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There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research. In this study, we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes: Actb, Ankrd27, CypA, Gapdh, Hprt1, Mrpl10, Pgk1, Rictor, Rn18s, Tbp, Ubc, Ubxn11, and Ywhaz. Our results show that the expression of Ankrd27, Ubc, and Tbp were stable after spinal cord injury, while Actb was the most unstable internal control gene. Ankrd27, Ubc, Tbp, and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression. Target gene expression levels and changes over time were similar when Ankrd27, Ubc, and Tbp were used as internal controls but different when Actb was used as an internal control. We recommend that Ankrd27, Ubc, and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research. This study was approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (approval No. 20180304-008) on March 4, 2018.

Peripheral nerve injury triggers sequential phenotype alterations in Schwann cells, which are critical for axonal regeneration. Long noncoding RNAs (lncRNAs) are long transcripts without obvious coding potential. It has been reported that lncRNAs participate in diverse biological processes and diseases. However, the role of lncRNA in Schwann cells and peripheral nerve regeneration is unclear. Here, we identified an lncRNA, loc680254, which is upregulated in rat sciatic nerve after peripheral nerve injury. The loc680254 knockdown inhibits Schwann cell proliferation, enhances apoptosis, and hinders cell cycle, while loc680254 overexpression has the opposite effect. Mechanically, we found that loc680254 might act as a microRNA sponge to regulate the expression of mitosis-related gene, spindle and kinetochore associated complex subunit 1 (Ska1) and proline/serine-rich coiled-coil 1 (Psrc1). Silencing of Psrc1 or Ska1 attenuates the effect of loc680254 overexpression on Schwann cell proliferation. Finally, we repaired the rat sciatic nerve gap with chitosan scaffolds loaded with loc680254-overexpressing Schwann cells and evaluated axon regeneration and functional recovery. Our results indicated that loc680254 is a new potential modulator for Schwann cell proliferation, which could be targeted to develop novel therapeutic strategies for peripheral nerve repair.

Background Growth factors execute essential biological functions and affect various physiological and pathological processes, including peripheral nerve repair and regeneration. Our previous sequencing data showed that the mRNA coding for betacellulin (Btc), an epidermal growth factor protein family member, was up-regulated in rat sciatic nerve segment after nerve injury, implying the potential involvement of Btc during peripheral nerve regeneration. Methods Expression of Btc was examined in Schwann cells by immunostaining. The function of Btc in regulating Schwann cells was investigated by transfecting cultured cells with siRNA segment against Btc or treating cells with Btc recombinant protein. The influence of Schwann cell-secreted Btc on neurons was determined using a co-culture assay. The in vivo effects of Btc on Schwann cell migration and axon elongation after rat sciatic nerve injury were further evaluated. Results Immunostaining images and ELISA outcomes indicated that Btc was present in and secreted by Schwann cells. Transwell migration and wound healing observations showed that transfection with siRNA against Btc impeded Schwann cell migration while application of exogenous Btc advanced Schwann cell migration. Besides the regulating effect on Schwann cell phenotype, Btc secreted by Schwann cells influenced neuron behavior and increased neurite length. In vivo evidence supported the promoting role of Btc in nerve regeneration after both rat sciatic nerve crush injury and transection injury. Conclusion Our findings demonstrate the essential roles of Btc on Schwann cell migration and axon elongation and imply the potential application of Btc as a regenerative strategy for treating peripheral nerve injury.

Cisplatin (DDP) is a commonly used chemotherapy drug, but DDP can cause skeletal muscle atrophy and cachexia. This study combined the DDP-induced C2C12 myotube atrophy model and the LLC tumour-bearing mouse cachexia model under DDP treatment to explore the effect and mechanism of naringenin (NAR) in alleviating skeletal muscle atrophy and cachexia. NAR alleviated the decreases in body weight, food intake, muscle, adipose tissue and kidney weight and forelimb grip strength in LLC tumour-bearing mice under DDP treatment and reduced the serum level of TNF-α. High dose NAR also enhanced the antitumour effect of DDP. NAR can prevent the cross-sectional area reduction of skeletal muscle fibres and prevent the change in fibre type ratio induced by DDP. NAR intervention inhibits the RIPK1/AMPK/NF-κB pathway in skeletal muscle, downregulates the expression of Atrogin1 and MuRF1, and inhibits the degradation of skeletal muscle protein. In C2C12 myotubes treated with DDP, NAR inhibited the RIPK1/AMPK/NF-κB pathway to reduce myotube cell atrophy, and the overexpression of RIPK1 reversed the therapeutic effect of NAR. These results indicate that NAR downregulates the expression of Atrogin1 and MuRF1 by regulating the RIPK1/AMPK/NF-κB pathway and alleviates DDP-induced skeletal muscle atrophy.

