| Objective1.To further study its anti fibrosis effect by observing the effects of Qizhu Granules on liver function,four items of liver fibrosis,liver elasticity,and TCM syndromes in patients with liver fibrosis due to Qi deficiency and blood stasis syndrome.2.To study the effect of Qizhu Granules on liver fibrosis in rats and its effect on autophagy of liver cells through animal experiments.3.To study the effect of Qizhu Granules on the activation of hepatic stellate cells(HSCs)and its effect on autophagy of activated HSCs through cell experiments.Methods1.Clinical trial:Patients with liver fibrosis of Qi deficiency and blood stasis syndrome who were hospitalized in the Department of Spleen and Stomach Diseases and the Hepatitis Clinic of Guang’anmen Hospital from January 2022 to February 2023 were selected as subjects and randomly divided into the experimental group(Qizhu Granules Group)and the control group(Compound Biejia Ruangan Tablet Group).The experimental group received oral administration of Qizhu Granules,while the control group received oral administration of Compound Biejia Ruangan Tablet for 12 weeks.During the treatment period,no other anti fibrosis drugs were given.Liver function indicators were measured before and after 12 weeks of treatment in both groups:alanine aminotransferase(ALT),aspartate aminotransferase(AST),glutamyltransferase(GGT),and total bilirubin(TBIL);Four items of liver fiber:hyaluronic acid(HA),laminin(LN),procollagen type Ⅲ(PC Ⅲ),and collagen type Ⅳ(Col-Ⅳ);Liver elasticity;And fill in the TCM symptom score table.2.Animal experiment:Forty male Wistar rats were randomly divided into blank group,model group,Qizhu Granules group,and Compound Biejia Ruangan Tablet group.The rats in the model group,the Qizhu Granules group,and the Compound Biejia Ruangan Tablet group were injected intraperitoneally with 40%carbon tetrachloride(CCl4)olive oil solution to create a model,while the blank group was injected intraperitoneally with olive oil.After the first modeling,preventive medication was administered.The Qizhu Granules group and the Compound Biejia Ruangan Tablet group were administered by gavage,while the model group and the blank group were given equal volume of sterile water for 4 consecutive weeks.After the course of treatment,blood was taken from the abdominal aorta under pentobarbital sodium anesthesia to detect liver function.Liver tissue was taken for Masson staining,HE staining,and immunohistochemical staining α-Smooth muscle actin(α-smooth muscle actin,α-SMA)and LN,Western blot(WB)detectionα-SMA,to study the effect of Qizhu Granules on liver fibrosis in rats.WB and real-time fluorescence quantitative polymerase chain reaction(QRT-PCR)methods were used to detect the protein and gene expression of autophagy related genes 3(Atg3),Beclinl,microtubule associated protein light chain 3(LC3-Ⅰ),LC3-Ⅱ,and p62.The autophagy phenomenon was observed under transmission electron microscopy,and LC3 was detected by immunofluorescence method,To study the effect of Qizhu Granules on autophagy in rats with liver fibrosis.3.Cell experiment:Forty male Wistar rats were randomly divided into a blank group of 10 rats,a Qizhu Granules group of 20 rats,and a Compound Biejia Ruangan Tablet group of 10 rats.The Qizhu Granules group and the Compound Biejia Ruangan Tablet group were administrated with drugs by gavage,while the blank group of rats were administrated with triple distilled water twice a day for 5 consecutive times.One hour after the last gastric infusion,pentobarbital sodium was used for anesthesia,blood was collected from the abdominal aorta,and serum was isolated to obtain Qizhu Granules drug serum,Compound Biejia Ruangan Tablet drug serum,and normal non drug containing serum.The primary HSCs were obtained from male Wistar rats using in situ liver perfusion,in vitro liver tissue digestion,and density gradient centrifugation.Cells were counted with trypan blue,and desmin(Des)was detected by immunofluorescence assay for identification.Using transforming growth factors-β1(Transforming growth factor-β1,TGF-β1)Stimulate passage cells to activate,and detect Collagen type Ⅰ(Col-Ⅰ)by QRT-PCR α-SMA,WB detectionα-SMA,immunofluorescence detection The activation degree of HSCs was identified by α-SMA.The passage cells were divided into 7 groups:blank group,model group,normal serum control group,Compound Biejia Ruangan tablet group,high,medium,and low dose groups of Qizhu Granules.TGF-β1 was administered to all patients