| Liver cirrhosis,including chronic hepatitis B virus nonalcoholic steatohepatitis and hepatolenticular degeneration(HLD),has a complex pathogenesis.HLD is an autosomal recessive disorder characterized by an imbalance in copper metabolism,resulting in impairment of the integrity and function of liver mitochondria.Mitochondrial damage leads to impaired hepatic energy metabolism and decreased cholesterol biosynthesis-related genes,which in turn induces liver fibrosis and cirrhosis,and leads to portal hypertension,secondary esophagogastric venous hemorrhage,splenomegaly,and hypersplenism.At this time,surgical intervention is required.On the basis of the previous research,this study identified the abnormal glucose and lipid metabolism in patients with HLD liver cirrhosis through clinical observation,and further conducted transcriptomic observation of the liver tissue of patients with HLD liver cirrhosis to clarify the biochemical metabolic pathways and signal transduction pathways of abnormal glucose and lipid metabolism.The LX-2 cell liver cirrhosis model was established and the spleen-invigorating and blood-activating traditional Chinese medicine was intervened to explore the mechanism of traditional Chinese medicine to reverse liver fibrosis,so as to provide a new treatment strategy and scheme for the treatment of HLD complicated with portal hypertension and hypersplenism by traditional Chinese and Western medicine.Objective:By analyzing the clinical data and transcriptomics of patients with HLD liver cirrhosis,it is clearly involved in the metabolism of glucose and lipids in patients with HLD,thereby affecting the signaling pathways and mechanisms of liver fibrosis;then using traditional Chinese medicine for strengthening the spleen and activating blood to intervene in the LX-2 cell model of liver cirrhosis to explore the mechanism of liver fibrosis reversal,in order to provide experimental evidence for clinical application.Methods:1.The findings of abnormal glucose and lipid metabolism in patients with HLD cirrhosis.Clinical data of 236 patients with hepatolenticular degeneration,286 patients with hepatitis cirrhosis and 5 patients with hepatic hemangioma admitted to the Department of General Surgery of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine from January 2020 to December 2021 were collected to analyze the glucose and lipid metabolism of HLD patients.Liver tissues were collected from 5 patients with HLD cirrhosis undergoing splenectomy,5 patients with HBV cirrhosis undergoing splenectomy,and 5 patients undergoing resection of hepatic hemangioma.The pathological changes of liver tissue were observed by Masson staining,the distribution of glycogen in the tissue was observed by PAS staining,and the content of glycogen in the tissue was detected by glycogen detection method.2.Transcriptome study in patients with HLD.Small RNA was sequenced from the liver tissues collected in the first part,the sequencing data was analyzed,and the GO function was analyzed by Top GO software.Based on the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,the biochemical metabolic pathways and signal transduction pathways involved in the target genes of differentially expressed mi RNAs were analyzed;Western blot was used to detect the expression of protein YAP & p-YAP,SREBP-1,SREBP-2,AMPK& p-AMPK,which are related to the specific activated signaling pathway in patients with Wilson’s disease.3.The molecular mechanism of spleen-invigorating and Blood-activating TCM in the intervention of hepatic fibrosis glucose and Lipid metabolism.Liver Fibrosis model of LX-2 cells was induced by TGF-β1,and the distribution and expression of α-SMA,Collagen I,Desmin and Timp-1 were detected by immunofluorescence staining and Western blot,the m RNA expressions of α-SMA,Collagen I,Desmin and Timp-1 were detected by RT-q PCR;screening the optimal effective concentration of the traditional Chinese medicine,intervening the LX-2 cell model by the traditional Chinese medicine,Western blot was used to detect the expression of hepatic fibrosis-related proteins α-SMA,Collagen I,Desmin,Timp-1,Hippo signaling pathway related proteins Yap&p-YAP,TAZ&p-TAZ,AMPK signaling pathway related proteins AMPK&p-AMPK after Chinese medicine intervention.Results:1.The findings of abnormal glucose and lipid metabolism in patients with HLD cirrhosis.The incidences of cholecystitis and gallstones in HLD patients were 49.53% and14.24%,respectively,which were significantly higher than those in the hepatitis and cirrhosis group(7.79% and 9.12%,respectively).The incidence of hyperglycemia in HLD patients was 8.47%,which was significantly lower than that in the hepatitis cirrhosis group(14.89%).These results indicate that both glucose metabolism and lipid metabolism are abnormal in HLD patients.The results of Masson staining showed degeneration of liver and liver tissue in patients with hepatitis cirrhosis group have similar degree of liver cirrhosis,liver cells were separated into false flocculus by fibrous tissue,false flocculus central vein dislocation or lack of fibrous tissue hyperplasia with inflammatory cells infiltration,with Scheuer classification S4.The liver tissue of the hepatic hemangioma group showed the morphology of normal liver tissue.The results of glycogen detection showed that the level of glycogen in liver tissue of patients with HLD was significantly higher than that of patients with hepatitis cirrhosis and hepatic hemangioma,and the results of glycogen PAS staining showed that the liver tissue of HLD patients had significantly more purple-red lumps than hepatitis patients.The