| Objective:(1)To sort out and supplement the theoretical connotation of the new theory of "toxic evil-toxic turbidity" in liver failure;(2)To systematically evaluate the efficacy and safety of detoxification and stasis removal combined with comprehensive treatment of Western medicine in the treatment of liver failure;(3)From the perspective of regulating the transmission of miRNA by Kupffer cells through exosome pathway,this paper discusses the therapeutic mechanism of Jiedu Huayu granule in the treatment of chronic and acute liver failure(ACLF),so as to provide more scientific basis for the treatment of ACLF.Methods:(1)Literature review methods to collect the relevant contents of the new theory of "toxic evil and toxic turbidity" of liver failure,sort out and supplement the theoretical connotation of the theory.Search the clinical research literature on the treatment of liver failure with integrated traditional Chinese and Western medicine from January 1,1990 to December 31,2021 in databases at home and abroad such as China How Net and Pub Med,and count the common clinical treatment methods of traditional Chinese medicine for liver failure;Meta analysis was used to evaluate the efficacy and safety of detoxification and blood stasis combined with comprehensive treatment of Western medicine in the treatment of liver failure.(2)Animal experiment to explore the therapeutic mechanism of Jiedu Huayu granule in the treatment of ACLF.Fifty healthy SPF rats were randomly divided into two groups: model group(25 rats)and traditional Chinese medicine group(25 rats in JDHY group);Normal group(10 rats)were set as control.At the beginning of the experiment,the traditional Chinese medicine group was given 6.18g/kg Jiedu Huayu granule by gavage,48 hours after modeling,twice a day,with an interval of 12 hours.The amount of gavage liquid was 4ml/100 g body weight per day.The normal group and the model group were given the same amount of distilled water by gavage at the same time,and the amount of gavage liquid was 4ml/100 g body weight per day.48 h after modeling,aseptic and non heat source blood was collected from rat abdominal aorta,centrifuged whole blood was collected,and serum was collected to detect liver function(ALT and AST);10mm at the same time × 5mm × 2mm liver tissue was fixed at 4 ℃ in 10% formaldehyde fixation solution for at least 24 hours,and then the pathological sections were observed(HE,Masson).Kupffer cell exosomes were isolated by cell experiment,and the exosome miRNA was labeled by chip.Then the labeled exosomes were co cultured with hepatocytes.The co cultured hepatocytes of each group were taken to detect the concentration of exosome NTA.The shape of exosomes in each group was observed by transmission electron microscope.The expression of key membrane proteins of exosomes in liver tissue of each group was detected by Western blot(WB),and the liver function,cell necrosis and cell apoptosis were detected at the same time.Then,the secrete miRNAs of each group were extracted for comprehensive analysis of miRNA SEQ and bioinformatics,and the differential miRNA expression between groups was detected by QRT PCR.After identifying the different miRNAs between groups,we used adenovirus miRNA analogues and inhibitors to analyze go and KEGG,focusing on the changes of cell phenotype involved in hepatocyte inflammatory stress,apoptosis,necrosis and regeneration related signal pathways.Finally,after identifying the differential miRNA and related signal pathways between groups,miRNA inhibitors and signal pathway agonists were used to detect the expression of miRNA and miRNA regulated signal transduction pathway related proteins and genes.Results:(1)The construction of the new theory of "toxic evil-toxic turbidity" in liver failure originated from the treatise on febrile diseases,which has a sufficient theoretical background.The theory puts forward a new viewpoint and new idea on the etiology and pathogenesis of "acute yellow" of "jaundice",and describes in detail the meaning and manifestation of "toxic" and "turbidity" in liver failure,as well as the evolution of etiology and pathogenesis.(2)A total of 120 literatures on common clinical treatment of liver failure were counted.According to the literature,there are seven commonly used treatment methods: detoxification and removing blood stasis,cooling blood and detoxification,warming yang and detoxification,clearing heat and detoxification,clearing heat and dampness,strengthening spleen and Qi,promoting blood circulation and removing blood stasis.Among them,detoxification and removing blood stasis are the most treatment methods(48articles),accounting for 40%,indicating that understanding the method of detoxification and removing blood stasis is the main method of traditional Chinese medicine in the treatment of liver failure.Meta analysis showed that the total clinical effective rate of detoxification and stasis removing method combined with western medicine in the treatment of liver failure was higher than that of Western Medicine(RR=1.30,95%CI [1.18,1.42],P<0.00001);It is suggested that the effect of detoxification and stasis removing method combined with western medicine in the treatment of liver failure is better than that of Western medicine.In terms of improving liver function,reducing prothrombin time,improving prothrombin activity and reducing patient mortality,the detoxification and stasis removing method combined with western medicine comprehensive treatment group compared with the western medicine comprehensive treatment group,P<0.05,the difference is statistically significant.From another point of view,it shows that the clinical efficacy of detoxification and stasis removing method combined with western medicine in the treatment of patients with liver failure is better than that of Western medicine comprehensive treatment.