except the blank group Stimulate for 48 hours to activate HSCs,and incubate them with corresponding drug serum for 24 hours.Add the blank group to the same culture medium for culture.WB test was performed in each group α-SMA,to study the effect of Qizhu Granules on activating HSCs.The passage cells were divided into 7 groups:blank group,model group,normal serum control group,Compound Biejia Ruangan tablet group,Qizhu Granules group,Baflomycin A1(Baf A1)inhibitor group,Baf A1 inhibitor+Qizhu Granules group.TGF-β1 was administered to all patients except the blank group.Stimulate for 48 hours to activate HSCs,and incubate them with corresponding drug serum for 24 hours,respectively.The Baf A1 inhibitor group and the Baf A1 inhibitor+Qizhu Granules group were incubated with Baf A1 for 4 hours,and Col-Ⅰ α-SMA,Atg3,Beclinl,LC3-Ⅰ,LC3-Ⅱ,p62,to study the effect of Qizhu Granules on autophagy of activated HSCs.Results1.Clinical trial:(1)General information:A total of 65 patients were included in this study,including 33 in the experimental group and 32 in the control group.Among them,1 patient in the experimental group fell off and 2 patients withdrew;Two patients in the control group fell off,and in the end,30 patients in each group completed the experiment.There were 16 males(53.33%)and 14 females(46.96%)in the experimental group,while 17 males(56.67%)and 13 females(43.33%)in the control group.The average age of the experimental group was 51.067 years old,with an average course of disease of 4.433 years.The average age of the control group was 48.633 years old,with an average course of disease of 4 years.There was no statistically significant difference in gender,age,and course distribution between the two groups of patients,P>0.05.(2)Effect of Qizhu Granules on liver function:Two groups of patients were compared before and after self treatment,and ALT,AST,GGT,and TBIL were significantly reduced,with a statistically significant difference(P<0.01).(3)Effect of Qizhu Granules on four items of liver fiber:There was no statistically significant difference in HA,PCⅢ,and Col-Ⅳ between the two groups of patients after treatment.The LN of the experimental group patients was lower than that of the control group after treatment,P<0.01.Two groups of patients were compared before and after self treatment,HA,PCⅢ,and Col-Ⅳ were significantly reduced after treatment compared to before treatment,with a statistically significant difference(P<0.01);the experimental group of patients,after treatment compared to before treatment,the LN of patients in the experimental group showed a significant decrease(P<0.01);the control group of patients,after treatment compared to before treatment,the LN of patients in the control group showed a significant decrease(P<0.05).(4)Effect of Qizhu Granules on liver elasticity:There was no statistically significant difference in liver stiffness measurement(LSM)between the two groups after treatment,P>0.05.The LSM of the two groups of patients before and after treatment was significantly lower than that before and after treatment,with a statistically significant difference(P<0.05).(5)Effect of Qizhu Granules on TCM syndromes:The total effective rate of both groups of patients was determined by the efficacy index to be 90%.There was no statistically significant difference in the efficacy evaluation of traditional Chinese medicine syndrome scores between the two groups of patients,P>0.05.According to the statistics of symptoms and signs of two groups of patients,there were 19 cases of thoracic and lateral pain in the experimental group,with a total effective rate of 73.68%.There were 21 cases of thoracic and lateral pain in the control group,with a total effective rate of 71.43%;23 people in the experimental group were fatigued and fatigued,with a total effective rate of 60.87%,while 27 people in the control group were fatigued and fatigued,with a total effective rate of 62.96%;There were 21 cases of anorexia in the experimental group,with a total effective rate of 77.78%,and 17 cases of anorexia in the control group,with a total effective rate of 57.89%.There were 21 cases of abdominal distension and discomfort in the experimental group,with a total effective rate of 80.95%.In the control group,there were 19 cases of abdominal distension and discomfort,with a total effective rate of 88.24%;21 people in the experimental group had dry and bitter mouth,with a total effective rate of 61.90%,while 19 people in the control group had dry and