results of glycogen detection showed that the content of glycogen in the liver tissue of HLD patients was significantly higher than that of patients with hepatocirrhosis and patients with hepatic hemangioma,and the results of glycogen-PAS staining showed that the number of purplish red masses in the liver tissue of HLD patients was significantly more than that of hepatocirrhosis patients,indicating that the content of glycogen in HLD patients was significantly more than that in hepatocirrhosis patients.2.Transcriptome study in patients with HLD.Compared with hepatic hemangioma patients,28 mi RNAs which significantly enriched in Hippo signaling pathway by bioinformatics analysis were screened for differential expression in HLD patients.Compared with patients with hepatitis and cirrhosis,56 mi RNAs significantly enriched in the AMPK signaling pathway were screened to be differentially expressed in HLD patients.The results of Western blot showed that compared with the hepatic hemangioma group and the hepatitis cirrhosis group,the ratio of Hippo signaling pathway-related protein p-YAP to YAP in the liver tissue of HLD patients was significantly lower than that of other groups,while the AMPK signaling pathway-related protein p-AMPK was significantly associated with The ratio of AMPK was significantly higher than that of other groups;the ratio of Hippo signaling pathway-related protein p-YAP to YAP in HBV liver tissue was significantly higher than that of other groups,and the ratio of AMPK signaling pathway-related protein p-AMPK to AMPK was significantly higher than that of NOR group;patients with HLD and The expression level of Hippo pathway downstream gene SREBP-2 in HBV group was significantly higher than those in NOR group and HBV group,while the expression level of SREBP-1 was significantly lower than those in NOR group and HBV group.3.The molecular mechanism of spleen-invigorating and blood-activating TCM in the intervention of hepatic fibrosis glucose and lipid metabolism.Liver cirrhosis model was established by LX-2 cells induced by TGF-β1,and the results of immunofluorescence,Western blot and RT-q PCR showed that the related indicators of liver cirrhosis,such as α-SMA,Collagen I,Desmin and Timp-1,were significantly increased,indicating that the model was successfully established.The better concentration of Chinese herbal medicine selected by CCK-8 experiment was0.625 mg/m L and 1.25 mg/m L,and the expression of α-SMA,Collagen I and Timp-1 m RNA was detected by RT-q PCR.The results showed that 1.25 mg/m L of traditional Chinese medicine had the best inhibitory effect on liver fibrosis,and finally 1.25 mg/m L of traditional Chinese medicine was selected to study the mechanism.Western blot results showed that after TGF-β1 treatment,the expressions of liver fibrosis related proteins α-SMA,Collagen I,Desmin and Timp-1 were significantly increased.The expressions of liver fibrosis related proteins α-SMA,Collagen I,Desmin and Timp-1 were significantly decreased after the intervention of traditional Chinese medicine compared with TGF-β1 group.The effects of traditional Chinese medicine on Hippo signaling pathway and AMPK signaling pathway were detected.The results showed that compared with the control group,the expression levels of p-YAP and p-TAZ in the model group(TGF-β 1 treatment group)were significantly decreased.,the expression levels of p-YAP and p-TAZ in the traditional Chinese medicine group were significantly increased.Compared with the control group,the expression of p-AMPK in the model group(TGF-β1-treated group)was significantly decreased,and compared with the model group,the expression of p-AMPK in the traditional Chinese medicine group was significantly increased.Conclusion 1.Abnormal glucose and lipid metabolism was found in patients with HLD cirrhosis.2.The activation of Hippo signaling pathway in patients with HLD cirrhosis leads to the dephosphorylation of YAP protein,which inhibits the expression of SREBP-1,promotes the expression of SREBP-2,thereby enhancing the synthesis of fatty acids and cholesterol,and promoting the progression of liver fibrosis.3.AMPK signaling pathway is activated in patients with HLD,resulting in the phosphorylation of AMPK protein,which leads to the increase of glycogen synthesis,the accumulation of glycogen in the liver and the progression of liver fibrosis.4.Traditional Chinese medicine for invigorating spleen and activating blood circulation can improve the degree of liver fibrosis by promoting the phosphorylation of YAP protein,inhibiting the activity of Hippo signaling pathway,inhibiting the synthesis of fatty acids and cholesterol,and improving the degree of liver fibrosis.On the other hand,the traditional Chinese medicine can promote the expression of P-AMPK,activate AMPK signaling pathway,regulate glucose metabolism,promote glycogen metabolism,and improve the degree of liver fibrosis.5.Hippo pathway and AMPK pathway,which regulate lipid metabolism and glucose metabolism,are intrinsically associated with four classical liver fibrosis markers(α-SMA,Timp-1,Desmin,Collagen I).The traditional Chinese medicine for strengthening the spleen and promoting blood circulation not only inhibit the process of liver fibrosis by regulating the Hippo pathway and AMPK pathway,but also interferes with the four classic liver fibrosis indicators.It shows that the traditional Chinese medicine for strengthening the spleen and promoting blood circulation can exert the multi-link and multi-target pharmacological effects of traditional Chinese medicine. |
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