| ObjectiveInjection "Chuankezhi" is an extracted from Yinyanghuo (Epimedium brevicornu Maxim) and Bajitian (Morinda officinalis How), had been approved for treating asthma in China. Application Chuankezhi treated on chronic SIV infected rhesus monkey model, to explore immunoregulatory effects of Chuankezhi and the mechanism of Th17cell differentiation. For the study of warming kidney-yang drugs for laying a foundation for the role of immune regulation, for development from the perspective of immune regulation new treatment of HIV/AIDS program, research and development of new drugs to provide experimental basis.Method12simian immunodeficiency virus (SIV) chronic infection (infection9-22months) in Chinese rhesus monkeys were divided into treatment and control groups, each group6, respectively intramuscular Chuankezhi and saline injections2months. On day0,4weeks,8weeks collecting blood and lymph node biopsies to detect the following indicators:First, the application of real-time PCR detection of SIV monkey plasma load and flow cytometry to detect T lymphocytes CD4, CD8subsets, and using "quantitative score table of rhesus monkeys signs and symptoms" observed AIDS clinical symptoms and weight change of treatment group and control group monkeys, an objective evaluation of the efficacy of the treatment of AIDS Chuankezhi monkeys.Secondly, to explore Chuankezhi for AIDS monkey model of T cell subsets, phenotype and lymphoid tissue structure.①Detected Th17(IL17A mark) and Treg (CD25/Foxp3mark) cell ratio, the number of dynamic change and Thl7/Treg ratio in PBMC by flow cytometry;②Detected T cells initial/memory subsets in PBMC (CD28/CD95mark), T cell proliferation (Ki67marker) and activation (HLA-DR and CD69markers) ratio, and calculate the number of cells to explore its variation by flow cytometry;③pplication lymph node biopsy for HE staining; Ki67immunohistochemical staining to observe the lymph node cell proliferation; reticular fiber staining (silver staining), Masson staining and collagen (Collagen Ⅰ) staining to observe the lymph node tissue and fibrous structure; Desmin immunohistochemical staining to observe f ibroblast reticular cells within the lymph nodes of the network, so as to assess the system Chuankezhi therapy on aspects of lymphoid tissue structure. Finally, focus on Chuankezhi monkey model of AIDS regulating mechanism Th17cell differentiation.①Lymph nodes biopsy separation cell suspension by flow cytometry method for detection of Th17and Treg subsets changes, to verify whether conform to the change in peripheral blood;②To study Th17cell differentiation regulatory mechanisms of Chuankezhi treated on AIDS monkey model, cytokine TGF-0, IL-10, IFN-γ changes of lymph node cells were detected by flow cytometry, and TGF-β expression of lymph node is detected by immunohistochemical methods;③Application Realtime-PCR assay Th17transcription factor IL-17A, RORc, RORa, IL-21, AhR, IFN-r, IL-2and T-box21change, to search signaling pathways of Chuankezhi regulating Th17cells differentiation.④Application of flow cytometry technique to detect the change of B cell and its subsets, ELISA to detect the contents of plasma endotoxin (LPS), in order to understand B cells and the impact of gut microbes transposition after Chuankezhi treated, the monkey SIV IgG antigen to detect sivs IgG antibody. Results(1) Efficacy evaluation results of Chuankezhi treated AIDS monkey model:①Clinical symptoms observed in the monkey model of AIDS:treatment group symptom scores from the beginning of the4week lower than before treatment, rhesus symptoms improved mental activity status is good, anorexia, diarrhea, better than before; symptom scores in the control group gradually increased, rhesus poor mental state, decreased appetite, diarrhea occur repeatedly. Time and treatment interaction effect was significant difference between the value of proximity (P-0.082). Chuankezhi can improve HIV/AIDS monkey spirit, appetite, diarrhea and other symptoms.②Plasma SIV viral load changes:During the observation period, each animal plasma viral load most of the treatment group and control group number between104-106copies/ml, remained stable, no significant difference between the two groups.③T lymphocytes CD4, CD8subsets in peripheral blood:4-8weeks after treatment, the treatment group and the control group the proportion of CD4and CD4/CD8ratio has tended to increase, while CD8counts were dropped, CD8ratio is basically flat. Slightly lower CD4counts in treatment group, a slight increase in the control group, no significant difference between the two groups.