCSMCs was over 95%. Compared with the blank control group, the expression levels of α-SMA, SMMHC, and calponin mRNA were significantly decreased (P < 0.05) while that of OPN mRNA remarkably increased (P < 0.05) in the PDGF-BB groups. The 20 ng/ml PDGF-BB group also showed significantly downregulated expressions of α-SMA, SMMHC, and calponin mRNA (P < 0.05) and upregulated expression of OPN mRNA (P < 0.05) at 24, 48, and 72 hours., Conclusion: PDGF-BB can induce the transformation of the phenotype of CCSMCs in SD rats from the contractile to the synthetic type.

Objective: To observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats., Methods: Thirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues., Results: Compared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure., Conclusion: These results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.

Objective: To study the expression of Notch1, Jagged1 and Notch intracellular domain (NICD)in epithelial ovarian carcinoma tissues and analyze the clinical significance. To explore the activity of γ-secretase in epithelial ovarian carcinoma cell line SKOV3 and the effect of N-[N-(3, 5-dil uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor on the activity of γ-secretase in SKOV3., Methods: Immunohistochemistry staining method was performed in 43 patients with epithelial ovarian carcinoma and 11 patients with benign epithelial ovarian tumor to detect the expression of Notch1, Jagged1 and NICD. The differences of expressionof Notch1, Jagged1 and NICD between malignant and benign ovarian tumors was compared and alsoanalyzed the correlation with clinicopathological parameters of ovarian carcinoma. Human serous ovarian cancer cell line SKOV3 and immortalized nontumorigenic ovarian epithelial cell line T29 were incubated in vitro. The activities of γ-secretase in SKOV3 and T29 with dimethyl sulfoxide (DMSO) and DAPT were detected respectively by Gal4-VP16/UAS and dual luciferase reporter assay system., Results: (1) The immunohistochemical composite scores (ICS) of Notch1 in epithelial ovarian carcinoma (6.7 ± 2.2) were not significantly different with those in benign epithelial ovarian tumor (5.4 ± 2.7, P = 0.153), while the ICS of Jagged1 and NICD in epithelial ovarian carcinoma (5.3 ± 2.4, 5.3 ± 2.3)were higher than those in benign epithelial ovarian tumor (1.6 ± 1.4, 3.1 ± 1.7; all P < 0.01). The expression of Notch1, Jagged1 and NICD had no correlation with patients' aged, history of carcinoma, ascites, the level of serum CA125, maximum length of ovarian tumor, Federation International of Gynecology and Obstetrics (FIGO) stage, grade and pathology subtypes (all P > 0.05). The hazard ratio between the high expression of Notch1, Jagged1, or NICD and the moderate to low expression of Notch1, Jagged1, or NICD, and Jagged1 were 0.771, 1.648 and 1.316, respectively (all P > 0.05). The 5-year survival rate and median survival time between the high expression of Notch, Jagged1 or NICD in subgroup and moderate to low expression in subgroup were of no difference (all P > 0.05). The activity of γ-secretase in SKOV3 was significantly higher than that in T29([ 12.2 ± 1.4)%, P = 0.019]. (2) After DAPT treated, the relative activity of γ-secretase in SKOV3 (50 µmol/L) was declined from (100.0 ± 5.3)% to(6.6 ± 0.8)% (P = 0.001)., Conclusions: Jagged1 and NICD in Notch1 pathway may play a key role in the occurrence of ovarian carcinoma. The activity of γ-secretase in epithelial ovarian carcinoma was higher than that in ovarian epithelial cell which suggest that DAPT, γ-secretase inhibitor, may become the target of ovarian carcinoma treatment.