bitter mouth,with a total effective rate of 68.42%;The experimental group had 15 cases of loose stools,with a total effective rate of 80%,while the control group had 17 cases of loose stools,with a total effective rate of 88.24%.There was no statistically significant difference in the total effective rate of treatment for each syndrome between the two groups of patients,P>0.05.2.Animals’ Experiment:(1)Effect of Astragalus granule on liver fibrosis in rats:①Liver function:The results showed that compared with the blank group,the serum ALT,AST,ALB,and ALP in the model group of liver fibrosis rats were significantly increased,with a statistically significant difference(P<0.01).Compared with the model group,the expression of serum ALT,AST,ALB,and ALP in rats with liver fibrosis in the Compound Biejia Ruangan tablet group and Qizhu granules group were significantly reduced,with a statistically significant difference(P<0.01).② Masson staining:The liver structure and tissue of the blank group rats are clear,the cytoplasm of liver cells is intact,the nucleus is clear,there are a small amount of collagen fibers distributed in the portal vein area,the liver sinuses are not dilated,and the liver cells are arranged radially and neatly around the central vein.Inflammatory cells infiltrate the liver tissue of the model group rats,with a large amount of collagen fibers deposited and extending outward from the periphery of the portal area,with thicker fiber strands.The liver tissue of rats in the Qizhu Granules and Compound Biejia Ruangan Tablets groups showed lighter blue staining compared to the model group,while the fibrous tissue in both groups decreased compared to the model group.③ HE staining:In the normal group,there is no abnormality in the liver tissue structure,and the hepatocytes are radially distributed around the central vein.In the model group,inflammatory cells infiltrated and slightly expanded in the portal area,liver lobular structures were damaged,hepatocytes degenerated and necrotic,with vacuolar changes and disordered arrangement.Compared with the model group,the vacuoles in the Qizhu Granules group and the Compound Biejia Ruangan Tablet group were significantly reduced,and the structure of liver lobules was improved and arranged neatly.No pseudolobular structure was observed.④ Immunohistochemical test α-SMA and LN:α-SMA normal group showed weak positive expression,while the model group showed strong positive expression,which was distributed radially along the fibrous tissue.The expression in the Qizhu Granules group and the Compound Biejia Ruangan Tablet group was weaker.LN was distributed around the cells,with weak positive expression in the normal group,strong positive expression in the model group,and weak expression in the Qizhu Granules group.⑤ WB detection α-SMA:Compared with the blank group,the model group exhibited a significant increase inα-SMA protein expression in rat liver tissue,with a statistically significant difference(P<0.01).In comparison with the model group,the Compound Biejia Ruangan tablet group showed a significant decrease in α-SMA protein expression in the liver(P<0.01),and the Qishu Granules group also showed a decrease in α-SMA protein expression with a statistically significant difference(P<0.05).(2)Effects of Qizhu Granules on autophagy of liver cells:① mRNA expression of Atg3,Beclin1,LC3-Ⅱ,and p62 in liver tissue of rats with liver fibrosis in each group:Compared with the blank group,the mRNA expression levels of Atg3,Beclinl,and LC3-Ⅱ in rat liver tissue were increased,while the mRNA expression level of p62 was decreased in the model group.Compared with the model group,the mRNA expression of Atg3 was significantly decreased in the Qizhu Granules group and Compound Biejia Ruangan Tablet group(p<0.05).The mRNA expression of Beclinl was also significantly decreased,while the mRNA expression of p62 was increased in these two treatment groups.Additionally,the mRNA expression of LC3-Ⅱ was decreased in the Qizhu Granules group and the Compound Biejia Ruangan tablet group(p<0.05).