Mechanism Research Of Bushen Jianpi Jiedu Prescription And Its Constituents On Immune Resistance In Chronic HBV Carriers

Objective(1) Through the clinical trials in vivo, before and after the treatment of Bushen Jianpi Jiedu Prescription in chronic HBV carriers, the correlation between the key cellulars of immune resistance CD4+CD25+Treg expression frequency and functions、DC mature defect and immune function、the cellular immune functions and serum HBV DNA、HBsAg quantitation was analyzed. Explored the main mechanism of Bushen Jianpi Jiedu Prescription clearing HBV and the relationship of Bushen Jianpi Jiedu Prescription with those key cellulars and their functions in chronic HBV carriers. (2) Through the drug serum in vitro, to explore the target and strength of Bushen Jianpi Jiedu Prescription and its constituent inhibiting immune resistance in chronic HBV carriers, as well as the synergies of each constituent. Providing idears and theories from microcosmic area for the Chinese medicine treatment in chronic HBV carriers.MethodsFirst part experiment:The correlation between the CD4+CD25+Treg expression frequency and functions、DC mature defect and immune function^ the cellular immune functions and serum HBV DNA、HBsAg quantitation before and after treatment.30 cases of chronic HBV carriers were applied with Bushen Jianpi Jiedu Prescription. After 48 weeks the therapeutic effects were assessed, and the following indexes were observed. (1) Divided the chronic HBV carriers baseline serum HBV DNA quantitation into three levels:≤61gIU/ml、61g-81gIU/ml、≥ 81gIU/ml and HBsAg quantitation into two levels:≤51gIU/ml、>51gIU/ml, analyzed the correlation between the CD4+CD25+Treg expression frequency and functions (IL-10, TGF-β)、DC mature defect (HLA-DR、CD80、CD86、CD1 α) and immune functions (MLR、IL-12、IFN-γ)、the cellular immune functions (CD3+、 CD4+、CD8+及CD4+/CD8+) and serum HBV DNA、HBsAg quantitation before treatment。 (2) After 48 weeks treatment, divided the changes of serum HBV DNA quantitation into two levels:≥11gIU/ml、<11gIU/ml and the changes of serum HBsAg quantitation into two levels:≥ 0.51gIU/ml、<0.51gIU/ml, analyzed the correlation between the changes of CD4+CD25+Treg expression frequency and functions、DC mature defect and immune functions、the cellular immune functions and serum HBV DNA、HBsAg quantitation after treatment。(3) Observed the occurrence of adverse effects.Second part experiment:The effect of Bushen Jianpi Jiedu Prescription and its constituents on CD4+CD25+Treg expression frequency and functions、DC mature defect and immune functions in chronic HBV carriers.Randomly divided 25 normal Wistar rats into 5 groups, including saline group、Bushen Jianpi Jiedu Prescription group、Bushen group、Jianpi group and Jiedu group, each group had 5 rats. Preparated the drug serums, and used 5 kinds of drug serums to intervene the chronic HBV carriers’serum in vitro. (1) Observed the effect of 5 drug serums intervening on CD4+CD25+Treg expression frequency and functions (IL-10、TGF-β). (2) Observed the effect of 5 drug serums intervening on DC mature defect (HLA-DR、CD80、CD86、CD1 α) and immune functions (MLR、IL-12、IFN-γ)ResultsFirst part experiment:(1)The correlation between the CD4+CD25+Treg expression frequency and functions、DC mature defect and immune function、the cellular immune functions and serum HBV DNA、HBsAg quantitation in baseline.Divided the chronic HBV carriers baseline serum HBV DNA quantitation into three levels:≤61gIU/ml、61g-81gIU/ml、≥81gIU/ml. Compared the correlation between each HBV DNA quantitation level and with their corresponding CD4+CD25+Treg expression frequency and functions (IL-10、TGF-β)、DC mature defect (HLA-DR、CD80、CD86、CD1 α) and immune functions (MLR、IL-12、IFN-γ)、the cellular immune functions (CD3+、CD4+、CD8+及CD4+/CD8+),which was no significant respectively (P>0.05). Divided the chronic HBV carriers baseline serum HBsAg