Quantitative assessment of the recovery of nerve function, especially sensory and autonomic nerve function, remains a challenge in the field of nerve regeneration research. We previously found that neural control of vasomotor activity could be potentially harnessed to evaluate nerve function. In the present study, five different models of left sciatic nerve injury in rats were established: nerve crush injury, nerve transection/suturing, nerve defect/autografting, nerve defect/conduit repair, and nerve defect/non-regeneration. Laser Doppler perfusion imaging was used to analyze blood perfusion of the hind feet. The toe pinch test and walking track analysis were used to assess sensory and motor functions of the rat hind limb, respectively. Transmission electron microscopy was used to observe the density of unmyelinated axons in the injured sciatic nerve. Our results showed that axonotmesis-evoked vasodilatation in the foot 6 months after nerve injury/repair recovered to normal levels in the nerve crush injury group and partially in the other three repair groups; whereas the nerve defect/non-regeneration group exhibited no recovery in vasodilatation. Furthermore, the recovery index of axonotmesis-evoked vasodilatation was positively correlated with toe pinch reflex scores and the density of unmyelinated nerve fibers in the regenerated nerve. As C-fiber afferents are predominantly responsible for dilatation of the superficial vasculature in the glabrous skin in rats, the present findings indicate that axonotmesis-evoked vasodilatation can be used as a novel way to assess C-afferent function recovery after peripheral nerve injury. This study was approved by the Ethics Committee for Laboratory Animals of Nantong University of China (approval No. 20130410-006) on April 10, 2013.

Following severe spinal cord injury (SCI), dysregulated neuroinflammation causes neuronal and glial apoptosis, resulting in scar and cystic cavity formation during wound healing and ultimately the formation of an atrophic microenvironment that inhibits nerve regrowth. Because of this complex and dynamic pathophysiology, a systemic solution for scar- and cavity-free wound healing with microenvironment remodeling to promote nerve regrowth has rarely been explored. A one-step solution is proposed through a self-assembling, multifunctional hydrogel depot that punctually releases the anti-inflammatory drug methylprednisolone sodium succinate (MPSS) and growth factors (GFs) locally according to pathophysiology to repair severe SCI. Synergistically releasing the anti-inflammatory drug MPSS and GFs in the hydrogel depot throughout SCI pathophysiology protects spared tissues/axons from secondary injury, promotes scar boundary- and cavity-free wound healing, and results in permissive bridges for remarkable axonal regrowth. Behavioral and electrophysiological studies indicate that remnants of spared axons, not regenerating axons, mediate functional recovery, strongly suggesting that additional interventions are still required to render the rebuilt neuronal circuits functional. These findings pave the way for the development of a systemic solution to treat acute SCI.

Background Complex cryptoglandular perianal fistula (CPAF) is a kind of anal fistula that may cause anal incontinence after surgery. Minimally invasive surgery of anal fistula is constantly emerging. Over the past 20 years, there are several sphincter-sparing surgeries, one of which is autologous adipose-derived stem cell (ADSC) transplantation. However, to date, there is no study regarding the treatment of complex CPAF with ADSC in China. This is the first study in China on the treatment of complex CPAF with ADSC to evaluate its safety and efficacy. Methods Totally, 24 patients with complex CPAF were enrolled in this prospective case-control study from January 2018 to December 2019 in the National Colorectal Disease Center of Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine. Patients were divided into ADSC group and endorectal advancement flap (ERAF) group according to their desire. The healing of fistulas (healing of all treated fistulas at baseline, confirmed by doctor’s clinical assessment and magnetic resonance imaging or transrectal ultrasonography) was evaluated at week 12 after treatment. In addition to their safety evaluation based on adverse events monitored at each follow-up, the patients were also asked to complete some scoring scales at each follow-up including pain score with visual analog score (VAS) and anal incontinence score with Wexner score. Results The closure rates within ADSC group and ERAF group at week 12 were 54.55% (6/11) and 53.85% (7/13), respectively, without significant difference between them. VAS score in ADSC group was significantly lower than that in ERAF group at the 5th day postoperatively [1(0,2) VS 2(2,4), p = 0.011], but no differences were observed at the other time. Wexner score of all patients was not increased with no significant differences between the two groups. Adverse events were observed fewer in ADSC group (27.27%) than that in ERAF group (53.85%), but there was no significant difference between them. Conclusion This study indicated safety and efficiency of ADSC for the treatment of complex CPAF in the short term, which is not inferior to that of ERAF. ADSC may provide a promised and potential treatment for complex CPAF conforming to the future of the treatment, which is reconstruction and regeneration. Trail registration ChiCTR, ChiCTR1800014599. Registered 23 January 2018—retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=24548

