Huoxue Tongjiang decoction-resisted reflux esophagitis by activation stem cell factor/c-kit/interstitial cell of cajal pathway and regulating the T-helper 17/regulatory T-cells balance in rats

Background:Huoxue Tongjiang decoction(HXTJD)is an effective prescription for treating reflux esophagitis(RE).We investigated the effects of HXTJD on esophageal motility and mucosal inflammation in a rat RE model.Methods:Chemical composition of HXTJD was analyzed by ultrahigh-performance liquid chromatography Q-Orbitrap mass spectrometry(MS).The change rates of mean contraction tension forces,mean amplitudes,and mean frequencies for the lower esophageal sphincter(LES)were recorded using the isolated tissue bath system,mechanical tension transducer,and PowerLab physiological recorder.After weighing the stomach,the phenol red labeling method was used to measure the gastric emptying rate.The LES ultrastructure was observed through transmission electron microscopy.Immunofluorescence and western blotting were used to detect the number of interstitial cells of Cajal(ICC)and the expression levels of c-kit protein,connexin43(Cx43),and stem cell factor(SCF).Flow cytometric analysis and enzyme-linked immunosorbent assay were conducted to detect the percentages of T helper 17(Th17)cells and regulatory T(Treg)cells and the serum concentrations of interleukin 6(IL-6),interleukin 17(IL-17),and interleukin 10(IL-10)in the rats.Results:We identified 28 chemical constituents in HXTJD.Regarding esophageal motility,we revealed that HXTJD increased the mean contraction tension forces,mean amplitudes,and mean frequency change rate of LES and the gastric emptying rate;decreased stomach weight;and improved the LES ultrastructure.Additionally,HXTJD increased the number of ICC-positive cells,and c-kit,Cx43,and SCF expression levels.Regarding esophageal inflammation,HXTJD significantly decreased the percentage of Th17 cells,and IL-6 and IL-17 concentrations,and increased the percentage of Treg cells and IL-10 concentration.Conclusion:HXTJD was found to be efficacious in the rat RE model.It may promote esophageal motility and alleviate the inflammatory response by activating the SCF/c-kit/ICC pathway and regulating the Th17/Treg cell balance.

Anti-inflammatory effect of miR-125a-5p on experimental optic neuritis by promoting the differentiation of Treg cells

Methylprednisolone pulse treatment is currently used fo r optic neuritis.It can speed visual recovery,but does not improve the ultimate visual outcomes.Recent studies have repo rted that miR-125 a-5 p has immunomodulatory effects on autoimmune diseases.However,it remains unclear whether miR-125 a-5 p has effects on optic neuritis.In this study,we used adeno-associated virus to overexpress or silence miR-125 a-5 p in mice.We found that silencing miR-125 a-5 p increased the latency of visual evoked potential and aggravated inflammation of the optic nerve.Ove rexpression of miR-125 a-5 p suppressed inflammation of the optic nerve,protected retinal ganglion cells,and increased the percentage of Treg cells.Our findings show that miR-125 a-5 p exhibits anti-inflammatory effects through promoting the diffe rentiation of Treg cells.

Th1 cells inducing IFNγ response improves immunotherapy efficacy in gastric cancer

