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ObjectiveTo investigate the association of the polymorphisms of the FGB gene rs4220 and rs1044291 loci with plasma fibrinogen (Fg) level and gallstones in Xinjiang, China. MethodsBlood samples were collected from 150 Uygur and Han patients with gallstones and 150 Uygur and Han individuals without gallstones who were hospitalized or underwent physical examination in The People’s Hospital of Xinjiang Uygur Autonomous Region from December 2017 to May 2020. Plasma Fg level was measured, and based on the previous results of whole exon sequencing of the FGB gene, the SNaPshot method was used to identify the genotype at rs4220 and rs1044291 loci of the FGB gene. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups; a multivariate logistic regression analysis was used to investigate the association between each variable and gallstones. ResultsFor the Chinese Han population, the gallstones group had a significantly higher plasma Fg level than the control group ［2.90 (2.43-3.49) g/L vs 2.47 (2.06-3.02) g/L, Z=-3.62, P＜0.01), while there was no significant difference in the Uyghur population. There were no significant differences in the frequencies of genotypes and alleles at the rs4220 and rs1044291 loci of the FGB gene between the gallstones group and the control group in both Uyghur and Han populations (all P＞0.05). For the Chinese Han population, the subjects carrying GG genotype at the rs4220 locus in the gallstones group had a significantly higher plasma Fg level than those in the control group ［2.84(2.32-3.61) g/L vs 2.34(2.05-2.75) g/L, Z=-3.04, P＜0.05］, and the subjects carrying T genotype at the rs1044291 locus had a significantly higher plasma Fg level than those in the control group (3.08±0.75 g/L vs 2.48±0.48 g/L, t=2.80, P＜0.05). For the Uyghur population, only the subjects carrying A genotype at the rs4220 locus in the gallstones group had a significantly lower plasma Fg level than those in the control group ［2.84(2.08-3.06) g/L vs 3.10(2.85-3.98) g/L, Z=-2.41, P＜0.05］. There was no significant difference in plasma Fg level between the subjects carrying different genotypes within the gallstones group or the control group for both Uyghur and Han populations (all P＞0.05). ConclusionThe influence of FGB gene polymorphism on plasma Fg level may be associated with race, and FGB gene polymorphisms at the rs4220 and rs1044291 loci may be involved in the pathogenesis of gallstones by regulating Fg level in the population in Xinjiang.

We previously demonstrated that Pleurotus ferulae polysaccharide (PFPS) promoted dendritic cell (DC) maturation through the TLR4 signaling pathway. To improve PFPS activity and bioavailability, gold nanoparticles with PFPS (PFPS-Au NPs) were synthesized. Of note, although the polysaccharide content of PFPS-Au NPs was only one tenth of PFPS, PFPS-Au NPs enhanced the immunostimulatory activities of PFPS in the maturation and function of dendritic cells (DCs) by TLR4 and NLRP3 signaling pathways, evidenced by stronger activation of the down-stream MAPK and NF-κB pathways and NLRP3 inflammasome pathway. More importantly, PFPS-Au NPs enhanced DC migration and murine immunity, particularly in type 1 T-helper cell responses. Moreover, the half-life of PFPS-Au NPs (2.217 ± 0.187 h) was longer than that of PFPS (1.39 ± 0.257 h) in the blood and the distribution of PFPS-Au NPs (19.8 %) in the spleen was significantly increased compared with PFPS (13.3 %), indicating the improved bioavailability in vivo. PFPS-Au NPs as an adjuvant promoted antigen-specific cellular immune responses to an HPV DC-based vaccine, which significantly inhibited the growth of TC-1 tumors in mice. All results suggest that the prepared Au NPs could enhance PFPS-immunostimulatory activity, which will pave the way for PFPS-Au NPs to be applied in clinical trials.

