| Objective:1.To study the clinical efficacy and evaluate the safety of Qizhu granules in the treatment of hepatitis B compensated cirrhosis with QiXu and XueYu in patients with liver function,serological score,HBV-DNA negative rate,liver elasticity,portal vein internal diameter,spleen thickness,and TCM symptoms.2.To study the mechanism of Qizhu Granules inhibiting liver fibrosis in rats by transcriptomics.3.To study the effects of Astragalus polysaccharide and Curcuma oil on the morphology and Young’s modulus of injured LSECs in rats by establishing a rat model of LSECs.Methods:1.Clinical Trials:Eighty-two patients with hepatitis B compensated cirrhosis who attended the hepatitis clinic of Guang’anmen Hospital of the Chinese Academy of Traditional Chinese Medicine,the gastroenterology clinic of the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,and the liver disease clinic of Foshan City Hospital of Traditional Chinese Medicine between June 2020 and August 2021 and met the inclusion and exclusion criteria were selected and randomly divided into treatment and control groups,with the treatment group treated with Qizhu granules combined with entecavir and the control group treated with entecavir.The patients in both groups were examined for blood routine,liver and kidney function,HBV-DNA,liver hardness,and upper abdominal ultrasound before and after treatment,respectively,and the TCM symptom scores were recorded before and after treatment,and followed up at the end of 24 weeks of treatment.2.Animal expermients:Thirty-five SPF Wistar male rats,aged 6-8 weeks and weighing 200±20g,were randomly divided into blank group,model group,Qizhu granules group and colchicine group.The rats in the blank group were intraperitoneally injected with pure olive oil,and the other groups were intraperitoneally injected with 40%CCl4 olive oil mixed solution at a dose of 3ml/kg,2 times/week.The model was established for 8 weeks,and the rats in each group were perfused on the 14th day of modeling.Stomach,after modeling,the liver tissues of rats in each group were taken for HE staining and Masson staining,and then 3 liver tissue samples were randomly selected from the blank group,model group and Qizhu granules group for transcriptomic analysis.3.Cell experiments:primary normal rat LSECs were extracted,and serum from fibrotic rats was used as an intervention to establish a rat LSECs injury model.The cells were grouped into normal control group(normal LSECs+cell culture medium),injury group(injured LSECs+cell culture medium),Astragalus polysaccharide low,medium and high dose group(injured LSECs+culture medium containing low,medium and high concentrations of Astragalus polysaccharide),and Curcuma oil low,medium and high dose group(injured LSECs+culture medium containing low,medium and high concentrations of Curcuma oil),and the morphology of LSECs in each group was observed by electron microscopy and the fenestration,and Young’s modulus of LSECs was measured by atomic force microscopy in each group of rats.Results:1.Clinical Trials(1)Baseline data:41 patients in the treatment group(31 males and 10 females)and 41 patients in the control group(31 males and 10 females),and there was no statistical significance(P>0.05)of baseline data such as gender,age,and course of disease between the two groups.(2)Liver function:After 24 weeks of treatment,ALT and AST in the treatment group decreased compared with those before treatment,and the difference was statistically significant(P<0.05);the difference between ALT and AST in the control group compared with those before treatment was not statistically significant(P>0.05).(3)FIB-4,APRI,GPR serological scores:After 24 weeks of treatment,the APRI and GPR indices in the treatment group decreased compared with those before,and the difference was statistically significant(P<0.05),and the FIB-4 index was not statistically significant compared with that before treatment(P>0.05);all indices in the control group were not statistically significant compared with those before treatment(P>0.05).(4)HBV-DNA negative rate:After 24 weeks of treatment,there were 12 negative cases in the treatment group,with a negative rate of 66.66%,and 10 negative cases in the control group,with a negative rate of 62.5%,and the difference between the two groups was not statistically significant(P>0.05).(5)Liver stuffness measurement:After 24 weeks of treatment,LSM decreased from 17.41± 4.41 Kpa before treatment to 13.20 ± 3.08 Kpa in the treatment group and from 17.21± 5.11 Kpa before treatment to 15.01 ± 4.61 Kpa in the control group;the difference was statistically significant(P<0.01)in the intra-group comparison between the two groups,indicating that after 24 weeks of treatment,the liver hardness of both groups was improved;the difference between the groups was statistically significant(P<0.05),indicating that the treatment group was better than the control group in improving the liver hardness value after 24 weeks of treatment.