Objective: To investigate the relationship between sarcoplasmic reticulum Ca²⁺ modulation proteins and postresuscitation myocardial dysfunction., Methods: Thirty-eight SPF male Sprague-Dawley (SD) rats were randomly divided into control group (n=12) and cardiac arrest (CA) group (n=26). CA was induced by intravenous bolus of potassium chloride (40 μg/g), and cardiopulmonary resuscitation (CPR) was conducted 8 minutes later. No CA was induced in control group except catheter placement for monitoring cardiopulmonary parameters after anesthesia. Invasive hemodynamic parameters were monitored for 1 hour after CPR. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after restoration of spontaneous circulation (ROSC), and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a), phosphorylated phospholamban (p-PLB) and rynodine receptor (RyR) were determined by Western Blot., Results: ROSC rate of CA group was 92.3% (24/26), and mean recovery time was (68 ± 39) seconds. Cardiac function was significantly impaired in CA group at 1 hour after resuscitation, and ejection fraction, fraction shortening (FS), the maximal rate of left ventricular pressure increase/decline (± dp/dt max) were significantly decreased compared with those in control group [ejection fraction: 0.548 ± 0.060 vs. 0.809 ± 0.043, F=71.692, P=0.000; FS: (34.4 ± 4.4)% vs. (46.0 ± 3.5)%, F=55.443, P=0.000; + dp/dt max (mmHg/s): 4 718 ± 43 vs. 7 098 ± 394, P<0.01; - dp/dt max (mmHg/s): -3 824 ± 612 vs. -6 187 ± 473, P<0.01]. Compared with control group, the expression levels of p-PLB (gray value) was significantly decreased at 5 minutes and 60 minutes (5 minutes: 0.64 ± 0.15 vs. 1.29 ± 0.13, P<0.01; 60 minutes: 0.95 ± 0.08 vs. 1.30 ± 0.09, P<0.05) after resuscitation in CA group, while the level of sarcoplasmic SERCA2a (gray value) and RyR (gray value) showed no significant differences (SERCA2a 5 minutes: 1.01 ± 0.18 vs. 1.24 ± 0.07, 60 minutes: 1.03 ± 0.14 vs. 1.25 ± 0.06; RyR 5 minutes: 0.96 ± 0.13 vs. 0.97 ± 0.13, 60 minutes: 0.88 ± 0.14 vs. 0.99 ± 0.11, all P>0.05)., Conclusions: The impairment of the p-PLB is closely related to postresuscitation myocardial dysfunction.

The aim of the present study was to investigate the abnormal calcium re-uptake function of myocardium sarcoplasmic reticulum (SR) in rabbits with heart failure, as well as potential mechanisms. Heart failure model was established in rabbits through aortic insufficiency and constriction of abdominal aorta. The SR Ca(2+) re-uptake function was measured with a calcium imaging device. The activity of myocardium SR calcium adenodine triphosphatase 2a (SERCA2a) was measured by inorganic phosphate. The protein expressions of SERCA2a, CaMKII, PKA, PP1α, phospholamban (PLB), PLB-Ser(16) and PLB-Thr(17) were evaluated by Western blot. The activities of PKA and CaMKII were detected by γ-(32)P substrate incorporation. The results showed that, compared with the sham operation group, the heart failure group exhibited reduced Ca(2+) re-uptake amount (P < 0.01) and the expression and activity of SERCA2a (P < 0.05 or P < 0.01), decreased expression of PLB and its phosphorylation status in sites of Ser(16) and Thr(17) (P < 0.05), increased expressions and activities of PKA and CaMKII (P < 0.05 or P < 0.01), and increased expression of PP1α (P < 0.05). These results suggest that the abnormal Ca(2+) re-uptake function in heart failure is related with reduced expression and activities of SERCA2a, as well as reduced expression of PLB and its phosphorylation status. Both PLB-Ser(16) and -Thr(17) may be involved in the regulation of myocardium SR calcium pump activity in heart failure.