Objective: Cancer immunotherapy has made remarkable advances in recent years, but its effectiveness in treating gastric cancer is often limited by the complexity of the tumor microenvironment and the lack of effective biomarkers. This study aimed to identify effective biomarkers for immunotherapy treatment by characterizing the tumor microenvironment.Methods: We retrieved the RNA-seq data from gastric cancer patients treated with the programmed death 1(PD-1) blockade pembrolizumab. Differentially expressed genes associated with clinical outcomes were identified and further analyzed using gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Gene signature scores were calculated by single sample Gene Set Enrichment Analysis(ssGSEA). The infiltration levels of immune cells were quantified using the xCell website. Cell type enrichment analysis was performed to compare treatment response and non-response groups, and regression analysis was used to investigate the relationship between interferon gamma(IFNγ) immune response and immune cell infiltration. Biomarkers were identified using least absolute shrinkage and selection operator(LASSO) analysis.Results: Compared to normal tissues, cytokine activity and interleukin-6 production were highly activated in gastric tumors. Responders to pembrolizumab showed significantly up-regulated expression of IFNγ responserelated genes. Cell type enrichment analysis revealed that Th1 cells were significantly enriched in the tumor microenvironment of responders. Regression analysis indicated that Th1 cells induced IFNγ response more efficiently than other cell types. Using signatures of Th1 cells, stromal cells and IFNγ response, a set of eight genes were identified that effectively predicted the efficacy of immunotherapy treatment and patient prognosis.Conclusions: Th1 cells promote therapeutic efficacy of PD-1 blockade by promoting IFNγ immune response in gastric cancer. The identified biomarkers have the potential to improve the effectiveness of immunotherapy treatment for gastric cancer patients.

MBD2 promotes Th2 differentiation in ovalbumin-induced CD4+T cells

Introduction:Allergen-specific CD4+T cells play a central role in autoimmune disorders,allergies and asthma,with Th2-type immunity being the typical functional response of CD4+T cells.This study aimed to investigate the role of MBD2 in regulating Th2 cell differentiation.Methods:Splenic mononuclear cells were extracted from C57BL/6 mice,and CD4+T cells were isolated using magnetic beads and confirmed through flow cytometry.Lentivirus was employed to construct MBD2-silenced CD4+T cells.In vitro experiments were performed to treat splenogenic mononuclear cells and CD4+T cells with Ovalbumin(OVA),and Th2 cell ratios and IL-4 levels were assessed using flow cytometry and ELISA.Results:The purity of the isolated CD4+T cells was 95.73%,confirming successful isolation of primary CD4+T cells.Compared to the control group,the Th2 cell ratio exhibited an increase in the Th2-induced group.Treatment with 5-Aza(concentrations,1-100μM)promoted Th2 cell differentiation and increased IL-4 levels.Notably,when combined with Th2 induction and 10μM 5-Aza treatment,silencing MBD2 further amplified Th2 cell ratios and elevated IL-4 levels in cell supernatants.Furthermore,OVA(concentration,200μg/mL)induced the differentiation of CD4+T cells into Th2 cells and increased IL-4 secretion.Interestingly,silencing MBD2 significantly increased the Th2 cell ratio and IL-4 levels in OVA-treated CD4+T cells.Conclusion:In summary,OVA promoted CD4+T cell differentiation into Th2 cells and enhanced IL-4 levels.MBD2 was identified as a mediator of Th2 cell differentiation in splenic-derived CD4+T cells,influenced by OVA or 5-Aza treatment.

Integration of scRNA-Seq and Bulk RNA-Seq to analyze the heterogeneity ofcolorectal cancer immune cells and establish a molecular risk model

Background:Colorectal cancer(CRC)is a highly heterogeneous malignant tumor that significantly impacts clinical diagnosis and treatment.Single-cell RNA sequencing is an innovative method for exploring tumor heterogeneity and understanding its role at cellular and genetic levels.Method:The colorectal cancer Single-cell RNA sequencing data were analysed on the immune.RNA-seq data in bulk form was utilized to assess the major genes of the immune cell subsets linked to CRC.We conducted an analysis of the abundance of immune cells in the microenvironment of CRC,and also performed weighted gene co-expression network analysis.Gene set enrichment analysis helped perform two analytical procedures of subtype groups.Furthermore,Least absolute shrinkage and selection operator regression was employed to analyse and screen for a gene signature.Finally,quantitative PCR Was performed to detect the expression levels of signature genes in CRC.Results:The Single-cell RNA sequencing(GSE146771)dataset was integrated to obtain 9 cell clusters.The Single-sample gene set enrichment analysis showed that the related gene expression of T-cell subsets of different functional statuses could vary greatly between patients with GSE146771.Immune cell analysis of TCGA-CRC indicated an improved overall survival rate for patients with elevated Th2 cell abundance.Five-gene signature(Risk Score=-0.205×CDC25C-0.231×GSTCD-0.010×KPNA2-0.002×KIF15-0.171×ORC1)was developed by weighted correlation network analysis,and lasso Cox regression.Then,the risk prediction efficacy of the signature was validated in four GSE datasets.Furthermore,the expression of five genes was reduced in CRC tissue by quantitative PCR.Conclusion:Five-gene signature based on CRC heterogeneity was developed as a prognosis predictor,which can serve as a potential treatment target.

