CXCR5/CD40/IL-21-based Immune Network Study Of The Viral Suppression And Immune Improvement Effects And Mechanisms Of Bushen Jiedu Formula No.2 On SIV Infection Models

Objectives1.To observe the inhibition of SIV/HIV activity and the in vitro modulation of CXCR5/CD40/IL-21 immune network by Bushen Jiedu Formula No.2(BJF-2).2.To establish an SIV-infected BALB/c nude mouse model based on intraperitoneal injection of MT-2 cells and to apply it to the in vivo efficacy evaluation of HAART.3.To investigate the effects and mechanisms of SIV inhibition,immune improvement and regulation of CXCR5/CD40/IL-21 immune network in SIV-infected BALB/c nude mice in vivo by BJF-2.Methods1.In vitro pharmacodynamic assay of BJF-2:Extract and preparation of BJF-2;CCK8 colorimetric assay to detect the cytotoxicity of BJF-2 on CEMx174/MT=2;establishment of an in vitro model of HIV/SIV-infected CEMx174/MT-2 cells to observe the cytopathic effect(CPE),real-tiime fluorescence quantitative RT-qPCR method to determine the intracellular viral The relative amount of intracellular RNA and viral load in the supernatant.2.In vitro regulation of CXCR5/CD40/IL-21 immune network by BJF-2:SIV-infected Jurkat cell model was constructed,and the effects of BJF-2 on CXCR5,CXCL13,IL-21,IL-21R,CD40 and CD40L mRNA expression were measured by RT-qPCR;Western Blot and immunofluorescence staining were used to detect the effects of BJF-2 on mRNA expression.Western Blot and immunofluorescence staining to detect the effect of BJF-2 on CXCR5 and CXCL13 protein expression and protein fluorescence intensity.3.Construction of an animal model of SIV infection based on BALB/c nude mice:in vitro culture and preparation of SIV-infected MT-2 cells;implantation of SIV-infected MT2 cells into the peritoneal cavity of BALB/c nude mice by disposable intraperitoneal injection;determination of viral load and dynamic changes in peripheral blood and tissues of nude mice by RT-qPCR;detection of MT-2 cells in nude mice by flow cytometry and immunofluorescence staining.The distribution and colonization of MT-2 cells in nude mice by flow cytometry and immunofluorescence staining;detection of plasma IgG content and dynamic changes in peripheral blood of nude mice by ELISA;detection of pathological changes in major organs and lymph nodes of BALB/c nude mice by hematoxylin-eosin(HE)staining;detection of changes in the percentage and number of lymphocytes,NK cells,B cells and activated B cells in peripheral blood of BALB/c nude mice by flow cytometry and biochemical analyzer.4.SIV-infected BALB/c nude mice model for HAART in vivo evaluation:construction of SIV-infected BALB/c nude mice model;animal grouping and HAART administration;determination of peripheral blood viral load and dynamic changes in each group of nude mice by RT-qPCR;determination of plasma IgG content and dynamic changes by ELISA;detection of pathological changes in major organs and lymphatic tissues by HE staining.Flow cytometry and biochemical analyzer to detect changes in the percentage and number of peripheral blood lymphocytes,NK cells,B cells and activated B cells in nude mice.5.Acute toxicity study of BJF-2:LD50 and acute oral toxicity test of BJF-2;observation of toxic reactions,gross condition of major organs,organ index,serum ALT,AST,GGT and histopathological changes of major organs after administration of BJF-2.6.In vivo efficacy study on SIV-infected BALB/c nude mice:construction of SIVinfected BALB/c nude mice model;animal grouping and administration of BJF-2;determination of peripheral blood viral load and its dynamic changes by RT-qPCR;determination of plasma IgG content and its dynamic changes by ELISA;detection of pathological changes of major organs and lymphatic tissues by HE staining.Flow cytometry and biochemistry analyzer were used to determine the percentage and number of peripheral blood lymphocytes,NK cells,B cells,activated B cells,CD40+B cells and IL21R+B cells in nude mice;RT-qPCR and Western Blot were used to determine the mRNA and protein expression levels of CXCR5,CXCL13,IL-21,IL-21R,CD40 and CD40L in lymph node tissues of nude mice in each group;immunofluorescence staining was performed to detect the fluorescence intensity of CXCR5 and CXCL13 proteins in lymph node tissues of each group.Results1.In vitro pharmacodynamic assay of BJF-2:The proportion