(3)(1)Animal experimental researchALT and AST: compared with the model group,ALT and AST in the traditional Chinese medicine group decreased significantly(P = 0.014,P =0.008),and the difference was statistically significant,suggesting that Jiedu Huayu granule has a better effect on protecting the liver function of ACLF rats than the model group.Liver histopathology: Compared with the model group,the results of HE staining and Masson staining in the traditional Chinese medicine group suggest that the structural disorder of liver lobules in rats has been improved to some extent,and the aspects of liver congestion,liver degeneration and liver necrosis have been improved to varying degrees,indicating that this formula can alleviate the pathological injury of liver tissue in ACLF rats.(2)Cell experiment showed that Jiedu Huayu granule could alleviate the damage of hepatocytes through Kupffer cell exosome pathway [ALT and AST(16.23±1.72,32.23±3.46)in traditional Chinese medicine group were significantly lower than those in model group(25.7±7.18,44.6±5.37)(P=0.049,P=0.013)],improve the survival rate of hepatocytes and reduce hepatocyte apoptosis,and this mechanism may be related to reducing the overexpression of key marker protein CD63 in exosomes(P<0.05),It shows that this differential expression may be the specific expression of Jiedu Huayu granule in the treatment of ACLF through exosomes.(3)After bioinformatics comprehensive analysis of Kupffer cell exosome miRNA and PCR detection of differential miRNA,it was found that the difference of up-regulated mir-9a-5p level in traditional Chinese medicine group was statistically significant compared with model group(P=0.048),indicating that Jiedu Huayu granule antagonizing ACLF disease process may be related to up-regulated mir-9a-5p expression.(4)Then,the biological mechanism information of Jiedu Huayu granule regulating the expression of mir-9a-5p was constructed by using the reverse interference of mir-9a-5p antagonist;Through go and KEGG pathway analysis,it was found that the down regulated "HIF-1 signal pathway" in the traditional Chinese medicine group was consistent with the previously expected signal pathway related to hepatocyte apoptosis,necrosis and regeneration,and the expression difference of this pathway between the groups was statistically significant(P<0.05),suggesting that the specific effect of Jiedu Huayu Granule on up regulating mir-9a-5p may be related to HIF-1 signal pathway.(5)Finally,mir-9a-5p antagonist and HIF-1 signal pathway agonist were used for group intervention.The results of liver function test showed that when mir-9a-5p inhibitor was added in the traditional Chinese medicine group,alt,AST and TBIL(272.34±27.52,496.85±27.08,30.72±3.71)were significantly higher than those in the traditional Chinese medicine group(122.12±10.66,238.88±40.84,14.56±1.26)(P=0.007,P<0.001,P=0.001);After adding mir-9a-5p antagonist and HIF-1 signal pathway agonist at the same time,the levels of ATL,AST and TBIL(372.85±16.29,821.85±35.56 and 40.23±2.89)increased more significantly(P<0.001);At the same time,WB results showed that the expression of HIF-1 protein in traditional Chinese medicine group was significantly higher than that in traditional Chinese medicine group when mir-9a-5p antagonist was added(P<0.001),and the expression of HIF-1 protein was more significantly higher than that in traditional Chinese medicine group when mir-9a-5p antagonist and HIF-1signal pathway agonist were added at the same time(P<0.001);The PCR results were consistent with WB results.After adding mir-9a-5p antagonist,the expression of HIF-1 gene in the traditional Chinese medicine group was significantly higher than that in the traditional Chinese medicine group(P=0.009),while the expression of HIF-1 gene increased more significantly after adding mir-9a-5p antagonist and HIF-1 signal pathway agonist at the same time(P<0.001).This proves that one of the therapeutic mechanisms of Jiedu Huayu Granule on ACLF may be achieved by up regulating the exosomal mir-9a-5p gene and then inhibiting the overexpression of HIF-1pathway.(6)TUNEL staining also found that Jiedu Huayu granule can reduce the apoptosis caused by mir-9a-5p antagonist and HIF-1 signal pathway agonist,which further proved that the biological mechanism of Jiedu Huayu granule in intervening ACLF is to inhibit the over activation of HIF-1 signal pathway by up regulating the expression of mir-9a-5p,so as to reduce the apoptosis of hepatocytes in ACLF rats.Conclusion:(1)The new theory of "toxic evil-toxic turbidity" in liver failure is the inheritance and innovation of the "acute yellow" theory of "jaundice" in traditional Chinese medicine.The new concept put forward by this theory is more suitable for the etiology,pathogenesis and treatment of "acute yellow";(2)Through meta-analysis,it is found that the clinical efficacy of detoxification and stasis removing method combined with western medicine in the treatment of liver failure is better than that of simple western medicine,and the safety is good;(3)Experimental studies have confirmed that Jiedu Huayu granule can antagonize ACLF through exosome transmission miRNA pathway of Kupffer cells,which is specifically manifested in protecting liver function,alleviating liver tissue injury and reducing apoptosis and necrosis of hepatocytes in ACLF rats;Its mechanism is to up regulate the expression of mir-9a-5p gene in hepatocytes,inhibit HIF-1 signal pathway,improve the hypoxia state of ACLF hepatocytes and block the continuous deterioration of hepatocyte apoptosis.At this level,it explains the treatment and utility path of Jiedu Huayu Granule on ACLF. |
PDF Full Text Request