Detection and Clinical Significance of Th17/Treg Cell-Related Factors in Patients with Gestational Diabetes Mellitus

Objective:To investigate the detection of Th17/Treg cell-related factors in patients with gestational diabetes mellitus(GDM)and its clinical significance.Methods:In this study,a retrospective cohort research method was used to collect the clinical data of 42 patients who were hospitalized in the Affiliated Hospital of Hebei University and received the diagnosis of GDM from January 2018 to December 2022,as well as 42 patients with normal pregnancies during the same period.The Th17/Treg expression levels and metabolism-related indexes in the peripheral blood of patients were detected by radioimmunoassay.Results:The relative expression of Th17 in the serum of patients in the GDM group was significantly higher than that of the control group,and the level of Treg was significantly lower than that of the control group(P<0.05);the levels of FBG,FINS,2hBG,TC,TG and HOMA-IR of the patients in the GDM group were significantly higher than that of the control group,and the level of HOMA-βwas significantly lower than that of the control group(P<0.05).Conclusion:The imbalance of the Th17/Treg cell ratio in patients with GDM may be related to their disease progression and prognosis,providing new ideas and strategies for the clinical treatment of GDM.

Modulating the crosstalk between macrophage and Th17: potential mechanism of natural products on acute lung injury

Sepsis is a life-threatening multiple organ dysfunction syndrome caused by the imbalance of the immune response to infection,featuring complex and variable conditions,and is one of the leading causes of mortality in ICU patients.Lung injury is a common organ damage observed in sepsis patients.Macrophages and Th17 cells,as crucial components of innate and adaptive immunity,play pivotal roles in the development of sepsis-induced acute lung injury(ALI).This review summarizes the alterations and mechanisms of macrophages and Th17 cells in sepsis-induced ALI.By focusing on the“cross-talk”between macrophages and Th17 cells,this review aims to provide a solid theoretical foundation for further exploring the therapeutic targets of traditional Chinese medicine formulas in the treatment of sepsis complicated with ALI,thereby offering insights and guidance for the clinical application of traditional Chinese medicine in managing sepsis-associated ALI.

GATA-3 promotes Th2 responses through three different mechanisms： induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Thl cell-specific factors

Naive CD4 T cells can differentiate into at least two different types ofT helpers, Thl and Th2 cells. Th2 cells, capable of producing IL-4, IL-5 and IL-13, are involved in humoral immunity against extracellular pathogens and in the induction of asthma and other allergic diseases. In this review, we summarize recent reports regarding the transcription factors involved in Th2 differentiation and cell expansion, including StatS, Gfi- 1 and GATA-3. Stats activation is necessary and sufficient for IL-2-mediated function in Th2 differentiation. Enhanced Stats signaling induces Th2 differentiation independent of IL-4 signaling; although it does not up-regulate GATA-3 expression, it does require the presence of GATA-3 for its action. Gfi-1, induced by IL-4, promotes the expansion of GATA-3-expressing cells. Analysis of conditional Gata3 knockout mice confirmed the critical role of GATA-3 in Th2 cell differentiation （both IL-4 dependent and IL-4 independent） and in Th2 cell proliferation and also showed the importance of basal GATA-3 expression in inhibiting Thl differentiation.