BackgroundVasomotor regulation of dermal blood vessels, which are critical in the function of the skin in thermoregulatory control, involves both neural and non-neural mechanisms. Whereas the role of sympathetic nerves in regulating vasomotor activities is comprehensively studied and well recognized, that of sensory nerves is underappreciated. Studies in rodents have shown that severance of the sciatic nerve leads to vasodilatation in the foot, but whether sympathetic or sensory nerve fibers or both are responsible for the neurogenic vasodilatation remains unknown.ResultsIn adult Sprague–Dawley rats, vasodilatation after transection of the sciatic nerve gradually diminished to normal within 3–4 days. The neurotmesis-induced neurogenic vasodilatation was not detectable when the sciatic nerve was chronically deafferentated by selective resection of the dorsal root ganglia (DRGs) that supply the nerve. Specific activation of C-afferents by intra-neural injection of capsaicin resulted in neurogenic vasodilatation to a magnitude comparable to that by neurotmesis, and transection of the sciatic nerve pre-injected with capsaicin did not induce further vasodilatation.ConclusionsOur results collectively indicate that vasodilatation after traumatic nerve injury in rats is predominantly mediated by C-fiber afferents.

The effect of subclinical hyperthyroidism (SH) on bone mineral density (BMD) remains unclear, as do the linking mechanisms. This review aims to investigate the relationship between SH and bone loss in terms of the gender-dependent effects of SH on BMD. The PUBMED, EMBASE, OVID, MEDLINE, SINOMED and COCHRANE LIBRARY databases (inception to August 12, 2019) were searched for cohort studies investigating the effects of SH on BMD. Eligible studies were subjected to qualitative and quantitative analysis using a random-effects model meta-analysis with the Cochrane systematic evaluation method. Twelve cohort studies involving 275,086 participants who were followed for 3 months to 13 years were included based on predefined inclusion and exclusion criteria. The results indicated that SH did not affect lumbar spine BMD in females or males. However, a significant reduction in femoral neck BMD was observed in females, but not in males. Further, there was a significant increase in hip fractures events in both females and males with SH. The present findings indicate that SH is significantly associated with hip fracture risk, and therefore, it is important to assess the risk of fractures in patients with SH. Future studies should focus on methods for accurately determining this risk in patients with SH and providing them with timely and efficient diagnosis and treatment.

Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.

As the major glia in PNS, Schwann cells play a critical role in peripheral nerve injury repair. Finding an efficient approach to promote Schwann cell activation might facilitate peripheral nerve repair. Long noncoding RNAs (lncRNAs) have been shown to regulate gene expression and take part in many biological processes. However, the role of lncRNAs in peripheral nerve regeneration is not fully understood. In this study, we obtained a global lncRNA portrayal following sciatic nerve injury in male rats using microarray and further investigated one of these dys-regulated lncRNAs, TNXA-PS1, confirming its vital role in regulating Schwann cells. Silencing TNAX-PS1 could promote Schwann cell migration and mechanism analyses showed that TNXA-PS1 might exert its regulatory role by sponging miR-24–3p/miR-152–3p and affecting dual specificity phosphatase 1 (Dusp1) expression. Systematic lncRNA expression profiling of sciatic nerve segments following nerve injury in rats suggested lncRNA TNXA-PS1 as a key regulator of Schwann cell migration, providing a potential therapeutic target for nerve injury repair. SIGNIFICANCE STATEMENT The PNS has an intrinsic regeneration capacity after injury in which Schwann cells play a crucial role. Therefore, further exploration of functional molecules in the Schwann cell phenotype modulation is of great importance. We have identified a set of dys-regulated long noncoding RNAs (lncRNAs) in rats following sciatic nerve injury and found that the expression of TNXA-PS1 was significantly downregulated. Mechanically analyses showed that TNXA-PS1 might act as a competing endogenous RNA to affect dual specificity phosphatase 1 (Dusp1) expression, regulating migration of Schwann cells. This study provides for the first time a global landscape of lncRNAs following sciatic nerve injury in rats and broadens the known functions of lncRNA during nerve injury. The investigation of TNXA-PS1 might facilitate the development of novel targets for nerve injury therapy.