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Objective: To detect the expression of SCUBE3 in human osteosarcoma cell lines and surgical specimens of osteosarcomas and investigate its association with the patients' prognosis., Methods: The expression of SCUBE3 protein was detected in 5 osteosarcoma cell lines using Western blotting. CCK8 assay was used to assess the effect of SCUBE3 suppression mediated by two specific small interfering RNAs (siRNAs) on the proliferation of U2OS osteosarcoma cells, and the cell cycle changes were detected using flow cytometry. Immunohistochemistry was performed to detect the expression of SCUBE3 in 60 osteosarcoma tissues, and Kaplan-Meier method was performed for survival analysis of the patients., Results: Compared with osteoblast hFOB1.19 cells, the osteosarcoma cell lines all showed significantly higher expressions of SCUBE3. In U2OS cells, suppression of SCUBE3 expression by siRNA significantly inhibited the cell proliferation (P<0.05). Kaplan-Meier survival analysis indicated that patients with high SCUBE3 expression had worse prognosis than those with low SCUBE3 expression (P=0.036)., Conclusion: SCUBE3 is up-regulated in the 5 osteosarcoma cell lines and in primary osteosarcoma tissues to promote the proliferation of osteosarcoma cells. A high SCUBE3 expression in osteosarcoma tissues is associated with a poor prognosis of the patients, suggesting that SCUBE3 can serve as a potential therapeutic target for osteosarcoma.

Objective: To establish model of dental pulp cells with activated Notch signaling pathway, and investigate the effect of activating Notch signaling pathway on senescence of human dental pulp cells in vitro., Methods: Human dental pulp cells were isolated, cultured as usual, and used from the 4(th) passage. The cells were divided into the activated group and the negative control group. In the activated group, the way of coating dishes with Jagged1 protein (10 mg/L) was used to activate Notch signaling pathway. The negative control group cells received no treatment. In the 4(th), 8(th), and 10(th) passages, the expression levels of the Notch signaling pathway downstream gene Hes1 were verified by real-time quantitative PCR (RT-qPCR). The cell changes after activating Notch signaling pathway were observed at three levels: (1) The cell morphology changes were observed through invert phase contrast microscope. The cell activity was detected with MTT assay. (2) The alkaline phosphatase (ALP) expression and its activity, and senescence-associated &bgr;-galatosidase (SA-β-Gal) expression were observed with the kit. (3) The expression changes of senescence related genes were verified using RT-qPCR. The difference between the negative control group and the activated group was analyzed using student's t test., Results: The expression level of the downstream gene Hes1 of Notch signaling pathway increased after coating the dishes with Jagged1 protein, indicating the establishment of the model of dental pulp cells with activated Notch signaling pathway. Compared with the negative control group, the aging cells of the activated group appeared relatively late. In the 8(th) and 10(th) passage, the cell activity increased. In the 10th passage, ALP activity increased, but SA-β-Gal expression decreased. p16 gene expression decreased in each passage, and p53 gene expression decreased in the 8(th) and 10(th) passages., Conclusion: Jagged1 could activate Notch signaling pathway effectively. Through activating Notch signaling pathway, the dental pulp cells showed a trend of senescence delay at different levels, such as cell morphology, metabolic enzyme expressions and related gene expressions.

Objective: To observe the changes of pulmonary function and Notch signaling pathway of lung tissues in adjuvant-induced arthritis rats, and to investigate the mechanism of reduced lung function., Methods: A total of 30 rats were randomly divided into a normal group and a model group. Rats in the model group were induced to establish the adjuvant arthritis AA model by intradermally injecting 0.1 mL Freund's complete adjuvant into the right paw. After 30 days, we observed the paw edema volume, arthritis index, pulmonary function, histomorphology, and Notch receptor/ligand of the lung tissue., Results: Compared with the normal group, the paw edema volume, arthritis index, average expiratory flow within 0.3 s (FEV0.3/FVC), and the level of Notch3, Notch4 and Jagged2 of the lung tissue in the model group was significantly increased, while maximum expiratory flow at 50% of vital capacity (FEF50), maximum expiratory flow at 75% of vital capacity (FEF75), forced expiratory flow (PEF) and the expression of Notch1 of Jagged1 and Delta1 in the lung were significantly decreased (P<0.05, P<0.01). There were significant positive correlations between FEV0.3/FVC and Notch4. FEV0.3/FVC, FEF25, FEF50 and Notch3, Delta1 were negatively correlated, respectively (P<0.05, P<0.01)., Conclusion: While arthritis occurs in AA rats, pulmonary function declines and significantly correlates with the expression of Notch receptor/ligand. The deposition of immune complex in the lung after the injection of CFA activates the Notch signaling pathway, and results in further decline of pulmonary function by signaling cascades.

