The Study On Immunomodulatory Mechanism Of Fuzheng Huayu Recipe Combined With Entecavir To Effectively Reverse HBV Cirrhosis Based On The Relationship Between Liver Natural Killer Cells And Liver Stellate Cells

Objective:To investigate the phenotypic variation characteristics and relevant pathological molecules of liver NK cells in liver tissue of HBV cirrhosis and cancer.To analyze the immunoregulatory effects of Chinese medicine Fuzheng Huayu recipe（FZHY）combined with anti-viral drug Entecavir（ETV）on hepatic NK cells of HBV liver fibrosis mice.After further removing NK cells in vivo,the effect of combined drug regimen on the efficacy anti-HBV liver fibrosis was observed.To illustrate the immunoregulation mechanism of FZHY anti-fibrosis via improving the NK cells killing activity to inhibit the activation of HSC in vitro.The paper is divided into four parts.Methods:1.Liver tissue samples and preoperative clinical baseline data were collect from Surgical Inpatients in the Liver Transplantation Center of Renji Hospital affiliated to Shanghai Jiaotong University from November 2017 to October 2018.The inflammation and fibrotic degree were detected with HE and Sirius red pathological staining.The number and subtypes of NK cells and the expression of active receptors NKG2D,NKp30,NKp44 and NKp46 were detected by flow cytometry（FCM）.Pearson correlation analysis was used to observe the correlation between NK related indicators and clinical baseline data,and also the hepatic Sirius red staining area parameters.The co-localization and expression difference of activated NK cells and activated HSC was performed by immunohistochemical double staining.2.HBV transgenic（HBV-Tg）mice were combined with 10%CCl₄intraperitoneal injection to induce HBV background hepatic fibrosis mouse model.Wild type（WT）mice and only HBV-Tg mice were used as controls.After 2 weeks model building,FZHY,ETV and FZHY+ETV for treatment and continued 10%CCl₄intraperitoneal injection for 4 weeks.General condition of mice and organ index were then observed and calculated.HBs Ag,HBe Ag and HBV-DNA and other HBV related virology index,the hepatic function ALT,AST,AKP,TBil,Alb in serum were detected.The hepatic inflammation and fibrotic degree were observed.The content of hydroxyproline（Hyp）in liver tissues was detected,the lymphocyte subpopulation in liver and the expression of NKG2D on the surface of NK were detected by FCM,and the expression of NKG2D ligand Rae-1 on HSC were observed by immunofluorescence staining（IF）.3.HBV background hepatic fibrosis mouse model was established with the methods above.Meanwhile,ASGM1 antibody intraperitoneal injection was used to clear NK cells.The mice were divided into 5 groups:WT group,Model+Rabbit Ig G group（MI）,Model+Anti-ASGM1 group（MA）,Model+FZHY+ETV+Rabbit Ig G group（EFI）,Model+FZHY+ETV+Anti-ASGM1 group（EFA）.The model was prepared and the drug was administered simultaneously.After 4 weeks treatment,the mice were sacrificed,the body weight and general condition were recorded.Indicators related to liver fibrosis and NK cells were observed in the same way as the second part.4.In vitro,human peripheral blood derived NK92 cells line and human hepatic stellate cells line LX-2 was used.Firstly,the maximum non-toxic concentration of FZHY on NK92 cells and LX-2 cells were determined by CCK8.The apoptosis of NK92 cells was detected by Annexin V/7-AAD staining method.The m RNA of NKG2D,NKp30,NKp44 and NKp46 on NK92 cells were detected by q PCR after incubating with FZHY for 8h and 16h.The expression of NKG2D were further detected with FCM.The secretion levels of IFN-γand granzyme B were evaluated by ELISA.K562 cells line were used to evaluate immune killing function of NK92 in co-culture system,while Poly I:C was treated as a positive control.Finally,FZHY pre-incubated NK92 cells were co-cultured with LX-2 cells for 5h.Then the expression of NKG2D on NK92cells were detected by FCM,the activation of LX-2 cells and the expression of RAE-1were observed by IF.Results:1.Through the statistical analysis of the preoperative data of 9 patients in normal donor group,8 patients in HBV cirrhosis group,and 12 patients in HBV liver cancer group,we found that the average age of the three groups increased successively.Compared with normal donor group,the HBV cirrhosis group patient had significantly lower Hb and PLT,significantly higher neutrophils percentage,significantly longer PT,and significantly higher INR,while the HBV liver cancer group patient had significantly lower PLT,significantly higher neutrophils percentage andγ-GGT.Compared with HBV cirrhosis group,the level of PLT andγ-GGT in HBV liver cancer group was significantly higher.Through pathological observation,we found that a large number of inflammatory cells infiltrated into liver tissue with obvious fibrosis in the HBV cirrhosis group,while the active proliferation of cancer cells and inflammatory cells infiltration with diffuse fibrosis were observed in HBV liver cancer group.FCM data showed that the number of NK cells,the expression of NKG2D in HBV cirrhosis group were decreased significantly compared with the normal donor group,while the expression of NKp46 and NKG2D were decreased significantly in HBV liver cancer groups.Pearson correlation analysis showed that the proportion of liver NK cells was positively correlated with Alb level.The expression of NKG2D,NKp44,NKp46 was negatively correlated with PT and INR.The expression of NKG2D was also negatively correlated with the sirius red positive staining area of liver tissue.Besides,the lower expression of NKp46 was observed from the immunohistochemical double staining images in HBV cirrhosis