The proliferation and migration of Schwann cells are critical for the repair and regeneration of injured peripheral nerves. Noncoding RNAs, especially microRNAs (miRNAs), have been demonstrated to participate in regulating the biological behaviors of Schwann cells. Numerous differentially expressed novel miRNAs have been identified in the injured sciatic nerve stumps previously by Solexa sequencing. In the current research, we studied the biological function of a novel miRNA, miR-sc4, in detail. Outcomes from proliferation and migration assays suggested that miR-sc4 played an inhibitory role on the proliferation and migration of Schwann cells. Results from bioinformatic analysis, luciferase reporter assay, and rescue experiments suggested that miR-sc4 executed its effect through directly targeting cyclin-dependent kinase 5 activator 1 (Cdk5r1). Collectively, our current study revealed the biological functions of a novel miRNA, showed the effect of miR-sc4 in Schwann cell phenotypic changes, and thus indicated the involvement of miRNAs in peripheral nerve repair and regeneration.

Increasing the intrinsic regeneration potential of neurons is the key to promote axon regeneration and repair of nerve injury. Therefore, identifying the molecular switches that respond to nerve injury may play critical role in improving intrinsic regeneration ability. The mechanisms by which injury unlocks the intrinsic axonal growth competence of mature neurons are not well understood. The present study identified the key regulatory genes after sciatic nerve crush injury by RNA sequencing (RNA-Seq) and found that the hub gene Vav1 was highly expressed at both early response and regenerative stages of sciatic nerve injury. Furthermore, Vav1 was required for axon regeneration of dorsal root ganglia (DRG) neurons and functional recovery. Kruppel-like factor 2 (Klf2) was induced by retrograde Ca2+ signaling from injured axons and could directly promote Vav1 transcription in adult DRG neurons. The increased Vav1 then promoted axon regeneration by activating Rac1 GTPase independent of its tyrosine phosphorylation. Collectively, these findings break through previous limited cognition of Vav1, and first reveal a crucial role of Vav1 as a molecular switch in response to axonal injury for promoting axon regeneration, which might further serve as a novel molecular therapeutic target for clinical nerve injury repair.

Spinal cord injury (SCI) is a challenging clinical problem worldwide. The cellular state and molecular expression in spinal cord tissue after injury are extremely complex and closely related to functional recovery. However, the spatial and temporal changes of gene expression and regulation in various cell types after SCI are still unclear. Here, we collected the rostral and caudal regions to the lesion at 11 time points over a period of 28 days after rat hemisection SCI. Combining whole-transcriptome sequencing and bioinformatic analysis, we identified differentially expressed genes (DEGs) between spinal cord tissue from injured and sham-operated animals. Significantly altered biological processes were enriched from DEGs in astrocytes, microglia, oligodendrocytes, immune cells, and vascular systems after SCI. We then identified dynamic trends in these processes using the average expression profiles of DEGs. Gene expression and regulatory networks for selected biological processes were also constructed to illustrate the complicate difference between rostral and caudal tissues. Finally, we validated the expressions of some key genes from these networks, including α-synuclein, heme oxygenase 1, bone morphogenetic protein 2, activating transcription factor 3, and leukemia inhibitory factor. Collectively, we provided a comprehensive network of gene expression and regulation to shed light on the molecular characteristics of critical biological processes that occur after SCI, which will broaden the understanding of SCI and facilitate clinical therapeutics for SCI.