(6)Portal vein internal diameter and spleen thickness:After 24 weeks of treatment,the internal diameter of portal vein in both groups was not statistically significant compared with that before treatment(P>0.05);the spleen thickness in the treatment group decreased from 42.58±5.01 mm to 40.63 ± 6.19 mm before treatment,and the difference was statistically significant(P<0.05),and the difference in spleen thickness in the control group was not statistically significant compared with that before treatment(P>0.05).(7)Points of various TCM symptoms:After 24 weeks of treatment in both groups,chest pain,distension,tiredness,loss of appetite,liver palm and spider nevus,skin nail fault,and tongue image were improved to different degrees,(P<0.01).Among them,the treatment group was better than the control group in terms of improvement of chest pain,tiredness and weakness,loss of appetite,skin nail fault and tongue image after treatment(P<0.05).(8)Total TCM symptom scores:At 24 weeks of treatment,the total symptom score decreased from 17.65±3.31 to 8.04±3.33 in the treatment group,and the total symptom score decreased from 18.48±4.23 to 11.65±4.91 in the control group.The differences were statistically significant(P<0.01)when comparing the total symptom scores within the above two groups,indicating that the total symptom scores of patients in both groups decreased significantly after treatment compared with those before treatment,and the differences were statistically significant(P<0.01)when comparing the two groups at 24 weeks of treatment,indicating that the treatment group was better than the control group in reducing the total symptom scores of TCM.(9)Evidence efficacy:In the treatment group,there were 1 case of clinical cure,7 cases of significant effect,29 cases of effective,and 4 cases of invalid,with a total effective rate of 90.24%;in the control group,there were 0 cases of clinical cure,1 case of significant effect,25 cases of effective,and 15 cases of invalid,with a total effective rate of 63.41%,and the difference in total effective rate between the two groups was statistically significant(P<0.01).(10)Follow-up:36 people in the treatment group completed 24 weeks of followup and 32 people in the control group completed 24 weeks of follow-up.Analysis of the efficacy indexes of 68 people treated for 24 weeks and followed up for 24 weeks showed that the LSM in the treatment group decreased from 13.18±3.13 Kpa to 12.43±2.75 Kpa,with statistically significant differences(P<0.01),and the LSM in the control group decreased from 15.20±4.52 Kpa to 15.08±4.95 Kpa in the control group,with no statistically significant difference(P>0.05);the spleen thickness decreased from 40.26±6.11 mm to 39.91±5.92 mm in the treatment group,with a statistically significant difference(P<0.01),and the spleen thickness decreased from 44.36±10.35 mm to 43.78±8.05 in the control group,with no statistically significant(P>0.05);the total TCM symptom score in the treatment group decreased from 8.16 ± 3.29 to 6.94±2.55,with a statistically significant difference(P<0.01),and the total TCM symptom score in the control group increased from 11.25 ± 4.73 to 12.50 ± 3.76,with a statistically significant difference(P<0.01).In the treatment group,the TCM symptoms of distension,tiredness and weakness,and tongue image were further improved,while the TCM symptoms of chest pain and tongue image in the control group increased compared with those at 24 weeks of treatment.2.Animal experiments(1)HE staining of liver tissues:After HE staining of normal rat liver,hepatocytes could be observed with the central vein as the center and arranged in a radial pattern,and the structure of liver lobules and confluent area was clear.In the model group,inflammatory cell infiltration and mild dilatation in the confluent area,structural damage of liver lobules,degeneration and necrosis of hepatocytes,vacuolar changes and disorder of arrangement were seen.In the group of Qizhu granules and colchicine,the structure of liver lobules was improved,and the vacuoles of hepatocytes were reduced and arranged neatly.It indicates that Qizhu granules have the effect of treating liver fibrosis.(2)Masson staining of liver tissue:After Masson staining of normal rat liver,normal hepatocytes with obvious hepatic sinusoidal structure and no collagen deposition and inflammatory cell infiltration were seen.Inflammatory cell infiltration,hepatocyte necrosis,fibrous septum formation,collagen fiber deposition and pseudobullet formation were seen in the model group,while improved hepatic lobule structure and reduced collagen fiber deposition were seen in the Qizhu granules and colchicine groups.Under 20x field of view,5 fields of view were randomly selected in each group,and the ratio of fibrotic area area in each group to the whole field of view was calculated using Image J software,and the difference between groups was calculated using ANOVA one-way ANOVA,which showed that the ratio of fibrotic fibrotic area in each group was statistically significant compared with the model group(P<0.01),indicating that Qizhu granules could reduce the collagen fiber deposition and alleviate liver fibrosis.