| Objective:1.To analyze the clinical efficacy of Kangxian Ruangan Granules（KXRG）in preventing and treating Intestinal Endotoxemia（IETM）of liver cirrhosis caused by chronic hepatitis B（CHB）,and to provide clinical evidence-based basis for KXRG to prevent and treat IETM of liver cirrhosis.2.Through cell model and animal model experiments,from the LPS-TLR4signal transduction pathway to study the role of KXRG in improving IETM in liver cirrhosis and the related molecular mechanism.Methods:1.The literature research method was adopted to discuss the latest research progress of IETM in liver cirrhosis by modern medicine and traditional Chinese medicine,and provide a theoretical basis for KXRG treatment of IETM in liver cirrhosis.2.The clinical efficacy of KXRG in preventing and treating CHB-related cirrhosis IETM was analyzed by the method of clinical retrospective control study.Through the hospital electronic medical record system,the information of patients who were treated in the Huayuanshan District and Guanggu District of Hubei Provincial Hospital of Traditional Chinese Medicine from January 2018 to December 2021 was collected,and the confirmed CHB-related liver cirrhosis was collected.Cases with persistently negative Hepatitis B Virus（HBV）DNA after analog anti-HBV treatment were divided into KXRG group and control group according to whether KXRG was taken in the doctor’s order.The distance between the two observation time nodes was 6 months,and the APRI model score,FIB-4 model score,Child-Pugh grading score,serum liver fibrosis,inflammation-related indexes and liver elasticity test values were compared between the two groups before and after treatment.3.Cell experiment:The CCK-8 method was used to determine the administration concentration of KXRG,and the effect of the selected concentration on the TLR4 signaling pathway of normal Ana-1 cells was detected;then LPS was used to stimulate Ana-1 cells to establish a liver cirrhosis macrophage inflammation model,the effects of different concentrations of KXRG on the expression levels of TLR4 and its downstream Myd88,TRIF,TIRAP,TRAF-6,ERK,and NF-κB molecules in model cells were detected by q RT-PCR and Western Blot.At the same time,the contents of IL-1β,IL-6 and TNF-αin the supernatant of cell culture medium were detected by ELISA;the expression of TLR4 molecule in Ana-1 cells was down-regulated and up-regulated by siRNA and lentiviral vector,respectively,and the above modeling was repeated and dosed.The effects of different concentrations of KXRG on down-regulation and up-regulation of the expression levels of TLR4 and its downstream Myd88,TRIF,TIRAP,TRAF-6,ERK,and NF-κB molecules in model cells were detected by q RT-PCR and Western Blot.4.Animal experiment:The IETM animal model of liver cirrhosis was induced with CCl4and ethanol,and KXRG aqueous solution was administered by gavage at a dose of 3.125 g/kg/d.The weight gain,liver index,spleen index and serum biochemical indexes of the rats were observed.The expression levels of TLR4 and its downstream Myd88,TRIF,TIRAP,TRAF-6,ERK,NF-κB molecules in rat liver tissue were detected by q RT-PCR and Western Blot,and the levels of LPS,IL-1β,IL-6 and TNF-αin serum were detected by ELISA.Histopathological changes of rat liver were observed by HE staining and Masson staining.Results:1.Clinical study results:A total of 85 cases were collected and included in this study,including 46 cases in the KXRG group and 39 cases in the control group.There were no significant differences in gender,age,and past medical history between the two groups,and they were comparable（P>0.05）.（1）Before treatment,there were no significant differences in APRI,FIB-4 and Child-Pugh scores between the two groups.After treatment,the KXRG group was compared with the group before treatment,the three scores were improved,and compared with the control group,it was also significantly decreased,and the differences were all statistically significant（P<0.05）;the comparison between the control group and the group before treatment,the above three scores were lower than before,but the difference was not statistically significant（P>0.05）.（2）Before treatment,there were no significant differences in the four indexes of serum liver fibrosis between the two groups.After treatment,compared with those in the group before treatment,the 4 indexes were improved in the two groups（P<0.05）,but the improvement in the KXRG group was more significant,and the difference was statistically significant（P<0.05）.（3）Before treatment,there were no significant differences in serum CRP,PCT,IL-6 and LPS between the two groups.After treatment,compared with those in the group before treatment,the four indexes were significantly improved in the two groups,and the differences were all statistically significant（P<0.05）,but the improvement in the KXRG group was more significant,and the difference was statistically significant（P<0.05）.（4）Before treatment,there was no significant difference in LSM value between the two groups（P>0.05）.After treatment,the LSM value in the KXRG group was significantly decreased compared with the group before treatment,and compared with the control group,the difference was statistically significant（P<0.05）.There was a statistical difference（P>0.05）.2.Experimental Research ResultsExperiment 1:The effect of KXRG on LPS-activated TLR4 signaling pathway in Ana-1 cell line（1）The maximum non-toxic concentration of KXRG to Ana-1 cells:Analysis of the results of the CCK-8 method showed that when the administration concentration was in the range of 0.25-2.0 mg/mL,KXRG had no significant effect on the survival rate of Ana-1 cells.influence.There was no significant difference in the mRNA transcription levels of TLR4 and TRAF6 in normal Ana-1 cells with three concentrations of 2.0,1.0,and 0.5 mg/mL KXRG compared with the blank control group（P>0.05）.