Potential role of Th17 cells in the pathogenesis of inflammatory bowel disease

The etiopathology of inflammatory bowel disease （IBD） remains elusive. Accumulating evidence suggests that the abnormality of innate and adaptive immunity responses plays an important role in intestinal inflam- mation. IBD including Crohn＇s disease （CD） and ulcerative colitis （UC） is a chronic inflammatory disease of the gastrointestinal tract, which is implicated in an inappropriate and overactive mucosal immune response to luminal flora. Traditionally, CD is regarded as a Thl- mediated inflammatory disorder while UC is regarded as a Th2-1ike disease. Recently, Th17 cells were identified as a new subset of T helper cells unrelated to Thl or Th2 cells, and several cytokines [e.g. interleukin （IL）-21, IL-23] are involved in regulating their activation and differentiation. They not only play an important role in host defense against extracellular pathogens, but are also associated with the development of autoimmunity and inflammatory response such as IBD. The identification of Th17 cells helps us to explain some of the anomalies seen in the Thl/Th2 axis and has broadened our understanding of the immunopathological effects of Th17 cells in the development of IBD.

Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.

Increase of peripheral Th17 lymphocytes during acute cellular rejection in liver transplant recipients

BACKGROUND: Although many human inflammatory and autoimmune diseases were previously considered to be mediated by T helper type 1 (Th1) cells, the recently described Th17 cells play dominant roles in several of these diseases. We and others speculated that allograft rejection after organ transplantation may also involve Th17 cells. Episodes of acute rejection occur in 30% of liver transplants. This study aimed to determine the frequency of circulating Th17 cells in patients who had received liver transplants for benign end-stage liver disease and to identify any association between acute rejection episodes and levels of Th17 cells in the peripheral blood. METHODS: A prospective study compared Th17 cells from 76 consecutive benign end-stage liver disease patients who had undergone orthotopic liver transplantation from 2007 to 2011 with those from 20 age-matched healthy individuals. Peripheral blood samples were collected at different time points within one year after transplant. Blood samples and liver biopsies were also collected at the diagnosis of acute rejection. Percentages of circulating CD4+ IL-17+ cells were measured by flow cytometry The transplant patients were classified into two groups: a rejection group consisting of 17 patients who had an episode of acute rejection, and a non-rejection group comprising the remaining 59 patients with no acute rejection episodes Percentages of circulating Th17 cells were compared between the two groups and controls. RESULTS: The levels of circulating CD4+ IL-17+ T cells in the rejection group were higher during acute rejection than those in the non-rejection group (2.56±0.43% versus 1.79±0.44% P<0.001). The frequency of CD4+ IL-17+ cells in peripheral blood was positively correlated with the rejection activity index (r=0.79, P=0.0002).CONCLUSION: Circulating Th17 cells may be useful as a surrogate marker for predicting acute rejection in liver transplant recipients.

Correlation between circulating myeloid-derived suppressor cells and Th17 cells in esophageal cancer