Polysaccharides have various bioactivities including anti-tumour, antioxidant and immunoregulation, which are correlated with their structural characteristics. Here, four polysaccharides from Glycyrrhiza uralensis (GUPS), Pleurotus ferulae (PFPS), Triticum aestivum (TAPS) and Oryza sativa (OSPS) were purified to compare their structural characteristics and immunoregulatory activities. The data showed that TAPS, OSPS, GUPS and PFPS contained different monosaccharide components and had distinctly structural characteristics. All the four polysaccharides promoted the maturation of dendritic cells (DCs) through TLR4 with different activities. GUPS and PFPS also reduced the phagocytosis of DCs but not for TAPS and OSPS. Importantly, GUPS and PFPS enhanced the Th1 skewing immune response in mice without inflammation. TAPS and OSPS only showed weak immunostimulatory activity in vivo. The immunostimulatory activities of the four polysaccharides were ranked as PFPS = GUPS > TAPS > OSPS.

Background Marchantia polymorpha L. is a kind of Chinese herbal medicine and has various biological activities including antioxidant and antifungal. However, it is not clear about the antitumor effect and mechanism of M. polymorpha. We prepared M. polymorpha ethanol extract (MPEE) and investigated its antitumor effect on hepatocellular carcinoma cells both in vitro and in vivo. Methods The viability of hepatocellular carcinoma cells was detected by MTT assay. The distribution of cell cycle was analyzed by propidium iodide (PI) staining. The morphology of nuclei was observed by Hoechst 33258 staining. Apoptosis was detected by Annexin V/PI staining. JC-1 fluorescent probe and DCFH-DA were used to detect the mitochondrial membrane potential (ΔψM) and the level of reactive oxygen species (ROS), respectively. Caspase inhibitors were used to test the function of caspase in the induction of apoptosis. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the levels of mRNA and protein, respectively. Differentially expressed genes and signaling pathways were identified by transcriptome analysis. The H22 tumor mouse model was used to detect the antitumor effect of the extract. Results MPEE significantly suppressed the migration and growth of BEL-7404, HepG2 and H22 cells in a dose- and time-dependent manner through induction of apoptosis characterized by chromosomal condensation and cell cycle arrest at G0/G1 and G2/M phases. MPEE induced mitochondria-dependent apoptosis via upregulation of Bax and downregulation of Bcl-2 to reduce mitochondrial membrane potential and increase the release of cytochrome c. The levels of cleaved caspase-8 and -9 were significantly increased, which sequentially activated caspase-3 to cleave PARP. We further found that MPEE significantly increased ROS production and activated endoplasmic reticulum (ER) stress associated-apoptotic signaling pathway. Moreover, MPEE significantly inhibited H22 tumor growth in mouse model and improved the survival of tumor mice. Conclusion These results suggested that MPEE suppressed hepatocellular carcinoma cell growth through induction of apoptosis via intrinsic- and ER stress-associated pathways.

Cistanche tubulosa is a type of Chinese herbal medicine and exerts various biological functions. Previous studies have been demonstrated that Cistanche tubulosa phenylethanoid glycosides (CTPG) exhibit antitumor effects on a variety of tumor cells. However, the antitumor effects of CTPG on HepG2 and BEL-7404 hepatocellular carcinoma (HCC) cells are still elusive. Our study showed that CTPG significantly inhibited the growth of HepG2 and BEL-7404 cells through the induction of cell cycle arrest and apoptosis, which was associated with the activation of MAPK pathways characterized by the up-regulated phosphorylation of p38, JNK, and ERK1/2 and mitochondria-dependent pathway characterized by the reduction of mitochondrial membrane potential. The release of cytochrome c and the cleavage of caspase-3, -7, -9, and PARP were subsequently increased by CTPG treatment. Moreover, CTPG significantly suppressed the migration of HepG2 through reducing the levels of matrix metalloproteinase-2 and vascular endothelial growth factor. Interestingly, CTPG not only enhanced the proliferation of splenocytes but also reduced the apoptosis of splenocytes induced by cisplatin. In H22 tumor mouse model, CTPG combined with cisplatin further inhibited the growth of H22 cells and reduced the side effects of cisplatin. Taken together, CTPG inhibited the growth of HCC through direct antitumor effect and indirect immunoenhancement effect, and improved the antitumor efficacy of cisplatin.