quantitation into two levels:≤51gIU/ml、>51gIU/ml. Compared the correlation between each HBsAg quantitation level and with their corresponding CD4+CD25+Treg expression frequency and functions (IL-10、TGF-β)、DC mature defect (HLA-DR、CD80、CD86、CD1 α) and immune functions (MLR、IL-12、IFN-γ)、the cellular immune functions (CD+、CD4+、CD8+及CD4+/CD8+), which was no significant respectively (P>0.05)(2) The correlation between the changes of CD4+CD25+Treg expression frequency and functions、DC mature defect and immune functions、the cellular immune functions and serum HBV DNA、HBsAg quantitation after treatment。After 48 weeks treatment, divided the changes of serum HBV DNA quantitation into two levels:≥11gIU/ml、<11gIU/ml. Compared the correlation between each HBV DNA quantitation level and with their corresponding changes of CD4+CD25+Treg expression frequency and functions (IL-10, TGF-J3β)、DC mature defect (HLA-DR、CD80、CD86、CD1 α) and immune functions (MLR、IL-12、IFN-γ)、the cellular immune functions (CD3+、CD4+、CD8+及CD4+/CD8+). When the serum HBV DNA descline≥11gIU/ml, the correlation was significant with the change of IL-10 and CD8+respectively (P<0.05). For the left groups, the correlation was no significant respectively (P>0.05). Divided the changes of serum HBsAg quantitation into two levels:≥0.51gIU/ml、< 0.51gIU/ml. Compared the correlation between each HBsAg quantitation level and with their corresponding changes of CD4+CD25+Treg expression frequency and functions (IL-10、TGF-β), DC mature defect (HLA-DR、CD80、CD86、CD1 α) and immune functions (MLR、IL-12 IFN-γ)、the cellular immune functions (CD3+、CD4+、CD8+及CD4+/CD8+). When the serum HBsAg descline≥0.51gIU/ml, the correlation was significant with the change of CD1 α、IL-12 and CD8+　respectively (P<0.05). For the left groups, the correlation was no significant respectively (P>0.05). Second part experiment:(1) After the intervention of drug serums, in Bushen Jianpi Jiedu Prescription group,Bushen group, Jianpi group and Jiedu group, the CD4+CD25+Treg expression frequency were 5.27%、6.32%、7.08%　and 9.34%　respectively; the level of IL-10 were 8.34pg/ml、12.08pg/ml、10.97pg/ml and 17.27pg/ml respectively; the level of TGF-　β were 237.45pg/ml、307.59pg/ml、264.32pg/ml and 349.72pg/ml. Except for Jiedu group, the CD4+CD25+Treg expression frequency, the level of IL-10 and TGF- β were all declined in the other three groups, especially in the Bushen Jianpi Jiedu Prescription group (P< 0.05). Compared with the saline group, there was a significant difference (P <0.05). (2) Observed the effect of 5 drug serums intervening on DC mature defect (HLA-DR、CD80、CD86、CD1α) and immune functions (MLR、IL-12、IFN-γ) .(2) After the intervention of drug serums, in Bushen Jianpi Jiedu Prescription group. Bushen group、Jianpi group and Jiedu group, the HLA-DR expression rates were 90.94%、89.45%、86.25%　and 83.28%　respectively. Except for Jiedu group, the HLA-DR expression rates were all improved in the other three groups, especially in the Bushen Jianpi Jiedu Prescription group (P< 0.05).Compared with the saline group, there was a significant difference (P <0.05);The CD86 expression rates were 86.35%、85.29%、84.37% and 79.62% respectively. Except for Jiedu group, the CD86 expression rates were all improved in the other three groups, which compared with the saline group, there was a significant difference(P<0.05). In the Bushen Jianpi Jiedu Prescription group, the CD86 expression rate was improved most, but with no significant difference compared with Bushen group and Jianpi group (P>0.05);The CD80 expression rates were 58.07%、55.69%、52.41% and 50.21% respectively, which were improved in Bushen Jianpi Jiedu Prescription group and Bushen group, especially in the Bushen Jianpi Jiedu Prescription group (P< 0.05).Compared with the saline group, there was a significant difference (P <0.05).For the Jianpi group and Jiedu group, there was