(3)Transcriptome analysis:The mechanism of Qizhu granules inhibiting liver fibrosis may be related to the regulation of Focal adhesion,ECM-receptor interaction,PIK-Akt signaling pathway,Regulation of actin cytoskeleton,Human papillomavirus infection,Calcium signaling Pathway,Gap Junction,Prostate cancer,EGFR Tyrosine kinase inhibitor Resistance Proteoglycans in Cancer and several target genes such as ItgbI1,Spp1,Lamc2,Col6a1,Col1a2,Itgb6,Itgb4,Bmp4,Erbb2,Epcam and so on.3.Cell experiments.(1)The effect of astragalus polysaccharide on the fenestration opening rate of injured LSECs:the difference between normal and injured groups was statistically significant(P<0.05),and the difference between injured and astragalus polysaccharide medium and high dose groups was statistically significant(P<0.05).Comparing the median between groups,it can be seen that liver fibrosis injury serum can cause LSECs to lose fenestration,and cell cultures containing astragalus polysaccharide have a good alleviating effect,but the improving effect of astragalus polysaccharide only becomes obvious when the dose of astragalus polysaccharide reaches medium dose,and the fenestration opening rate of medium and high dose cells almost reaches normal level.(2)Effect of astragalus polysaccharide on Young’s modulus of injured LSECs:the differences were statistically significant(P<0.01)in the normal group compared with the injured group and the high and low dose groups of astragalus polysaccharide;the differences were statistically significant(P<0.01)in the injured group compared with the high and low dose groups of astragalus polysaccharide,and there was no statistical significance(P>0.05)between the groups of astragalus polysaccharide.Combined with the median data,it is clear that injury serum significantly affects the Young’s modulus of cells in normal LSECs,causing a decrease in Young’s modulus,while astragalus polysaccharide has a significant effect on Young’s modulus of fibrosis-injured cells,but it is not dose-dependent and does not reach the level of normal cells.(3)The effect of curcuma oil on the fenestration opening rate of injured LSECs:the difference between the normal group and the injured group was statistically significant(P<0.01),and the difference between the injured group and the mediumdose group of curcuma oil was statistically significant(P<0.05).The difference between the high-dose group of curcuma oil and the normal group was statistically significant(P<0.01).Comparing the median between the groups,it can be seen that the serum of liver fibrosis injury can cause loss of fenestration in LSECs,and the cell culture solution containing curcuma oil has a good alleviating effect,but only the medium dose of curcuma oil can exert the effect,and the differences between the low and high-dose groups of curcuma oil and the injury group were not statistically significant(P>0.05).(4)The effect of curcuma oil on Young’s modulus of injured LSECs:the difference was statistically significant(P<0.05)in the normal group compared with the injured group and the high and low dose groups of curcuma oil;the difference was statistically significant(P<0.05)in the injured group compared with the high and low dose groups of curcuma oil,and there was no statistical significance(P>0.05)in the low dose group of curcuma oil compared with the medium dose group,and compared with the high dose group.statistically significant(P<0.05),and curcuma oil medium dose compared with high dose group(P<0.05).Combined with the median data,it can be seen that the effect of curcuma oil on Young’s modulus of fibrosis damaged cells was significant and dose-dependent,and the Young’s modulus of curcuma oil high dose group was close to the level of normal cells.Conclusion:1.Qizhu granules combined with entecavir has good efficacy in treating patients with hepatitis B compensated cirrhosis with the evidence of QiXu and XueYu,which can alleviate the symptoms of QiXu and XueYu,reduce liver inflammation,improve serological score,reduce liver hardness and spleen thickness without adverse effects.Therefore,Qizhu granules is a safe and effective herbal compound for the treatment of hepatitis B compensated cirrhosis,which is worthy of clinical promotion and application.This study provides new evidence-based medical evidence for the treatment of hepatitis B compensated cirrhosis with Qizhu granules.2.Qizhu granules may play a role in inhibiting liver fibrosis by regulating focal adhesion,PI3K-Akt,ECM-receptor interaction and other signaling pathways and several target genes such as ITGBL1,SPP1,and LAMC2.3.Astragalus polysaccharide and zedoary curcuma oil showed obvious promoting effects on the fenestration and Young’s modulus of injured LSECs,and the effects on the microscopic morphology and mechanical properties of LSECs showed the same trend.Therefore,it is speculated that astragalus polysaccharide and curcuma oil may affect LSECs by affecting the cytoskeleton of LSECs,thereby affecting the fenestration and Young’s modulus of cells to exert anti-fibrotic effects. |
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