BackgroundPrevious studies have been demonstrated that Cistanche tubulosa phenylethanoid glycosides (CTPG) exhibit antitumor effects on a variety of tumor cells. However, the antitumor effects of CTPG on HepG2 and BEL-7404 hepatocellular carcinoma (HCC) cells are still elusive.ResultsOur study showed that CTPG significantly inhibited the growth of HepG2 and BEL-7404 cells through the induction of cell cycle arrest and apoptosis, which was associated with the activation of MAPK pathways characterized by the up-regulated phosphorylation of p38, JNK, and ERK1/2 and mitochondria-dependent pathway characterized by the reduction of mitochondrial membrane potential. The release of cytochrome c and the cleavage of caspase-3, -7, -9 and PARP were subsequently increased by CTPG treatment. Moreover, CTPG significantly suppressed the migration of HepG2 through reducing the levels of matrix metalloproteinase-2 and vascular endothelial growth factor. Interestingly, CTPG not only enhanced the proliferation of splenocytes but also reduced the apoptosis of splenocytes induced by cisplatin. In H22 tumor mouse model, CTPG combined with cisplatin further inhibited the growth of H22 cells and reduced the side effects of cisplatin.ConclusionTaken together, CTPG inhibited the growth of HCC through direct antitumor effect and indirect immunoenhancement effect, improved the antitumor efficacy of cisplatin.

Additional file 5: Table S2. Main active ingredients identified under positive ESI mode (ESI +)by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF–MS) and their Contents in the MPEE.

Additional file 4: Table S1. Main active ingredients identified under negative ESI mode (ESI-)by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF–MS) and their Contents in the MPEE

Pleurotus feruleus is an edible and medicinal mushroom. We previously demonstrated that the crude and purified polysaccharides from cultivated P. feruleus (PFCP-C and PFPS-C) had the immunostimulatory activity. Here, the crude and purified polysaccharides of wild P. feruleus (PFCP-W and PFPS-W) were compared with PFCP-C and PFPS-C on the immunostimulatory activity. PFCP-W and PFCP-C showed similar activity on dendritic cell (DC) maturation in vitro, whereas the activity of PFCP-W was higher than that of PFCP-C in vivo. The monosaccharide compositions of PFPS-W and PFPS-C were different. PFPS-W was sulfated polysaccharides with crude surface. PFPS-W and PFPS-C promoted the activation of DCs and macrophages through TLR4 signaling pathway, whereas the activity of PFPS-W was higher than that of PFPS-C. The maturation and migration of DCs in vivo were significantly increased by PFPS-W and PFPS-C. Moreover, PFPS-W and PFPS-C significantly enhanced the antigen-specific cellular immune responses induced by DC-based vaccine.

// Jinyao Li 1, 2, 3 , Adila Aipire 2 , Jinyu Li 2 , Hongge Zhu 1 , Yanping Wang 4 , Wenjia Guo 1 , Xiaoqin Li 1 , Jia Yang 1 , Chunling Liu 1 1 Affiliated Tumor Hospital of Xinjiang Medical University, Urumqi 830011, China 2 College of Life Science and Technology, Xinjiang University, Urumqi 830046, China 3 XinJiang DingJu Biotech CO., LTD, Urumqi 830000, China 4 Bayin Guoleng Vocational and Technical College, Korla 841000, China Correspondence to: Jinyao Li, email: ljyxju@qq.com Jinyu Li, email: lijinyu234@163.com Chunling Liu, email: liudeyouxiang66@sina.com Keywords: λ-carrageenan, TLR4, dendritic cell-based vaccine, cancer immunotherapy, cellular responses Received: November 10, 2016 Accepted: January 24, 2017 Published: February 22, 2017 ABSTRACT In this study, we investigated the effect of λ-carrageenan on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We found that λ-carrageenan dose-dependently decreased the endocytosis of DCs, promoted DC maturation and increased cytokine production through TLR4 mediated signaling pathway. λ-carrageenan treatment also enhanced the ability of DCs in the stimulating allogenic splenocyte proliferation. In TC-1 tumor mouse model, HPV peptides pulsed λ-carrageenan-DC (HPV-CGN-DC) significantly inhibited tumor growth compared with control group. The frequencies of CD4 + and CD8 + T cells in spleens of tumor mice and their activation status were significantly increased in HPV-CGN-DC group, but the frequencies of natural regulatory T cells and CD11b + Gr-1 + cells were significantly decreased. Further, HPV-CGN-DC induced strong CD8 + T cell responses, which are negatively correlated with tumor volumes. The results suggested that λ-carrageenan promoted DC maturation through TLR4 signaling pathway and could be used as the adjuvant in DC-based vaccines.