of BJF-2 was determined based on the half effective concentration of each extract;the maximum non-toxic concentration of BJF-2 acting on CEMx174/MT-2 cells was 40μg·mL-1;CPE results showed that BJF-2 was free of viral syncytia at concentrations of 0.32～40 μg.mL-1 showed no viral syncytia（"-")or only a small amount of syncytia formation(CPE "-"～"+");the results of RT-qPCR showed that BJF-2 at 0.32-40 μg.mL-1 had a significant downregulation effect on the relative amount of intracellular HIV/SIV RNA and the viral load in supernatant in the CEMx-174 model of HIV/SIV infection(P<0.05,P<0.01 or P<0.001),and its viral inhibitory effect showed a certain quantity-effect relationship,with the effect of the high concentration group being comparable to that of the AZT-positive control group;the effect of BJF-2 The IC50 of BJF-2 in four in vitro models of HIV-1/SIV infection CEMx-174/MT-2 ranged from 0.184 to 0.526tg·mL-1.2.In vitro regulation of CXCR5/CD40/IL-21 immune network by BJF-2:compared with normal control group,CXCR5,CXCL13,IL-21,IL-21R mRNA and CXCR5 and CXCL13 protein expression were differentially up-regulated after SIV infection;compared with the SIV group,the administration of BJF-2 showed different degrees of CXCR5 and CXCR5 and CXCL13 mRNA and CXCR5 protein expression were particularly significantly up-regulated in the SIV group(P<0.01 or P<0.05).3.The animal model of SIV infection based on BALB/c nude mice was constructed:the modeling success rate was 100%,and SIV replicated continuously in the model group of nude mice,which could be measured in the peripheral blood of nude mice for 15-16 weeks and could be consistently stabilized at 4.26±0.33-5.49±0.24 log10 SIV RNA copies/mL;the inoculated SIV infected MT-2 may cause a stressful increase in total plasma IgG in BALB/c nude mice,followed by a gradual decrease and stabilization at significantly lower than normal levels;after 16 weeks of modeling,pathological changes such as reduced lymphocytes and structural changes in spleen and lymph node tissues were observed in the model nude mice;SIV infection may SIV infection may cause a stressful increase in peripheral blood lymphocyte count,NK lymphocyte count,B lymphocyte count,B cell count,activated B lymphocyte count and activated B cell count in BALB/c nude mice(P<0.05.P<0.01 or P<0.001),followed by a decline to below normal levels for several weeks(P<0.05,P<0.01 or P<0.0001),suggesting that an animal model of SIV infection based on BALB/c nude mice may induce immune activity of NK cells,B cells and possibly induce B cell activation and lead to depletion of activated B cells,resulting in immune disorders.4.In vivo evaluation of the efficacy of HAART in SIV-infected BALB/c nude mice:HAART treatment increased the body weight and viability of SIV-infected BALB/c nude mice to a certain extent;after 2 weeks of administration,the peripheral blood plasma SIV viral load of nude mice in the HAART group was significantly lower than that in the model group(P<0.001),and this downregulation effect persisted until 3 weeks after drug withdrawal(P<0.0001 or P<0.001).In the HAART group,the plasma total IgG was significantly higher than that in the model group(P<0.01)and lasted until 3 weeks after drug withdrawal(P<05).The number of peripheral blood lymphocytes(P<0.01),B lymphocytes(P<0.0001)and activated B lymphocytes(P<0.01)in the HAART group were significantly higher than those in the model group,and were still significantly higher than those in the model group after 2 weeks of drug withdrawal(P<0.05);this indicates that HAART treatment can significantly suppress the peripheral blood viral load in the SIVinfected animal model and has a certain modulating effect on the immune function of SIVinfected nude mice.5.Acute toxicity study of BJF-2:The LD50 of BJF-2 was>5000 mg/kg BW;mice did not show any toxic reactions after administration of BJF-2 at a concentration of 5000 mg/kg BW.After 14 days of observation,there were no significant abnormalities in the gross observations and organ indices of the main organs,serum ALT,AST and GGT activities of the mice,and no significant pathological changes were observed in the main organs of the mice.6.In vivo efficacy study of BJF-2 on SIV-infected BALB/c nude mice:BJF-2 significantly inhibited the peripheral blood viral load of SPV-infected animal model