AIM: To perform a comprehensive investigation into the potential correlation between circulating myeloidderived suppressor cells (MDSCs) and Th17 cells in esophageal cancer (ECA). METHODS: A total of 31 patients newly diagnosed with ECA and 26 healthy subjects were included in the current study. The frequencies of MDSCs and Th17 cells in peripheral blood were determined by flow cytometry. The mRNA expression of cytokines, arginase 1 (Arg1) and inducible NO synthase (iNOS) in peripheral blood mononuclear cells (PBMCs) and plasma Arg1 were assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: There was an increased prevalence of MDSCs in the peripheral blood from ECA patients (15.21% ± 2.25%) when compared with healthy control (HC) (1.10% ± 0.12%, P < 0.0001). The plasma levels of Arg1 in ECA patients were significantly higher than those in HC (28.28 ± 4.10 ng/mL vs 9.57 ± 1.51 ng/ mL, P=0.0003). iNOS mRNA levels in the peripheral blood of ECA patients also showed a threefold increase compared with HC (P=0.0162). The frequencies of Th17 cells (CD4 + IL-17A + ) were significantly elevated in ECA patients versus HC (3.50% ± 0.33% vs 1.82% ± 0.19%, P=0.0001). Increased mRNA expression of IL-17 and ROR-γt was also observed in ECA patients compared with HC (P=0.0041 and P=0.0004, respectively), while the mRNA expression of IL-6 and tumor necrosis factor-α (TNF-α) showed significant decreases (P=0.0049 and P < 0.0001, respectively). No obvious correlations were found between the frequencies of MDSCs and Th17 cells in the peripheral blood from ECA patients(r=-0.1725, P=0.3534). Arg1 mRNA levels were positively correlated with levels of IL-6 (r=0.6404, P=0.0031) and TNF-α (r=0.7646, P=0.0001). Similarly, iNOS mRNA levels were also positively correlated with levels of IL-6 (r=0.6782, P=0.0007) and TNF-α (r=0.7633, P < 0.0001). CONCLUSION: This study reveals the relationship between circulating MDSCs and Th17 cells, which may lead to new immunotherapy approaches for ECA based on the associated metabolites and cytokines.

ECT2通过调控PI3K/AKT信号通路对胰腺癌细胞恶性行为、糖酵解及TH细胞分化的影响

目的探讨上皮细胞转化序列2(ECT2)通过调控PI3K/AKT信号通路对胰腺癌细胞恶性行为、糖酵解及TH细胞分化的影响。方法RT⁃qRCR检测人脐静脉内皮细胞系HUVEC及胰腺癌细胞中ECT2 mRNA表达,将胰腺癌Panc⁃1细胞株分为胰腺癌组(Panc⁃1细胞株正常培养)、NC组(Panc⁃1细胞株转染ECT2阴性对照)、ECT2 siRNA组(Panc⁃1细胞株转染ECT2 siRNA)、抑制剂组(Panc⁃1细胞株转染加入PI3K/AKT抑制剂LY294002),ECT2 siRNA+抑制剂组(Panc⁃1细胞株转染ECT2 siRNA加入PI3K/AKT抑制剂LY294002),采用RT⁃qRCR各组细胞中ECT2 mRNA表达;Transwell法检测各组Panc⁃1细胞侵袭能力;划痕实验检测迁移;流式细胞仪检测各组细胞IFN⁃γ及IL⁃4表达;免疫印迹分别检测糖酵解代表蛋白及PI3K/AKT表达。结果胰腺癌组、NC组、ECT2 siRNA组、抑制剂组及ECT2 siRNA+抑制剂组的ECT2 mRNA比较分别为1.00±0.00、0.95±0.03、0.41±0.08、0.65±0.05及0.20±0.04,组间比较,差异有统计学意义(F=310.700,P<0.05);胰腺癌组、NC组、ECT2 siRNA组、抑制剂组及ECT2 siRNA+抑制剂组Panc⁃1细胞侵袭数目分别为(256.30±28.36)个、(241.18±24.05)个、(155.48±17.56)个、(178.90±18.44)个及(95.15±12.10)个,组间比较,差异有统计学意义(F=59.310,P<0.01);胰腺癌组、NC组、ECT2 siRNA组、抑制剂组及ECT2 siRNA+抑制剂组Panc⁃1细胞迁移距离分别为(14.02±1.36)mm、(13.42±1.29)mm、(9.25±0.85)mm、(8.50±0.45)mm及(4.25±0.53)mm,组间比较差异有统计学意义(F=101.200,P<0.05);ECT2 siRNA组细胞IFN⁃γ升高及IL⁃4占比降低,与NC组比较,差异有统计学意义(P<0.05),与抑制剂组相比,ECT2 siRNA+抑制剂组细胞IFN⁃γ升高及IL⁃4占比降低,组间比较差异有统计学意义(P<0.05);ECT2 siRNA组细胞HIF⁃1α、GLUT1、HK⁃Ⅱ和PFK蛋白均降低,与抑制剂组无意义(P>0.05),与抑制剂组相比,ECT2 siRNA+抑制剂组细胞HIF⁃1α、GLUT1、HK⁃Ⅱ和PFK蛋白降低,组间比较差异有统计学意义(P<0.05);ECT2 siRNA组细胞HIF⁃1α、GLUT1、HK⁃Ⅱ和PFK蛋白均降低,与NC组差异有统计学意义(P<0.05),与抑制剂组相比,ECT2 siRNA+抑制剂组细胞HIF⁃1α、GLUT1、HK⁃Ⅱ和PFK蛋白降低,组间比较差异有统计学意义(P<0.05)。结论抑制ECT2可通过降低胰腺癌细胞糖酵解,提高Th细胞中IFN⁃γ表达而抑制恶性侵袭及迁移,其机制可能与抑制PI3K/AKT信号通路活性相关。