Pleurotus ferulae is an edible mushroom and has been used in Uygur medicine for a long time. In this study, we purified polysaccharides from P. ferulae (PFPS) and investigated its structural characteristics. We obtained a homogeneous PFPS with a molecular weight of around 1600 kDa and prominent characteristic polysaccharide groups, which mainly contained glucose (97%), followed by mannose and galactose (3%). Both 1H and 13C NMR spectra indicated that PFPS contained both α- and β-anomeric configurations. Atomic force microscopy and Congo red-staining data further suggested that PFPS belonged to a linear branched structure that existed in flexible single chains at low concentrations and could form aggregates such as a triple-helical structure at high concentrations. Moreover, PFPS promoted the maturation of dendritic cells through a TLR4 mediated signaling pathway, which is characterized by the increased expressions of CD40, CD86, IL-12 and TNF-α and the decreased endocytosis. The results suggest that PFPS has immunoregulatory activities.

Although the underlying mechanism of stress remains unknown, it has been associated with the pathophysiology of gastroesophageal reflux diseases, the development of which appear to be accelerated by oxidative stress and fibrosis. The aim of the current study was to investigate the effect of chronic restraint stress on esophageal oxidative stress and fibrosis, as well as the impact of oxidative stress in a murine model whereby 8-week old C57BL/6J male mice were subjected to intermittent chronic restraint stress for a two-week period. The current study demonstrated that chronic restraint stress significantly reduced the body weight of mice compared with the control group. Although chronic restraint stress did not significantly alter the levels of triglycerides or cholesterol, free fatty acid concentration was significantly increased compared with the control group. Furthermore, chronic restraint stress significantly upregulated the expression levels of several fibrotic biomarkers including collagen type I, transforming growth factor β-1, α-smooth muscle actin and SMAD-3 compared with the control group. In addition, the expression levels of the reactive oxygen species (ROS) NADPH oxidase-4 and malondialdehyde were significantly increased, while the expression levels of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 were significantly decreased in esophageal tissue from mice in the chronic restraint stress group compared with the control group. In conclusion, chronic restraint stress may induce esophageal fibrosis by accumulating ROS and increasing fibrotic gene expression in a murine model.

Cistanche tubulosa phenylethanoid glycosides (CTPG) have been shown various biological activities including anti-allergy, hepatoprotective activity and bone regeneration. However, the anti-tumor activity of CTPG needs to be investigated. CTPG was used to treat B16-F10 cells both in vitro and in vivo. We found that CTPG dramatically changed the morphology of B16-F10 cells, and significantly reduced the viability of B16-F10 cells in a dose-dependent and time-dependent manner, which might be mediated by CTPG-induced apoptosis and cell cycle arrest. After CTPG treatment, the expressions of BAX and BCL-2 were up-regulated and down-regulated, respectively. Moreover, mitochondrial membrane potential was reduced and ROS generation was increased. Consequently, the levels of cytochrome c and cleaved-caspase-3 and -9 were up-regulated by CTPG treatment but not for cleaved-caspase-8. We further observed that CTPG significantly inhibited the tumor growth in vivo and improved the survival rate of tumor mice. We also observed that CTPG promoted the proliferation of splenocytes and increased the proportions of CD4+ and CD8+ T cells in spleens of tumor mice. The results showed that CTPG induced the apoptosis of B16-F10 cells through mitochondria-dependent pathway, suggesting that CTPG could be a potential candidate for treatment of cancer.