based on BALB/c nude mice during the administration period(P<0.0001,P<0.001 or P<0.05)in a dose-dependent manner,and its inhibitory effect on SIV could be sustained until 2 weeks after discontinuation of the drug(P<0.001 or P<0.05).The treatment with BJF-2 had a significant reversal effect on the decrease of total plasma IgG in SIV-infected nude mice(P<0.001,P<0.01 or P<0.05);the spleen tissue and lymph node histopathology of the nude mice in the BJF-2 administration group showed significant improvement;the treatment with BJF-2 had a significant reversal effect on the decrease of peripheral blood lymphocytes in SIV-infected nude mice(P<0.001,P<0.01 or P<0.05).The treatment with BJF-2 significantly reversed the decrease in peripheral blood lymphocytes of SIVinfected nude mice(P<0.001,P<0.01 or P<0.05)and increased the number of peripheral blood B cells(P<0.001,P<0.01 or P<0.05)and activated B lymphocytes(P<0.05)to different degrees,and had an upregulating effect on the decrease in NK lymphocytes.The proportion and amount of B cells expressing CD40 and IL-21R on the surface of peripheral blood of nude mice were significantly increased by BJF-2(P<0.0001,P<0.001,P<0.01 or P<0.05),which may help B cells exert antibody secretion and promote CD8+ T cells and NK cells to exert immune effects;CXCR5,CXCL13 and IL-21R mRNA expression were significantly increased(P<0.0001.P<0.001,P<0.01 or P<0.05)and IL-21 mRNA expression was significantly decreased(P<0.05)in the lymph nodes of BALB/c nude mice in the BJF-2 administration group;CXCR5,CXCL13,PL-21 R and CD40 protein expression in the BALB/c nude mice in the BJF-2 administration group were significantly(P<0.001,P<0.01 or P<0.05),and IL-21 protein expression was significantly downregulated(P<0.05);CXCR5 and CXCL13 protein fluorescence intensity in lymph nodes of BALB/c nude mice was enhanced by the administration of BJF-2(P<0.001 orP<0.05).Conclusion1.The drug composition of BJF-2 is reasonable,with appropriate ratio of each component,low toxicity and high efficiency,and has significant in vitro synergistic antiviral effects on HIV/SIV-infected CEMx174 and HIV/SIV-infected MT-2 cells in vitro models;BJF-2 can specifically activate CXCR5/CD40/IL-21 immune network after SIV infection,which may have a beneficial effect on SIV The formula can specifically activate CXCR5/CD40/IL-21 immune network after SIV infection,which may have a modulating and improving effect on the immune function of SIV infected cells.2.In this study,we propose an SIV-infected BALB/c nude mouse model based on intraperitoneal injection of MT-2 cells,which is capable of achieving high and stable levels of SIV replication in nude mice and is continuously measurable in peripheral blood;SIVinfected MT-2 cells are distributed and colonized in the peritoneal cavity and lymph nodes of nude mice,where these cells continue to proliferate and release free viral particles to maintain infection;free viral replication induces immune activity in B and NK cells in animals and may cause pathological changes similar to HIV-induced immunodeficiency in humans.Although we replaced the missing CD4+T cells in nude mice with the human Tlymphocytic leukemia cell line MT-2 cells,the nude mice still do not have a normal and intact immune system and SIV can only infect MT-2 cells in vivo,which does not fully mimic the host-virus interaction after HIV infection in humans.Despite the limitations of this model,it provides us with a practical small animal model with low modelling costs,easy handling,short modelling period,high success rate and good reproducibility,which can be used for primary screening of HIV drugs and preliminary in vivo eficacy assessment.3.SIV replication in a SIV-infected nude mouse model was significantly inhibited by BJF-2,and the immune abnormalities caused by SIV infection were significantly modulated and improved,reversing the B-cell reduction and depletion of activated Blymphocytes caused by SIV infection,stimulating B-cell secretion of antibodies,and promoting the immune effects of B-cells and NK cells.The mechanism of action may be related to the CXCR5/CD40/IL-21 immune network,through the regulation of CXCR5/CD40/IL-21 immune network-mediated immune clearance-related cellular chemotaxis,which may play a role in immune regulation,immune reconstitution,or immune rejuvenation.