(2) The impact of T cell subsets, phenotype and lymphoid tissue structure after Chuankezhi treated AIDS monkey model:①Thl7cells in peripheral blood and lymph node changes:four weeks of treatment the treatment group and the proportion of Th17cells have greatly reduced the number still remained on a declining trend in the treatment of eight weeks; homogenization treatment performed after the first four weeks of Thl7the number of cells was significantly lower than the control group (P=0.032). The proportion of Th17control group basically unchanged, Thl7there is a growing trend in the number. Also interesting finding, CD4negative groups did not significantly reduce IL17+cells. The Thl7result is consistent with peripheral lymph.②Treg cells in peripheral blood and lymph nodes:4-8weeks after treatment, the treatment of peripheral blood CD25+FoxP3-, the number of CD25-FoxP3+and CD25+FoxP3+(Treg) ratio of the three groups and have different degrees of decline in the first with CD25+FoxP3+and CD25+FoxP3-two groups decreased significantly during eight weeks, while the control group increased slightly, the differences between the two groups was statistically significant (P=0.028and0.043). CD25-FoxP3+cell count decreased in both groups, no significant difference between the two groups. Three groups of cells in the lymph nodes obvious change remained stable. Chuankezhi can reduce Treg cells.③Thl7/Treg ratio in peripheral blood and lymph:In4weeks, the treatment group of Th17/Treg ratio drops does not change significantly in the control group, no difference between the two groups statistically (P>0.05). Chuankezhi may reduce Th17/Treg ratio.④CD4initial/memory subsets:Treatment group and control group in the initial CD4, effect of memory CD4type and the central memory CD4does not change significantly over time, no difference between the two groups statistically (P>0.05). After treating Chuankezhi, the Th17cells reduction is not due to initial CD4cells decreased.⑤Early/late activation of CD4cells:Two early cell activation (CD4+HLA-DR+) are basically the same number of changes. The treatment group from the first four weeks beginning late activated cells (CD4+CD69+) tended to decrease,8weeks before treatment dropped by35.1%, while the late-activated cells in control group remained stable or slightly increased. No significant difference between the two groups. Chuankezhi did not cause increase CD4cell activation.⑥CD4cell proliferation:After4weeks of treatment, the treatment group a decrease in CD4cell proliferation, remains a decreasing trend during the first eight weeks, compared with a58.1%reduction in pre-treatment; while the control group after4weeks CD4cell proliferation increased by19.4%to the first eight weeks time and slightly reduced, but increased proliferation than before treatment. No statistical difference between the two groups.⑦Pathology HE results:After8weeks of treatment, the treatment group had five monkeys lymph node structure preserved, in proliferation, degraded state, only a monkey into the depletion of the lymph nodes; while the control group had five monkeys showed lymph node structure degradation and depletion state. Active treatment group germinal center follicular structure is more structured, vice cortex thickening; germinal centers of lymph node control group decreased or disappeared, follicular structure is not complete, vice cortical areas beginning follicular structure is incomplete.⑧Pathology immunohistochemistry and special staining Results:The lymph follicles surrounding normal reticular fibers and Desmin orderly structure and increased; collagen fibers decreased in high endothelial peri vascular vice cortex; collagen Ⅰ (Collagen Ⅰ) reduced. The control group follicles surrounding reticular fibers and Desmin incomplete and reduced; collagen fibers and Collagen Ⅰ did not change or increased.(3) Chuankezhi regulatory mechanisms and the impact of AIDS monkey model of Th17cell differentiation:①Lymph TGF-β expression:After4weeks of treatment, the treatment group TGF-β in T-cell and non-T cells in both groups were decreased, compared with before treatment to reduce up to74.5%, a decrease in non-T cell populations more obvious; TGF-β in the control group did not change significantly, or there is an upward trend. Lymph immunohistochemical expression of TGF-β treatment group decreased in the control group basically unchanged, consistent with the flow results.