BackgroundThe enhancement of immunity is very important for immunocompromised patients such as cancer patients with radiotherapy or chemotherapy.Glycyrrhiza uralensishas been used as food and medicine for a long history.G. uralensispolysaccharides (GUPS) were prepared and its immunostimulatory effects were investigated.MethodsHuman monocyte-derived dendritic cells (DCs) and murine bone marrow-derived DCs were treated with different concentrations of GUPS. The DCs maturation and cytokine production were analyzed by flow cytometry and ELISA, respectively. Inhibitors and Western blot were used to study the mechanism of GUPS. The immunostimulatory effects of GUPS were further evaluated by naïve mouse model and immunosuppressive mouse model induced by cyclophosphamide.ResultsGUPS significantly promoted the maturation and cytokine secretion of human monocyte-derived DCs and murine bone marrow-derived DCs through TLR4 and down-stream p38, JNK and NF-κB signaling pathways. Interestingly, the migration of GUPS treated-DCs to lymph node was increased. In the mouse model, GUPS increased IL-12 production in sera but not for TNF-α. Moreover, GUPS ameliorated the side effect of cyclophosphamide and improved the immunity of immunosuppressive mice induced by cyclophosphamide. These results suggested that GUPS might be used for cancer therapy to ameliorate the side effect of chemotherapy and enhance the immunity.

Glycyrrhiza uralensis is a Chinese herbal medicine with various bioactivities. Three fractions (GUPS-I, GUPS-II and GUPS-III) of G. uralensis polysaccharides (GUPS) were obtained with molecular weights of 1.06, 29.1, and 14.9 kDa, respectively. The monosaccharide compositions of GUPS-II and GUPS-III were similar, while that of GUPS-I was distinctively different. The results of scanning electron microscopy, FT-IR, and NMR suggested that GUPS-II and GUPS-III were flaky with a smooth surface and contained &alpha, and &beta, glycosidic linkages, while GUPS-I was granulated and contained only &alpha, glycosidic linkages. Moreover, GUPS-II and GUPS-III exhibited better bioactivities on the maturation and cytokine production of dendritic cells (DCs) in vitro than that of GUPS-I. An in vivo experiment showed that only GUPS-II significantly enhanced the maturation of DCs. These results indicate that GUPS-II has the potential to be used in combination with cancer immunotherapy to enhance the therapeutic effect.

We previously found that Pleurotus ferulae polysaccharides (PFPS) improved the maturation and function of dendritic cells (DCs). In this study, we investigated the effects of PFPS on the antitumor efficacy of therapeutic human papillomavirus (HPV) DC-based vaccine. PFPS stimulated DCs pulsed with HPV E6/E7 peptides were used to treat tumor mice on day 5 & 12 (HPV + PFPS-DCs early) and day 12 & 19 (HPV + PFPS-DCs late) after TC-1 cell injection. Compared to control group, both HPV + PFPS-DCs early and HPV + PFPS-DCs late strategies significantly inhibited tumor growth, which was significantly correlated with the increased activation status of both CD4(+) and CD8(+) T cells, the decreased frequencies of myeloid-derived suppressor cells, and the induction of HPV-specific CD8(+) T cell responses. The survival of tumor mice was also greatly improved by HPV + PFPS-DCs early. Moreover, HPV + PFPS-DCs early completely inhibited the growth of second challenged TC-1 cells in tumor free mice. The results showed that PFPS improved the antitumor efficacy of therapeutic HPV DC-based vaccine, suggesting that PFPS might be a potential adjuvant for DC-based vaccines. This study provides a potential strategy for developing the therapeutic DC-based vaccine against cervical cancer.