The intrinsic regeneration ability of neurons is a pivotal factor in the repair of peripheral nerve injury. Therefore, identifying the key modulators of nerve regeneration may help improve axon regeneration and functional recovery after injury. Unlike for classical transcription factors and regeneration-associated genes, the function of long noncoding RNAs (lncRNAs) in the regulation of neuronal regeneration remains mostly unknown. In this study, we used RNA-Seq-based transcriptome profiling to analyze the expression patterns of lncRNAs and mRNAs in rat dorsal root ganglion (DRG) following sciatic nerve injury. Analyses using the lncRNA-mRNA co-expression network, gene ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway databases indicated that the lncRNA Arrl1 decreases neurite outgrowth after neuronal injury. shRNA-mediated Arrl1 silencing increased axon regeneration both in vitro and in vivo and improved functional recovery of the sciatic nerve. Moreover, inhibiting an identified target gene of Arrl1, cyclin-dependent kinase inhibitor 2B (Cdkn2b), markedly promoted neurite outgrowth of DRG neurons. We also found that Arrl1 acts as a competing endogenous RNA that sponges a Cdkn2b repressor, microRNA-761 (miR-761), and thereby up-regulates Cdkn2b expression during neuron regeneration. We conclude that the lncRNA Arrl1 affects the intrinsic regeneration of DRG neurons by derepressing Cdkn2b expression. Our findings indicate a role for an lncRNA-microRNA-kinase pathway in the regulation of axon regeneration and functional recovery following peripheral nerve injury in rats.

Tract tracing with neuronal tracers not only represents a straightforward approach to identify axonal projection connection between regions of the nervous system at distance but also provides compelling evidence for axonal regeneration. An ideal neuronal tracer meets certain criteria including high labeling efficacy, minimal neurotoxicity, rapid labeling, suitable stability in vivo, and compatibility to tissue processing for histological/immunohistochemical staining. Although labeling efficacy of commonly used fluorescent tracers has been studied extensively, neurotoxicity and their effect on neural functions remains poorly understood. In the present study, we comprehensively evaluated motor and sensory nerve function 2–24 weeks after injection of retrograde tracer Fluoro-Gold (FG), True Blue (TB) or Fluoro-Ruby (FR) in the tibial nerve in adult Spague-Dawley rats. We found that motor and sensory nerve functions were completely recovered by 24 weeks after tracer exposure, and that FG lead to a more prolonged delay in functional recovery than TB. These findings shed light on the long-term effect of tracers on nerve function and peripheral axonal regeneration, and therefore have implications in selection of appropriate tracers in relevant studies.

Recent research has found that long noncoding RNAs (lncRNAs) were involved in various human cancers. However, the role of these lncRNAs in cervical cancer remains unexplored. Therefore, we aimed to investigate the biological function of maternally expressed gene 3 (MEG3), a cancer-related lncRNA, and its underlying mechanism in cervical cancer. In this study, MEG3 expression of 108 patients' cervical cancer tissues and adjacent normal tissues was detected by quantitative real-time PCR analysis (qRT-PCR), and the functional effect of MEG3 was determined in vitro assays. We observed that MEG3 was downregulated in cervical cancer tissues, compared to the adjacent normal tissues, and was negatively related with FIGO stages, tumor size, lymphatic metastasis, HR-HPV infection and the expression of homo sapiens microRNA-21 (miR-21). Furthermore, we focused on the function and molecular mechanism of MEG3, finding that overexpression of MEG3 reduced the level of miR-21-5p expression, causing inhibition of proliferation and increased apoptosis in cervical cancer cells. In summary, our findings indicate that MEG3 function as a tumor suppressor by regulating miR-21-5p, resulting in the inhibition of tumor growth in cervical cancer. As a result, this study improves our understanding of the function of MEG3 in cervical cancer and will help to provide new potential target sites for cervical cancer treatment.

Peripheral nerve injury is a common clinical problem. Nerve growth factor (NGF) promotes peripheral nerve regeneration, but its clinical applications are limited by several constraints. In this study, we found that the time-dependent expression profiles of eight let-7 family members in the injured nerve after sciatic nerve injury were roughly similar to each other. Let-7 microRNAs (miRNAs) significantly reduced cell proliferation and migration of primary Schwann cells (SCs) by directly targeting NGF and suppressing its protein translation. Following sciatic nerve injury, the temporal change in let-7 miRNA expression was negatively correlated with that in NGF expression. Inhibition of let-7 miRNAs increased NGF secretion by primary cultured SCs and enhanced axonal outgrowth from a coculture of primary SCs and dorsal root gangalion neurons. In vivo tests indicated that let-7 inhibition promoted SCs migration and axon outgrowth within a regenerative microenvironment. In addition, the inhibitory effect of let-7 miRNAs on SCs apoptosis might serve as an early stress response to nerve injury, but this effect seemed to be not mediated through a NGF-dependent pathway. Collectively, our results provide a new insight into let-7 miRNA regulation of peripheral nerve regeneration and suggest a potential therapy for repair of peripheral nerve injury.