and HBV cancer groups.It is mainly scattered expressed around theα-SMA positive staining area,while the expression ofα-SMA was very low in the normal donor group.2.Compared with WT control group,the serum HBV-DNA load was higher than 1×10⁷IU/ml in HBV-Tg and HBV-Tg+CCl₄mice.HBs Ag in serum and liver tissue,HBe Ag in serum and HBc Ag in liver tissue were positively expressed.Compared with HBV-Tg group,the levels of ALT AST,AKP and TBil of HBV-Tg+CCl₄group in serum were increased,Alb level was decreased.We also found hepatocyte necrosis,massive inflammatory cell infiltration in the portal area,marked collagen fibrosis,significantly increased Hyp content,and significantly increased expression ofα-SMA and Collagen-I in HBV-Tg+CCl₄group.Besides,the proportion of B cells increased,the proportion of NK cells and NKT cells decreased significantly,the proportion of CD4⁺T cells in T lymphocytes decreased significantly,and the proportion of CD8⁺T cells increased significantly in HBV-Tg+CCl₄group liver tissue.The expression of NKG2D and RAE-1 were significantly reduced.Compared with the HBV-Tg+CCl₄model group,the load of HBV-DNA in serum,HBe Ag level and liver function ALT,AST,AKP,TBil levels were decreased with different degrees in ETV,FZHY,FZHY+ETV groups.Although the degree of fibrosis was not significantly improved in the ETV group,the proportion of subpopulation of lymphocyte in the above three groups was changed in varying degrees.In ETV,FZHY,FZHY+ETV groups,the proportion of NK cells in lymphocytes,the expression of NKG2D on NK cells increased significantly in turn.The expression of RAE-1 in liver tissue increased significantly in FZHY and FZHY+ETV groups.3.After removing NK cells in vivo,compared with WT group,the proportion of intrahepatic NK cells decreased significantly,the load of HBV-DNA in serum was still higher than 1×10⁶IU/ml,the expression of HBs Ag,HBe Ag were positive,ALT,AST,AKP levels were significantly increased in MA and MI group.And a large number of inflammatory cells infiltrate into liver tissue,accompanied by local necrosis,hepatocyte balloonlike and fatty degeneration,obvious collagen deposition,increased content of Hyp,and disorganized proportion of immune cells in liver.Compared with the MI group,the proportion of NK cells in liver decreased significantly,and the fibrotic area and Hyp content showed an increasing trend in MA group.However,the proportion of intrahepatic NK cells was significantly increased,the load of HBV-DNA in serum,liver functional biochemical indicators,the degree of inflammation and fibrosis and Hyp level were significantly reduced in EFI group.Compared with MA group,the proportion of intrahepatic NK cells increased,the serum HBV-DNA load and liver function biochemical indexes decreased significantly,the degree of liver fibrosis slightly improved,and the content of Hyp slightly decreased in EFA group.4.The date in vitro showed that FZHY had no obvious toxicity to NK92 or LX-2 cells at a concentration of 100μg/ml or less.FZHY with lower concentration（0.1～10μg/ml）had protective effects on NK92 cells apoptosis.The expression of NKG2D,NKp30,NKp46 and the secretion level of IFN-γincreased at different degrees after incubating NK92 cells with FZHY at different concentrations for 16h.FZHY increased the killing function of NK92 cells to K562 cells in a dose-dependent manner.The expression of NKG2D on NK92 cells and RAE-1 on LX-2 cells were up-regulated,the expression ofα-SMA was decreased after FZHY pre-incubated NK92 cells co-cultured with LX-2cells for 5h.Conclusion:1.Under the condition of HBV cirrhosis,the proportion of NK cells decreased,the expression of NKG2D on NK cells surface decreased in liver.In the liver of HBV cancer,the expression of NKG2D and NKp46 was reduced.The number of NK cells was positively correlated with serum Alb level.The expression of NKG2D,NKp44and NKp46 was negatively correlated with PT and INR levels.The expression of NKG2D was also negatively correlated with liver fibrosis area.The data indicated that the number and function of NK cells in the liver were inhibited during the course of HBV-related chronic liver diseases.2.FZHY combined with ETV has the synergistic effect of anti-virus and anti-fibrosis in mice with HBV background.ETV has limited effect on improvement of liver fibrosis,but it can increase the proportion of NK cells in the liver and enhance the expression of NKG2D.FZHY can significantly increase the number of NK cells in the liver and further significantly promote the expression of NKG2D.At the same time,FZHY can also enhance the expression of NKG2D ligand RAE-1 on HSC surface.FZHY combined with ETV had a correctly regulation of intrahepatic immune lymphocyte population and promoted the activation of NK cells and played a role in anti-liver fibrosis with HBV.3.After NK cells were eliminated by injecting Anti-ASGM1,the number of NK cells was significantly reduced in the liver.The liver fibrosis was aggravated on the basis of HBV-Tg plus CCl₄model.FZHY combined with ETV could antagonize the eliminating effect of Anti-ASGM1 on NK cells in the liver of the composite model.However,inhibiting the number and function of NK cells,to a certain extent,limits the effect of this combined drug regimen on improving liver fibrosis.It suggested that FZHY combined with ETV could treat HBV liver fibrosis by increasing the number and activity of NK cells in the liver.4.FZHY can positively regulate the function of introhepatic NK cells.It played central roles in anti-fibrosis through inhibiting the activation of HSC via enhancing the direct contact between NK cells and HSC cells by surface molecule NKG2D-RAE-1activation.