no significant difference compared with the saline group (P>0.05); The CD1 a expression rates were 48.87%、49.31%、45.87% and 42.78% respectively, which were improved in Bushen Jianpi Jiedu Prescription group and Bushen group. Compared with the saline group, there was a significant difference(P<0.05). For the Jianpi group and Jiedu group, there was no significant difference compared with the saline group (P>0.05); The DC induced MLR abilities were 1.97、1.49、0.85 and 0.66 respectively. Except for Jiedu group, the DC induced MLR abilities were all improved in the other three groups, especially in the Bushen Jianpi Jiedu Prescription group (P<0.05).Compared with the saline group, there was a significant difference (P<0.05);The levels of IFN-γ in MLR serum were 59.4pg/ml、57.2pg/ml、50.6pg/ml and 32.7pg/ml respectively. Except for Jiedu group, the levels of IFN-γ in MLR serum were all improved in the other three groups, especially in the Bushen Jianpi Jiedu Prescription group (P< 0.05).Compared with the saline group, there was a significant difference (P <0.05); The levels of IL-12 in MLR serum were 288.7pg/ml、285.4pg/ml、 234.5pg/ml and 220.4pg/ml respectively, which were improved in Bushen Jianpi Jiedu Prescription group and Bushen group,especially in the Bushen Jianpi Jiedu Prescription group (P<0.05). Compared with the saline group, there was a significant difference (P<0.05).For the Jianpi group and Jiedu group, there was no significant difference compared with the saline group (P>0.05).ConclusionsFirst part experiment:The main mechanism of Bushen Jianpi Jiedu Prescription on treatment of chronic HBV carriers:(1) After the intervention of Bushen Jianpi Jiedu Prescription, which decreased the serum levels of TGF-β、IL-10, declined the CD4+CD25+Treg expression frequency, weakened the immune resistance dominated by CD4+CD25+Treg, provoked the HBV specific immune responses and suppressed /cleared HBV. And there is a positive correlation between the decrease of serum IL-10 level and decrease of HBV DNA load. The greater the decrease of HBV DNA load, the more significant positive correlation between the both. (2) After treatment, which improved DC mature, enhanced DC induced MLR ability, and so enhanced antigen presenting ability of DC, cleared the HBsAg. And there is a negative correlation between the increase of CDl a expression frequency, serum IL-12 level and decrease of HBsAg quantitation respectively. The greater the decrease of HBsAg quantitation,the more significant negative correlation between them. (3) After the intervention of Bushen Jianpi Jiedu Prescription, which increased the expression levels of CD3+　and CD4+T lymphocyte, decreased the expression levels of CD8+T lymphocyte, so that induced the increase of CD4+/CD8+　ratio, regulated the cellular immune function, enhanced the vires specific CTL ability and suppressed/cleared HBV and HBsAg. And there is a positive correlation between the decrease of the expression levels of CD8+T lymphocyte and decrease of HBV DNA 、 HBsAg quantitations respectively. The greater the decrease of HBV DNA、HBsAg quantitations, the more significant positive correlation between them. Second part experiment:For the chronic HBV carriers, the Bushen group as well as Jianpi group can decline the CD4+CD25+Treg expression frequency;Bushen group can improve the DC mature and enhance DC induced MLR ability, which is more significant than Jianpi group;Combining the Bushen group and Jianpi group, which can cooperate to enhance the effect of declining CD4+CD25+Treg expression frequency、improving the DC mature and enhancing DC induced MLR ability. In clinical therapy,we should pay attention to the application of tonifying kidney and spleen drugs, break the immune resistance in chronic HBV carriers, decrease the HBV DNA load and reduce the high risk of disease progression by high HBV DNA load in immune resistance.