②IL-10expression in lymph nodes:4weeks of treatment, a slight decrease in the expression of IL-10treatment group, the first eight weeks expressing increasing trend; whereas IL-10expression in the control group maintained a downward trend, the data homogenization treatment the first8weeks of treatment group compared with the control group IL-10expression was significantly increased (P=034).③Th17-related endogenous transcription factor Real-time PCR results: the treatment group T-box21and AhR in the first four weeks from the beginning has decreased, while the control group in a flat or rising trend; IL-17A, IL-21, RORa, RORC groups no significant difference in the first4weeks, but at8weeks treatment group decreased more than50%, while the control group did not change significantly. IL-2treatment groups with other indicators of changes in contrast, rose in the first four weeks, eight weeks after they fall. IFN-γ treatment did not change significantly. Tip downward effect RORa, RORc, AhR, T-box21, IL-17A, IL-21in varying degrees, on the contrary there is upregulation of IL-2,IFN-γ for no obvious effect.④Changes in peripheral blood B cells:After4weeks of treatment, the number of B cells in the treatment group decreased compared to eight weeks before the first treatment decreased by53%, mainly based on tissue-like memory B cells, activated mature B cells and resting memory B cells decrease mainly; than the control group reduced amplitude, but no significant difference between the two groups.⑤Toxins inside (LPS) Change:rhesus macaques infected with SIV uninfected and plasma endotoxin ELISA after SIV infection results show significantly higher levels of plasma LPS healthy monkeys (P<.05). Chuankezhi after LPS treatment is basically stable, no significant increase in the control group slightly increased tendency than before treatment, no statistically significant difference between the two groups. Gonclusion(1) Chuankezhi clinical symptoms of AIDS in monkeys improved after treatment, mental and activity status is good, anorexia, diarrhea, better than before, but the SIV viral load and CD4cell count change had no effect, suggesting that asthma may the efficacy of the treatment of AIDS treatment may not be reflected in lower viral load and CD4cell indicators.(2) Chuankezhi while reducing the blood and lymph nodes Th17and Treg cells, Th17and Treg cells reduce tips Chuankezhi is caused by the body’s systemic changes, because there is competition between Th17and Treg cell differentiation, suggesting that asthma may at least part of the role of governance in both the upstream and differentiation. Although Treg cells decreased, but did not lead to increased T cell activation and proliferation; Th17/Treg ratio decreased and that Th17cells decreased more significantly, suggesting Chuankezhi Th17cell therapy to decrease as the leading role.(3) Chuankezhi monkeys chronically infected with SIV make Th17cells decreased, not increased plasma endotoxin detection. Presumably due to AIDS can reduce inflammation, and does not cause intestinal microbial translocation. Reduce Chuankezhi Treg cells in SIV infected animals, suggesting that immune tolerance can reduce HIV induced immunosuppression tips to improve targets expected to develop new drugs and treatment strategies.(4) Chuankezhi mainly through the reduction of endogenous regulation of transcription factors RORa and RORc Th17differentiation; IL-21expression by down-regulating TGF-β, inhibition of key Th17cell differentiation; through increased expression of IL-10inhibited IL-17, play an anti-inflammatory effect; reduced expression of T-box21, may inhibit Th1and Th17cells upstream of the intermediate state; reduced hydrocarbon receptor (AHR), Treg and Th17cells may be the same reasons for the decline; reduced IL-2expression of Th17cells produce inhibition. Tip Chuankezhi mediated by TGF-β, IL-10and endogenous RORa, RORc, AhR, T-box21, IL-17A, IL-21, IL-2molecules reduce multiple signals differentiation of Th17cells.(5) Chuankezhi B cells reduced to tissue-like memory B cells and activated mature B cell subsets decrease mainly found in lymph follicles while regular morphology, because lymph follicles is induced B cell differentiation of Th17cells produce important parts of autoantibodies, suggesting Chuankezhi make Thl7cells decreased to improve the body’s own immune.(6) Chuankezhi make lymph follicles surrounding fibroblast reticular cells (FRC) ordered structure, collagen deposition and collagen fibers decreased, suggesting Chuankezhi monkey model of AIDS lymph nodes may improve fibrosis. |
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