（2）After LPS stimulation of Ana-1 cells,the mRNA and protein expressions of TLR4 and its downstream Myd88,TRIF,TIRAP,TRAF-6,ERK,and NF-κB molecules in the cells of the model group were increased compared with those of the blank control group.After KXRG intervention,compared with the model group,the expression level was significantly decreased（P<0.05）.At the same time,the contents of IL-1β,IL-6 and TNF-αin the supernatant of cell culture medium in the model group were significantly increased compared with those in the blank control group（P<0.05）,and the KXRG groups were significantly decreased compared with the model group（P<0.05）.（3）After the successful transfection of siRNA-TLR4 into Ana-1 cells,the cells were stimulated with LPS to create an inflammation model,and then KXRG was used to intervene.The results showed that the mRNA and protein expressions of TLR4 and its downstream Myd88,TRIF,TIRAP,TRAF-6,ERK,NF-κB molecules in the cells of the model group（siRNA-TLR4+LPS）were significantly higher than those of the siRNA-TLR4 group（P<0.05）.and after KXRG intervention,the expression level was significantly decreased（P<0.05）.（4）After Lentivirus-TLR4 was successfully transfected into Ana-1 cells,the cells were stimulated with LPS to create an inflammation model,and then KXRG was used to intervene.The results showed that the mRNA and protein expressions of TLR4 and its downstream Myd88,TRIF,TIRAP,TRAF-6,ERK,NF-κB molecules in the cells of the model group（Lentivirus-TLR4+LPS）were significantly higher than those of the Lentivirus-TLR4 group after LPS stimulation of the cells.（P<0.05）.After KXRG intervention,the expression level was significantly decreased（P<0.05）.Experiment 2:The effect of KXRG on TLR4 signaling pathway in liver of cirrhotic rats（1）The effect of KXRG on the general condition of rats:After 2 weeks of modeling,the average weekly body weight increase of rats was inhibited,and the average body weight increase of rats in the KXRG group began to be greater than that in the model group after 5 weeks of intervention（P<0.05）.Compared with the blank control group,the liver index of the model group was significantly increased（P<0.05）;compared with the model group,the liver index of the KXRG group decreased（P<0.05）;The index was significantly increased（P<0.05）;there was no statistical difference between the KXRG group and the model group（P>0.05）.（2）The effect of KXRG on serum biochemical indexes of rats:Compared with the blank control group,the serum levels of ALT,AST,ALP and TBA in the model group were significantly increased（P<0.05）,while the serum Alb level was significantly decreased（P<0.05）.compared with the model group,the serum levels of ALT,AST,ALP,and TBA in the KXRG group were significantly decreased（P<0.05）,while the serum Alb level was significantly increased（P<0.05）.（3）The effect of KXRG on serumLPS and inflammatory factors in rats:Compared with the blank control group,the serum levels of LPS,IL-1β,IL-6,TNF-αand CRP in the model group were significantly increased（P<0.05）;Compared with the model group,the serum levels of LPS,IL-1β,IL-6,TNF-αand CRP in the KXRG group were significantly decreased（P<0.05）.（4）Compared with the blank control group,the expressions of related molecules on the TLR4 signaling pathway in the liver tissue of the rats in the model group were significantly increased（P<0.05）;The mRNA and protein expressions of downstream Myd88,TRIF,TIRAP,TRAF-6,ERK and NF-κB molecules were significantly decreased（P<0.05）.（5）The effects of KXRG on the histopathology of the liver of rats in each group:HE staining showed that compared with the model group,the KXRG group had significantly less fatty degeneration,less inflammatory cell infiltration,and less hepatic lobular structural damage.Masson staining showed that compared with the model group,the deposition of blue collagen fibers in the liver tissue in the KXRG group was significantly reduced,and no obvious pseudolobule was found.Conclusion:1.KXRG can effectively improve the model score,liver fibrosis and stiffness indexes of patients with CHB-related liver cirrhosis,and at the same time has a certain regulatory effect on endotoxemia and the inflammatory response caused by it.2.KXRG has a significant inhibitory effect on LPS-activated TLR4 signaling pathway of macrophages,and the inhibitory efficiency is positively correlated with the concentration of KXRG.Its pharmacological action is selective and can inhibit the pathological process without affecting normal cells.Moreover,the target of KXRG on this pathway may be TLR4 molecules.3.By inhibiting the overexpression of TLR4 and its downstream molecules in liver macrophages in the IETM model of liver cirrhosis,KXRG reduces the release of pro-inflammatory factors such as IL-1β,IL-6,and TNF-αmediated by it,reducing the local Inflammatory response,thereby delaying the progression of liver cirrhosis and liver fibrosis,and at the same time playing a good preventive effect on IETM. |
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