Cistanche tubulosa has various biological functions. In the present study, the antitumor effect of water-soluble phenylethanoid glycosides of C. tubulosa (CTPG-W) on esophageal cancer was investigated. Eca-109 cells were treated with CTPG-W and the cell viability was measured by MTT assay. The apoptosis, cell cycle, mitochondrial membrane potential (Δψm) and reactive oxygen species were analyzed by flow cytometry. The levels of proteins in apoptotic pathways were detected by western blot analysis. It was determined that CTPG-W significantly reduced the viability of Eca-109 cells through the induction of apoptosis and cell cycle arrest. Following CTPG-W treatment, the Δψm of Eca-109 was notably decreased, which is associated with the upregulated levels of B-cell lymphoma-2 (Bcl-2)-associated X and downregulated levels of Bcl-2. Consequently, the levels of cytochrome c and c-Jun NH2-terminal kinase were increased, which upregulated the levels of cleaved-poly (ADP-ribose) polymerase and cleaved-caspase-3, −7 and −9, but not caspase-8. Correspondingly, the levels of reactive oxygen species in Eca-109 cells demonstrated notable changes. These results indicated that CTPG-W induced apoptosis of Eca-109 cells through a mitochondrial-dependent pathway.

Stress is a pivotal factor for inflammation, reactive oxygen species (ROS) production and formation of visceral hypersensitivity (VH) in the process of gastroesophageal reflux disease (GERD). In the present study, the effects of stress on esophageal inflammation, oxidative stress and VH were investigated in a chronic restraint stress mouse model. C57BL/6J male mice were subjected to 2 weeks of intermittent restraint stress, and histopathological analysis revealed that stress induced esophageal inflammation and fibrosis, while no distinct changes were detected in non‑stressed control mice. In addition, increased NADPH oxidase 4 expression was observed in the plasma and esophagus of stressed mice, indicating accumulation of ROS. The expression levels of antioxidants, including Mn‑superoxide dismutase (MnSOD), Cu/Zn‑SOD, catalase and glutathione peroxidase, were also analyzed using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In addition, transient receptor potential vanilloid 1 (TRPV‑1) and protease‑activated receptor 2 (PAR‑2), which are crucial receptors for VH, were measured by immunohistochemistry and RT‑qPCR. The results demonstrated that stress markedly reduced antioxidant expression, while it significantly upregulated TRPV‑1 and PAR‑2 expression levels in the mouse esophagus. Finally, 2 weeks of restraint stress significantly increased the esophageal and plasma levels of inflammatory cytokines, including interleukin (IL)‑6, IL‑8, interferon‑γ and tumor necrosis factor‑α. Taken together, the present study results indicated that stress‑induced esophageal inflammation and ROS generation involves VH.

Background Cistanche tubulosa (Schenk) R. Wight is a traditional Chinese medicine that parasitizes the roots of the Tamarix plant and has been used to treat male impotence, sterility, body weakness, and as a tonic. However, its antitumor effect on hepatocellular carcinoma is still elusive. Here, we investigated the antitumor effect of C. tubulosa phenylethanoid glycosides (CTPG) on H22 hepatocellular carcinoma cells both in vitro and in vivo and its mechanisms. Methods The morphology, viability, apoptosis, cell cycle and mitochondrial membrane potential (Δψm) of H22 cells were analyzed by inverted microscopy, MTT assay and flow cytometry, respectively. The expression and activation of proteins in apoptosis pathway were detected by Western blot. The in vivo antitumor effect was evaluated in tumor mouse model established using male Kunming mice. Results CTPG treatment significantly suppressed H22 cell growth in a dose- and time-dependent manner, which was correlated with the increased apoptosis and cell cycle arrest at G0/G1 and G2/M phases. Moreover, the chromosomal condensation was observed in CTPG-treated H22 cells. CTPG treatment significantly increased Bax/Bcl-2 ratio, reduced Δψm and enhanced the release of cytochrome c. The levels of cleaved caspase-8 and caspase-9 in both extrinsic and intrinsic signaling pathways were significantly increased that sequentially activated caspase-7 and -3 to cleave PARP. Finally, CTPG inhibited the growth of H22 cells in mice and improved the survival rate of tumor mice. Conclusions These results suggested that CTPG suppressed H22 cell growth through both extrinsic and intrinsic apoptosis pathways.