Objective: To investigate the effects of docosahexaenoic acid (DHA) on hypoxia-induced pulmonary arterial hypertension(PAH) and the mechanism., Methods: PAH was induced by chronic intermittent hypoxia for 21 days in vivo. Forty male Sprague-Dawley rats were randomly divided into 4 groups (n = 10, each):a normal control group, DHA-treated groups in normoxia and hypoxia, and a PAH group. At the end of study, mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy and the index of wall thickness of small pulmonary artery (WT% and WA%) among groups were compared. The changes of pulmonary arterial smooth muscle cell (PASMC) proliferation were determined by MTT in vitro. Migration assay was performed using the Boyden chamber. Real-time quantitative PCR was performed to quantify mRNA levels of the smooth muscle cell phenotype markers SM-α-actin, calponin and SM 22α under normoxic or hypoxic conditions, in the absence or presence of DHA., Results: DHA treatment significantly lowered mPAP [(22.7 ± 1.8) mmHg (1 mmHg = 0.133 kPa)], reduced thickening of small pulmonary artery wall [WT%:(21.6 ± 4.1)%, WA%: (52.0 ± 2.9)% ] and alleviated ventricular hypertrophy (34.2 ± 2.2) % compared to those of the hypoxic group (P < 0.05). DHA inhibited the proliferation, migration and phenotype switching of PASMCs induced by hypoxia in vitro., Conclusion: DHA therapy reduced mPAP in a rat model of hypoxia-induced PAH and this effect was linked with inhibition of pulmonary vascular remodelling.

Objective: To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system., Methods: UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1:1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of a-smooth muscle actin (a-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days., Results: The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of alpha-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of a-SMA could be observed in groups A and B; at 14 days, the positive expression of alpha-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of alpha-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining., Conclusion: UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.

Objective: To investigate the morphological features, immunohistochemical phenotype, diagnosis and differential diagnosis of collagenous spherulosis of the breast., Methods: Clinicopathologic observation, immunohistochemistry using EnVision method and histochemical staining were applied in 33 cases of collagenous spherulosis of the breast., Results: Collagenous spherulosis of the breast was a benign lesion, consisting of proliferative myoepithelial and ductal epithelial cells. These cells were arranged in a cribriform pattern with esinophilic, round, oval or star-shaped fibrillary spherules in the lumen.SMA, calponin and p63 by immunohistochemistry identified the proliferative myoepithelium, while E-cadherin identified the proliferative ductal epithelial cells. The esinophilic spherules were stained with collagen type IV, AB-PAS and reticulin. Collagenous spherulosis was often found in sclerosing adenosis., Conclusions: Collagenous spherulosis of the breast is often associated with other diseases. It has special morphological presentation and is easily confused with malignant tumors such as adenoid cystic carcinoma or cribriform carcinoma in situ, and needs to be differentiated from these disease entities.

Objective: To investigate the role of store-operated calcium channels (SOCs) in primary hepatocytes under conditions of calcium overload and ethanol-induced injury., Methods: The in vitro model of chronic ethanol-induced hepatocyte injury was established using primary hepatocytes isolated from Sprague-Dawley rats. Ethanol-induced changes (24, 48 and 72 h; 50, 100, 200, 400 and 800 mmol/L) in expression of the SOCs proteins stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Oria1) were detected by qualitative PCR analysis (mRNA) and western blotting (protein). The possible role of these two SOCs proteins in the ethanol-induced extracellular calcium influx and related liver cell injury was determined by treating the cell system with various channel blockers (EGTA, La3+, and 2-APB). Cell viability was determined by MTT assay and cytosolic free calcium ion concentration was determined by flow cytometry., Results: After 24 h of exposure to 0 (untreated) to 800 mM/L ethanol, the cell viability was reduced in a concentration-dependent manner. The 400 mmol/L concentration of ethanol decreased cell viability by 57.34% +/- 2.34%. and was chosen for use in subsequent experiments. Compared with the untreated control cells, the ethanol-treated cells showed significantly up-regulated mRNA and protein expression of both STIM1 and Orai1 at all times examined, suggesting that the ethanol-stimulated expression of STIM1 and Orai1 could persist for at least 72 h. The ethanol treatment induced increase in cytoplasmic calcium levels was significantly (and similarly) reduced by co-treatment with any of the three channel blockers., Conclusion: Chronic ethanol exposure can increase the expression of STIM1 and Orai1 in primary liver cells, suggesting that ethanol may increase extracellular calcium influx by up-regulating expression of these SOCs protein molecules, ultimately aggravating liver cell damage.