The repair of peripheral nerve laceration injury to obtain optimal function recovery remains a big challenge in the clinic. Misdirection of regenerating axons to inappropriate target, as a result of forced mismatch of endoneurial sheaths in the case of end-to-end nerve anastomosis or nerve autografting, represents one major drawback that limits nerve function recovery. Here we tested whether tubulation repair of a nerve defect could be beneficial in terms of nerve regeneration accuracy and nerve function. We employed sequential retrograde neuronal tracing to assess the accuracy of motor axon regeneration into the tibial nerve after sciatic nerve laceration and entubulation in adult Sprague-Dawley rats. In a separate cohort of rats with the same sciatic nerve injury/repair protocols, we evaluated nerve function recovery behaviorally and electrophysiologically. The results showed that tubulation repair of the lacerated sciatic nerve using a 3-6-mm-long bioabsorbable guidance conduit significantly reduced the misdirection of motor axons into the tibial nerve as compared to nerve autografting. In addition, tubulation repair ameliorated chronic flexion contracture. This study suggests that tubulation repair of a nerve laceration injury by utilizing a bioresorbable nerve guidance conduit represents a potential substitute for end-to-end epineurial suturing and nerve autografting.

Pharmacological treatment is a therapeutic approach to improving nerve regeneration and functional recovery after peripheral nerve crush injury. The objective of the present study was to investigate the effects of the polypeptides isolated from Achyranthes bidentata Blume (abbreviated as ABPP) on rat sciatic crush injury and to test the possible involvement of neurotrophic factors. After surgical crush injury, rats received daily intraperitoneal injection of 0.2 ml saline containing 2 mg ABPP, 1 μg nerve growth factor (NGF) or no additive. The results from walking track analysis, electrophysiological assessment and histological evaluation indicated that the repair outcomes by ABPP treatment were close to those by NGF treatment, but better than those by treatment with saline alone. The quantitative real-time RT-PCR was used to monitor the mRNA expression of growth associated protein in the crush nerves and the mRNA expression of NGF, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine kinase (Trk)A and TrkB in the dorsal root ganglia (DRGs) at L4-L6. The mRNA expression of these genes in the crush nerve sample and DRGs sample was higher after treatment with ABPP or NGF than after treatment with saline alone. Our findings suggest that ABPP might protect peripheral nerve against crush injury through stimulating release of neurotrophic factors and the other cytokines.

Following peripheral nerve injury, hypoxia is formed as a result of defects in blood supply at the injury site. Despite accumulating evidence on the effects of microRNAs (miRNAs) on phenotype modulation of Schwann cells (SCs) after peripheral nerve injury, the impact of hypoxia on SC behaviors through miRNAs during peripheral nerve regeneration has not been estimated. In this study, we confirmed our previous microarray data on the upregulation of miR-132 after sciatic nerve injury in rats and observed that overexpression of miR-132 significantly promoted cell migration of primary cultured SCs. Interestingly, hypoxia-increased expression of miR-132 also enhanced SC migration while inhibition of miR-132 suppressed hypoxia-induced increase in SC migration. miR-132 downregulated PRKAG3 through binding to its 3'-UTR, and PRKAG3 knockdown compromised the reducing effect of miR-132 inhibition on SC migration under normal or hypoxia condition. Moreover, in vivo injection of miR-132 agomir into rats with sciatic nerve transection accelerated SC migration from the proximal to distal stump. Overall, our results suggest that the hypoxia-induced upregulation of miR-132 could promote SC migration and facilitate peripheral nerve regeneration.