In vitro-derived insulin-producing cells modulate Th1 immune responses and induce IL-10 in streptozotocin-induced mouse model of pancreatic insulitis

Background: Insulitis is defined by the presence of immune cells infiltrating in the pancreatic islets that might progress into the complete β-cell loss. The immunomodulatory properties of bone marrow-derived mesenchymal stem cells(BM-MSCs) have attracted much attention. This study aimed to evaluate the possible immunomodulatory effects of rat BM-MSCs and MSCs-derived insulin-producing cells(IPCs) in a mouse model of pancreatic insulitis. Methods: Insulitis was induced in BALB/c mice using five consecuti ve doses of streptozotocin. MSCs or IPCs were directly injected into the pancreas of mice and their effects on the expression of Th subsetsrelated genes were evaluated. Results: Both BM-MSCs and IPCs significantly reduced the expression of pancreatic Th1-related IFN-γ( P < 0.001 and P < 0.05, respectively) and T-bet genes(both P < 0.001). Moreover, the expression of IL-10 gene was significantly increased in IPC-treated compared to BM-MSC-or PBS-treated mice( P < 0.001 both comparisons). Conclusions: BM-MSCs and IPCs could successfully suppress pathologic Th1 immune responses in the mouse model of insulitis. However, the marked increase in IL-10 gene expression by IPCs compared to BM-MSCs suggests that their simultaneous use at the initial phase of autoimmune diabetes might be a better option to reduce inflammation but these results need to be verified by further experiments.

Acquired pMHC I Complexes Greatly Enhance CD4^+ Th Cell's Stimulatory Effect on CD8^+ T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

CD4＋ helper T （Th） cells play pivotal roles in induction of CD8＋ CTL immunity. However, the mechanism of CD4＋ T cell help delivery to CD8＋ T cells in vivo is still elusive. In this study, we used ovalbumin （OVA）-pulsed dendritic cells （DCovA） to activate OT-II mouse CD4＋ T cells, and then studied the help effect of these CD4＋ T cells on CD8＋ cytotoxic T lymphocyte （CTL） responses＋ We also examined CTL mediated islet β cell destruction which leaded to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter （RIP）-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex （pMHC） I acquired from DCovA by DCovA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination ＋ ＋ ＋ with TCR transgenlc OT-I mouse CD8 T ceils, the tolerogenic dosage of CD4＋ Th cells with acquired pMHC I, but not CD4＋ （Kb-/-） Th cells without acquired pMHC I were able to cause diabetes in 8/10 （80%） RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4＋ T cell-mediated help in CD8＋ CTL induction. Cellular ＆ Molecular Immunology. 2008;5（6）：407-415.

