| Research purpose:In this study,PD-1/PD-L1 and CD28/CD80 signaling pathway were used as the entry point to observe the effect of ruanganyin on the function of i NKT cells in HBV transgenic mice,and to reveal the immunoregulation mechanism of ruanganyin in the treatment of HBV transgenic mice.Research methods:50 half male and half female SPF BALB/C strain HBV transgenic mice were randomly divided into Chinese medicine treatment group(high dose group,medium dose group,low dose group),positive control group,model control group and 10 half male and half female non HBV transgenic normal mice as blank control group.The treatment group was given different doses of Ruanganyin(including the crude drug concentration of Ruanganyin formula of 2,1,0.5g/ml),the positive control group was given entecavir dispersible tablet solution,the model control group and the blank control group were given normal saline,and the materials were taken after 4 weeks of continuous administration.The expression of HBV DNA in peripheral blood and liver tissue was detected by Q-PCR,and the pathology of liver tissue was observed by HE staining.The contents of alt,AST,TBIL,LN,HA,IV-C,PCIII,IL-2,IL-4,IL-10 and IFN-? in serum were detected by ELISA,the expression of PD-1,PD-L1,CD28 and CD80 in peripheral blood i NKT cells and their surfaces were detected by flow cytometry,and the expression of PD-1,PD-L1,CD28 and CD80 in liver i NKT cells and their surfaces were detected by immunofluorescence.Results:1.In the detection of HBV DNA expression in peripheral blood and liver tissues of mice by q-PCR: compared with the model group,the content of HBV DNA in serum and liver tissues of high dose,medium dose group and positive control group of Ruanganyin was significantly reduced(P<0.01/P<0.05);However,there was no significant difference in HBV DNA content in serum and liver tissue of the low dose group(P>0.05).2.In the HE staining results of liver pathology: the liver cells in the model group were poorly arranged,the cells were seriously swollen and deformed,even died,the shape of liver cord disappeared,and the inflammatory cells infiltrated;while the high dose,medium dose and positive control groups of ruanganyin all improved the lesions to varying degrees,and the low dose effect of ruanganyin was not significant.3.The content of alt,AST,TBIL,LN,HA,IV-C,PCIII,IL-2,IL-4,IL-10 and IFN-? in serum were detected by ELISA:(1)Compared with the blank control group,the alt,AST and TBIL levels in the model group were significantly higher than those in the blank control group(P<0.01);compared with the model group,the alt,AST and TBIL levels in the high-dose group and the positive control group were significantly lower than those in the model group(P<0.01/P<0.05);while the alt,AST and TBIL levels in the middle and low-dose groups after treatment were significantly higher(P<0.01/P<0.05)There was no significant difference between the two groups(P>0.05).(2)Compared with the blank control group,the level of LN,HA,IV-C and PCIII in the model group was significantly higher than that in the blank control group(P<0.01);compared with the model group,the level of LN,HA and IV-C in the high-dose group and the positive control group after treatment was significantly lower than that in the model group(P<0.01);the level of PCIII in the high-dose group and the positive control group after treatment was significantly lower than that in the model group(P<0.01)There was no significant difference in LN,HA,IV-C and PCIII levels between the two groups(P>0.05).(3)Compared with the blank control group,the level of IL-10,IL-2 and IFN-? in the model group was significantly lower than that in the blank control group(P<0.01);the level of IL-4 in the model group was significantly higher than that in the blank control group(P<0.01);compared with the model group,the level of IL-10 in the high dose,middle dose and positive control groups of ruangyin after treatment was significantly higher than that in the model group(P<0.01);There was no significant difference in the level of IL-10 between the low dose group and the model group(P>0.05);after treatment,the high dose group,the middle dose group and the positive control group had IL-2,IFN-? The level of IL-2 and IFN-? in the low dose group of ruanganyin was not significantly different from that in the model group(P>0.05);the level of IL-4 in the high dose,middle dose group and positive control group of ruanganyin was significantly lower than that in the model group(P<0.01);the level of IL-4 in the low dose group of ruanganyin was not significantly different from that in the model group(P>0.05).4.The expression of PD-1,PD-L1,CD28 and CD80 on the surface of peripheral blood i NKT cells was detected by flow cytometry:(1)Compared with the blank control group,the expression of i NKT cells in the model group was significantly lower(P<0.01);compared with the model group,the expression of i NKT cells in the high-dose group and the positive control group of Ruanganyin after treatment was significantly higher(P<0.01);the expression of i NKT cells in the middle dose group of ruanganyin increased(P<0.05);the low-dose group of Ruanganyin There was no significant difference in the expression of i NKT cells(P>0.05).(2)Compared with the blank control group,there was significant difference in the model group(P<0.01);compared with the model group,the expression level of PD-1and PD-L1 on the surface of i NKT cells in the high dose group and the positive control group of Ruanganyin after treatment decreased,with a certain difference(P<0.05);there was no significant difference in the expression level of PD-1 and PD-L1 on the surface of i NKT cells in the middle and low dose groups of Ruanganyin(P>0.05).(3)Compared with the blank control group,the expression level of CD28 and CD80 on the surface of i NKT cells in the model group had a certain down-regulation trend,with statistical significance(P<0.01/P<0.05);compared with the model group,the expression level of CD28 and CD80 on the surface of i NKT cells in the high dose,medium dose and positive control groups of Ruanganyin after treatment was significantly higher,with significant difference(P <0.01);the low dose group of Ruanganyin The expression level of CD28 and CD80 on the cell surface had no significant change and no significant difference(P>0.05).5.The expression of PD-1,PD-L1,CD28,CD80 on the surface of liver i NKT cells was detected by immunofluorescence: The expression of i NKT on the liver of HBV transgenic mice was significantly lower than that of the blank control group,and the expression of PD-1 and PD-L1 on the surface of i NKT cells was significantly higher than that of the blank control group,while the expression of CD28 and CD80 on the surface of i NKT cells was significantly lower than that of the blank control group(P<0.05).After 4 weeks of treatment,compared with the model group,the expression of i NKT cells in the liver cells of HBV transgenic mice in the high,middle and positive dose groups of Ruanganyin and the positive control group increased significantly,the expression of PD-1 and PD-L1 on the surface of i NKT cells decreased significantly,while the expression of CD28 and CD80 on the surface of i NKT cells increased significantly(P<0.05)There was no significant difference between the model group and the low dose group(P>0.05).Conclusion:1.A traditional Chinese medicine of Ruanganyin can improve the liver tissue damage of HBV transgenic mice,and has certain protective effect on liver function and liver fibrosis.2.Ruanganyin can inhibit HBV in transgenic mice,and its mechanism may be related to the regulation of IL-10,IL-2,IL-4 and IFN-? levels.3.The immune mechanism of Ruanganyin in the treatment of HBV transgenic mice may play an anti HBV role by blocking the PD-1 / PD-L1 signal pathway on the i NKT cells and activating the CD28/CD80 signal pathway on the i NKT cells. |
PDF Full Text Request