Objective: To investigate the role of micro RNA-451 (miRNA-451) in promoting the osteogenesis of mesenchymal stem cells (MSCs) by targeting regulatory calcium binding protein 39 (CAB39)., Methods: pMIR-report and pRL-TK vectors were selected to identify the relationship between miRNA-451 and CAB39 by using dual-luciferase reporter assay. pre-miRNA-451 (group A), anti-miRNA-451 (group C), pre-miRNA negative control (group B), and anti-miRNA negative control (group D) were transfected into the C3H10T1/2 cells, respectively. Then, the cells were collected after osteogenic induction for 7 and 14 days. At 7 and 14 days, the real-time fluorescent quantitative PCR and Western blot assays were performed to detect the related osteogenetic biomarkers [Runx2 and alkaline phosphatase (ALP) mRNA] and expressions of CAB39 protein. At 14 days, the extracellular calcium deposition during the osteogenesis of MSCs was tested by Alizarin red staining method., Results: CAB39 was the target gene of miRNA-451. At 7 and 14 days after osteogenic induction, the mRNA expressions of Runx2 and ALP in group A were significantly higher than those in group B (P < 0.05), and the expressions in group C was significantly lower than those in group D (P < 0.05). Furthermore, at 14 days after osteogenic induction, the protein expression of CAB39 in group A (0.55 +/- 0.05) was significantly lower than that in group B (1.00 +/- 0.07), and the protein expression in group C (1.21 +/- 0.05) was significantly higher than that in group D (1.00 +/- 0.04), all showing significant difference (P < 0.05). Finally, at 14 days after osteogenic induction, the extracellular calcium deposition in group A was obviously more than that in group B, and group C was downregulated when compared with group D., Conclusion: miRNA-451 can promote the osteogenesis process of MSCs by downregulating the CAB39.

Objective: To investigate effects of paraquat on the mRNA expression of key elements of Notch signaling (Notch1, Jagged1 and DTX1) during differentiation process of human neural stem cells (hNSCs)., Methods: hNSCs exposed to PQ at the concentrations 0.10, 1.00, 10.00 M. Cell proliferation ability was assessed using MTT assay and mRNA expressions of Notch1, Jagged1 and DTX1 were detected by Real-time RT-PCR at 2, 4, 8, 12 d of differentiation., Results: Compared with control group, NOTCH1, JAG1 mRNA expression levels exposed to PQ at the concentration of 0.10 M significantly reduced at 2, 4, 8 d and significantly went up at 12d (P < 0.01). Compared with control group, NOTCH1, JAG1 and DTX1 mRNA expression levels exposed to PQ at the concentration of 10.00 M significantly reduced at 2, 8, 12 d (P < 0.01). PQ could down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the early stage of differentiation, then up-regulate Notch1 mRNA expression, and down-regulate Notch1, Jagged1 and DTX1 mRNA expressions at the end of differentiation., Conclusion: Notch signaling pathway may be involved in differentiation of neural stem cell exposed to PQ.

Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.