Effects of the Overall Alkaloid of a Traditional Chinese Medicine “Tongbiling” on the Cytokine Expression in Th1 and Th2 Cells of Rheumatoid Arthritis and Their Signaling Pathway

To investigate the effects of overall alkali of a traditional Chinese medicine “Tongbiling” (brucine and strychnine alkaloids in main) on the cytokines expression in Th1 and Th2 cells in the synovial fluid of patients with rheumatism arthritis and their signal pathway, the mononuclear cells in the synovial fluid (SFMC) of patients were isolated by Ficoll-Hypaque gradient centrifugation, and the CD3 + CD69 + and CD3 + HLA-DR antigen were analyzed by flow cytometry in comparison with those of the peripheral blood. The rest of cells were cultured after resuspension with RPMI 1640 culture medium. Phorbol 12,13-dibutyrate (PDB) and ionomycin were added successively into the culture with various concentration of overall alkali Tongbiling (TBL). After 4 h of cultivation, the expression of IFN-γ and IL-4 in CD3 + cells were analyzed by flow cytometry. The influence of overall alkali TBL (100?mg/L) on the intracellular calcium was investigated after Fluo-3/AM labeling and stimulation with PDB and ionomycin at 1, 2, 4 and 10?min, and the influence of TBL on the expression of CD3 + CD69 + cells were determined with stimulation of PDB for 24?h in the whole blood lymphocytes culture. It was found that the percentage of T cells bearing CD69 was significantly up-regulated (77%), while that of T cells bearing HLA-DR was 44% in the synovial mononucleated cells. After PDB and ionomycin stimulation, the expression of IFN-γ in CD3 + cells were up-regulated, but there was no change on the expression of IL-4 in CD3 + cells, indicating that ratio of Th1/Th2 was significantly increased and Th cells differentiate to Th1 cells in mainly. Four concentrations of overall alkaloid of TBL (200?mg/L, 100?mg/L, 50?mg/L, 25?mg/L) could down-regulated the expression of IFN-γ in CD3 + cells and the Th1/Th2 ratio obviously, but all the concentrations of the overall alkaloids had no effect on the expression of IL-4 in CD3 + cells. 100?mg/L concentration of the overall alkaloid did not down-regulate the intracellular calcium level. Each concentration of the overall alkaloid could down-regulated the expression CD69 obviously on the PDB-activated mouse T cells. It concluded from the above observations that the overall alkaloid of TBL could relieve the inflammatory and immune damages by suppressing the expression of Th1 type cytokines and Th1 cell differentiation, regulating the imbalance of Th1/Th2 cells and inhibiting the early activation of the T lymphocytes bearing CD69. There was no remarkable influence on the intracellular calcium signaling transduction pathway. The inhibitory effected on T cells to express IFN-γ might be due to the suppression of PKC-MAPK signaling pathway. From the standpoint of traditional Chinese medicine, this might be due to the regulation of “Yin” and “Yang” imbalance of joints to modify the pathological status in rheumatoid arthritis. This study provided an experimental basis for the application of overall alkaloids of TBL in the treatment of rheumatoid arthritis.

HBV相关性肝衰竭发病中的Th细胞免疫调控机制及中医药干预的研究进展

HBV(乙型肝炎病毒)相关性肝衰竭发生、发展及演变主要是宿主、病毒作用的结果,其中宿主免疫紊乱、失衡是关键因素,由Th细胞介导的免疫应答在HBV相关性肝衰竭发病中的作用至关重要。从Th细胞介导的免疫调控角度对中医药治疗HBV相关性肝衰竭的相关靶点及其作用机制进行系统性梳理,有利于推动中医治疗HBV相关性肝衰竭的临床、基础研究等工作的开展。

Mechanism of Liancao-Xieli capsule in the treatment of ulcerative colitis through the differentiation of Th17 and Treg cells