Peripheral nerve injury triggers the dysregulation of a large number of genes at multiple sites, including neurons, peripheral nerve stump, and the target organ. Housekeeping genes were frequently used as reference genes to normalize the expression values of target genes. Suitable selection of housekeeping genes that are stably expressed after nerve injury minimizes bias elicited by reference genes and thus helps to better and more sensitively reflect gene expression changes. However, many housekeeping genes have been used as reference genes without testing the expression patterns of themselves. In the current study, we calculated the expression stability of nine commonly used housekeeping genes, such as 18S (18S ribosomal RNA), Actb (β-actin), CypA (cyclophilin A), Gapdh (glyceraldehydes-3-phosphate dehydrogenase), Hprt (hypoxanthine guanine phosphoribosyl transferase), Pgk1 (phosphoglycerate kinase 1), Tbp (TATA box binding protein), Ubc (ubiquitin C), YwhaZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation), and four newly identified housekeeping genes, including Ankrd27 (Ankyrin repeat domain 27), Mrpl10 (mitochondrial ribosomal protein L10), Rictor (rapamycin-insensitive companion of mTOR, Complex 2), and Ubxn 11 (UBX domain protein 11), in both distal sciatic nerve samples and dorsal root ganglion (DRG) samples after sciatic nerve injury. Our results suggested that following peripheral nerve injury, Mrpl10 and Tbp might be used as suitable reference genes for sciatic nerve stump and DRGs, respectively.

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

MicroRNAs (miRNAs or miRs) are involved in phenotype modulation of neural cells after peripheral nerve injury. The effects of miRNAs on the survival of dorsal root ganglion (DRG) neurons, however, have not yet been well understood. In this study, microarray profiling indicated that 13 miRNAs were differentially expressed in rat DRGs (L4-L6) during the initial 7d period post sciatic nerve transection, and that the expressions of miR-21 and miR-222 (2 out of the 13 miRNAs) were continually increased over the time period. Tissue inhibitor of metalloproteinase 3 (TIMP3), a pro-apoptotic protein in various cancer cells, was identified as a common target of miR-21 and miR-222. Over-expression of miR-21 and miR-222 inhibited cell apoptosis and enhanced cell viability in cultured DRG neurons. IL-6 could induce up-regulation of miR-21 expression. All the results showed that miR-21 and miR-222 inhibited neuronal apoptosis at least partially through suppressing TIMP3 after peripheral nerve injury.

Malignant gliomas are the most common and deadly primary brain tumors in adults. Increasing evidence has indicated that microRNAs (miRNAs) have an influence on the regulation of apoptotic cell signaling. Downregulation of miRNA 218 (miR-218) has been indicated in human glioma specimens. Here, we investigate the function of miR-218 in apoptosis and tumor growth of glioma cells.The expression of miR-218 was detected by real-time quantitative reverse transcriptase PCR. The effects of miR-218 on glioma cell proliferation and tumorigenicity were investigated by in vitro clonogenicity and in vivo xenograft assay. Apoptosis was evaluated by flow cytometric analysis and assay by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. The downstream targets of miR-218 were identified by bioinformatics analysis and further validated by Western blot and luciferase reporter assay.Overexpression of miR-218 induces glioma cell apoptosis and inhibits glioma cell viability, proliferation, and tumorigenicity. Epidermal growth factor receptor-coamplified and overexpressed protein (ECOP) was identified as a functional downstream target of miR-218, which can regulate transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and associated with apoptotic response. Ectopic expression of ECOP rescued the glioma cells from miR-218-induced apoptosis and increased NF-κB activity.These results suggest that miR-218 sensitizes glioma cells to apoptosis by regulating ECOP-mediated suppression of NF-κB activity, which may provide novel opportunities for glioma therapy.

OBJECTIVE To study the anti-tumor activity of SPS in vivo and in vitro and the cytotoxicity of CTL cells, NK cells of T739 lung cancer in mice. METHOD The transplanted tumor model of S180 Sarcoma was established with KM mouse. The SPS was adminished i.p. for 10 d, the tumor weight was detected. The transplanted tumor model of LA795 lung cancer was established with T739 mouse and SPS was adminished i.p. for 10 d and the tumor weight and the cytotoxicity of CTL cells, NK cells were detected. The Anti-tumor activity of SPS on three types of tumor cells in vitro was observed with trypan blue exclusion staining. RESULT SPS 40 mg x kg(-1) can significantly inhibit the growth of S180 Sarcoma in mice and inhibitory rate was 51.33% (P