Objective: To study the changes in diaphragmatic function and gene expressions of calcium regulatory proteins in diabetic rats and explore the mechanism of diaphragm dysfunction in diabetes mellitus., Methods: SD rats were randomly divided into normal control group and diabetic (induced by intraperitoneal STZ injection) group. After 4 and 8 weeks, the body weight and diaphragm to body weight ratio were measured, and the activities of succinic dehydrogenase (SDH) in the diaphragm and blood glucose were assayed. The diaphragm contractility was assessed and the alterations of diaphragm ultrastructure were observed. RT-PCR was used to detect the changes in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLB) mRNA expressions in the diaphragm., Results: The diabetic rats showed a significant weight loss with a lowered diaphragm to body weight ratio (P<0.01) and SDH activity (P<0.01). The peak twitch tension and maximum tetanic tension of the diaphragm were significantly lowered and the time to peak contraction and half relaxation time significantly prolonged (P<0.01) in the diabetic rats, which also exhibited a lowered tetanic force in response to stimulus (P<0.01). Transmission electron microscopy revealed obvious ultrastructural changes of the diaphragm in diabetic rats. RT-PCR showed significantly decreased SERCA and increased PLB mRNA expressions in diabetic rat diaphragm (P<0.01), and these changes intensified with time (P<0.01)., Conclusion: Diabetes can cause impairment of diaphragmatic ultrastructure, mitochondrial injuries, and lowered SDH activity and ATP production. Decreased SERCA and increased PLB mRNA expressions in diabetes result in reduced Ca(2+) uptake by the diaphragm sarcoplasmic reticulum to induce diaphragm dysfunction.

Objective: To investigate the effect of estrogen on expression of matrix GLA protein (MGP) in ovariectomized Sprague-Dawley (SD) rats and the role of estrogen in improving postmenopausal osteoporosis., Methods: Thirty-six SD female rats were allocated into 3 groups randomly, every 12 rats in ovariectomized group (OVX group), estrogen group (E group) and control group (sham group). Rats in OVX and E group all underwent bilateral ovariectomy, those rats in E group were given by estradiol benzoate intramuscularly after 3 weeks of ovariectomy. Rats in sham group underwent bilateral lipectomy near the ovary. All rats were kept the urine and the serum every three weeks and were sacrificed after 15 weeks. The pathology changes of uterus, lumbar vertebral bones were observed by immunohistochemistry. Bone mineral density (BMD) of lumbar vertebra of rats was determined by dual energy X ray absorptiometry (DEXA). The content of MGP in serum and urine was determined by ELISA. Expression of undercarboxylated matrix GLA Protein (MGP) was detected by immunohistochemistry. Relative quantification of MGP mRNA expression in lumbar vertebra bone was detected by Fluorescent real-time quantitative polymerase chain reaction., Results: (1) After 15 weeks of ovariectomized, the endometrium of uterus and lumbar vertebra exhibit remarkable pathologic changes in OVX group. The serum estrogen of (454 ± 66) pmol/L in OVX group were lower than in (527 ± 77) pmol/L in sham group and (556 ± 80) pmol/L in E group significantly (P < 0.05). The BMD of lumbar vertebra of (0.263 ± 0.030) g/cm(2) in OVX group were lower than (0.295 ± 0.024) g/cm(2) in sham group and (0.279 ± 0.024) g/cm(2) in E group significantly (P < 0.01). (2) The serum MPG protein in OVX group and E group showed decreased trends after ovariectomized, which were (104 ± 64) ng/L in OVX group and (134 ± 6) ng/L in E group at 9 weeks, which reached statistical difference (P < 0.05). However, MGP in urine in sham group did not exhibit significant difference after 15 weeks of surgery (P > 0.05). The MGP in urine of E group showed increased trends after 12 weeks of surgery, which reached (110.0 ± 3.4) ng/L at 15 weeks, in the mean time, it was found that (86.5 ± 2.5) ng/L of MGP in urine in OVX group, which showed significant difference (P < 0.05). (3) MGP could be observed in lumbar vertebra in OVX group by immunochemistry staining. In the other two groups, the expression of MGP was not dominant. (4) Relative quantification of MGP mRNA expression in lumbar vertebra was defined as 1 in OVX group, when compared with 0.289 ± 0.260 in E group and 0.103 ± 0.098 in sham group, the difference showed statistically significant (P < 0.01)., Conclusion: Estrogen could increase the expression of MGP mRNA and protein in ovariectomized rats and might play an important role in improving postmenopausal osteoporosis.