Objective:To observe the effect of Liancao-Xieli Capsule on STAT signal pathway and T cell differentiation in mouse model of ulcerative colitis;Methods:Forty BALB/c mice were randomly divided into the control group,model group,mesalazine group and Liancao-Xieli capsule group.Except the control group,the other three groups were treated with 3%dextran sodium sulfate free drinking water to construct the model of ulcerative colitis.During the modeling period,each group was given corresponding drugs for intervention,while the control group and the model group were given the same volume of distilled water by gavage as the control.After treatment,HE staining was used to observe the pathological changes of colon tissue,flow cytometry was used to detect the proportion of Th17 and Treg cells in spleen and mesenteric lymph nodes,and ELISA was used to detect TGF-β,IL-6 and IL-17A in colon tissue.Western blot was used to detect the expression levels of related proteins in STAT3/ROR-γt and STAT5/Foxp3 signaling pathways.Results:Compared with the model group,the body weight,colon length and the content of TGF-βin the colon tissue of the mice in the Liancao-Xieli capsule group increased significantly after the experiment,while the DAI score,colon histopathology score,and the contents of IL-6 and IL-17A in the colon tissue were significantly reduced,and the difference was statistically significant(P<0.01).At the same time,Liancao-Xieli capsule can reduce the ratio of Th17 cells and the ratio of Th17/Treg in the spleen and mesenteric lymph node tissues of UC mice,and increase the ratio of Treg cells,and the difference is statistically significant when compared with the model group(P<0.01).In addition,compared with the model group,the expression levels of p-STAT3 and RORγt protein in the colon tissue of the Liancao-Xieli capsule group were significantly reduced,and the expression levels of p-STAT5 and Foxp3 protein were significantly increased after treatment,and the differences are statistically significant(P<0.01),while the expression levels of STAT3 and STAT5 proteins in colon tissue did not change significantly,and the difference was not statistically significant(P>0.05);Conclusion:Liancao-Xieli Capsule can regulate the immune balance of Treg/Th17 and improve the intestinal inflammation of UC.Its mechanism of action is mainly through inhibiting STAT3/ROR-γt and promoting the activation of STAT5/Foxp3 signaling pathway.

All-transRetinoic Acid Regulates Th1/Th2 Balance in CD4+T cells When GATA-3 is Deficient

The essential effect of vitamin A on immune function occurs through various mechanisms including direct effect on ThloTh2 balance modulation. However, it is unclear whether or not vitamin A can regulate Thl-Th2 balance under a strong Thl-polarizing condition. Therefore, the purpose of our study was to examine the effect of vitamin A metabolite allotrans retinoic acid （ATRA） on ThloTh2 differentiation in CD4~ T cells under GATA-3 deficiency, which can induce Thl-polarizing condition. In the present study, GATA-3 deficiency T cells were induced by siRNA and checked by real-time quantitative PCR and western blot. GATA-3 deficiency CD4＋ T cells and normal CD4＋ T were treated for 48 h with or without ATRA.

Influence of the invasion of peripheral blood mononuclear cells by hepatitis B virus on immune response of the patients with chronic hepatitis B

Objective:To exploretheinfluenceof HBVinvasionintoperipheralbloodmononuclearcells(PBMC)on theimmuneresponseof patientswithchronichepatitisB.Method s:Thecytokinelevelsintheculturesupernatantof PBMCfrom56patientswithchronichepatitisB weredeterminedby ELISA,andPCRwasemployedto amplifythe HBVDNA.Results:Thelevelsof IFN-γinpatientswithhepatitisB waslowerthanthosetof thecontrol,butthe differencewasnotstatisticallysignificant,whilethelevelsof IL-4weresignificantlyhigherthanthoseof thecontrol(P<0.01).Theserumlevelsof HBVDNAwerenegativelycorrelatedwiththatof IFN-γin culturesupernatantsof PBMC.Thirty-fivepatientspositiveof HBVDNA inthePBMCswereidentifiedfrom56patientswithhepatitisB,andtheirIFN-γlevelprovedto be significantlydifferent.Conclusions:Th2cell-mediatedimmuneresponseis predo-minantin chronichepatitisB whichis associatedwiththechronicityof HBVinfection.HBVinvasionintothe PBMCsmayaffectTh1andTh2cell-mediatedimmuneresponseof thepatientswithchronichepatitisB.