② Expression of Atg3,Beclinl,LC3-II,and p62 proteins in the liver tissues of rats in each group:Compared with the blank group,the protein expression levels of Atg3 and LC3-Ⅱ were increased in rat liver tissue in the model group,and the differences were statistically significant(p<0.01).Additionally,the protein expression level of Beclinl was increased while the protein expression level of p62 was decreased in the model group.Compared with the model group,the protein expression levels of Atg3 and LC3-Ⅱ were significantly decreased in the Qizhu Granules group and the Compound Biejia Ruangan tablet group(p<0.01),and the protein expression level of p62 was increased in these treatment groups.Furthermore,the protein expression level of Beclinl was significantly decreased in the Qizhu Granules group(p<0.05),while the protein expression level of LC3-Ⅱ was significantly decreased in the Compound Biejia Ruangan tablet group(p<0.05).③Transmission electron microscopy observation of autolysosomes:Compared with the blank group,the number of autolysosomes and the level of autophagy were increased in the model group.Compared with the model group,the number of autolysosomes and autophagic bodies in the liver tissue of the Qizhu Granules group and the Compound Biejia Ruangan tablet group were decreased.④LC3 immunofluorescence in rat liver tissue of each group:Compared with the blank group,the expression level of LC3 was increased in the model group,and the difference was statistically significant(p<0.01).Compared with the model group,the expression level of LC3 was significantly decreased in the Qizhu Granules group and the Compound Biejia Ruangan tablet group,and the difference was statistically significant(p<0.05).3.Cell Experiment:(1)HSCs extraction and identification:① Observation and counting:The freshly isolated HSCs were in the form of dots and stars under the light microscope.Under the inverted fluorescence microscope 328 Bin ultraviolet light,the cells spontaneously emit blue-green fluorescence,but were quickly quenched.After passage,the cells grew rapidly,gradually fused into pieces,and their volume increased.Most of them were laid in a fibroblastic shape at the bottom of the bottle.The number of HSCs extracted from each rat is 1 ×107-3×107.②Immunofluorescence detection:Des positive rate>90%,HSCs purity>90%.(2)Activation of HSCs:① Immunofluorescence assay α-SMA:Model Group The positive rate of α-SMA was>90%,and the number of activated cells was>90%.②WB detection α-SMA:Compared with the blank group,the model group HSCs The expression of α-SMA was upregulated,with a statistically significant difference(P<0.05).(3)Effect of Qizhu Granules on Activated HSCs Impact of α-SMA:Compared to the blank group,the model group α-SMA was significantly upregulated,with a statistically significant difference(p<0.05);Compared with the model group,the high dose group of Qizhu Granules significantly decreased,with a statistically significant difference(p<0.05).(4)Effect of Qizhu Granules on Autophagy of Activated HSCs:①QRT-PCR detection of HSCs in each group mRNA expression of α-SMA,Col-I,LC3-I,LC3-II,p62,Atg3,and Beclin1:Compared with the blank group,the model group α-SMA significantly increased,with a statistically significant difference(p<0.01);Compared with the model group,the HSCs in the Compound Biejia Ruangan tablet group and the Qizhu granules group α-SMA decreased with statistically significant differences,P<0.01;Baf A1 inhibitor group α-SMA increased with statistically significant differences,P<0.01.Compared with the Baf A1 inhibitor group,the HSCs in the Qizhu Granules group and the Baf A1+Qizhu Granules groupα-SMA decreased with statistically significant differences,P<0.01.Compared with the blank group,the model group showed a significant increase in Col-I,with a statistically significant difference(p<0.01);Compared with the model group,the HSCs Col-Ⅰ in the Qizhu Granules group decreased with statistical significance(P<0.05);The Baf A1 inhibitor group showed an increase in HSCs Col-Ⅰ.Compared with the Baf A1 inhibitor group,the HSCs Col-I in the Qizhu Granules group and the Baf Al+Qizhu Granules group decreased with statistical significance,P<0.05.Compared with the Baf A1 inhibitor group,the Baf A1+Qishu Granules group showed an increase in HSCs Atg3,with a statistically significant difference(P<0.05),while the other groups showed no significant changes.Compared with the model group,the mRNA expression of LC3-Ⅰ in the Baf A1 inhibitor group significantly increased(P<0.01),while there was no significant change in other groups.Compared with the blank group,the model group showed an increase in LC3-Ⅱ,with a statistically significant difference(p<0.01);Compared with the model group,the mRNA expression of LC3-Ⅱ in the Compound Biejia Ruangan Tablet group and the Qishu Granule group decreased significantly(P<0.01),and the mRNA expression of LC3-Ⅱ in the Baf A1 inhibitor group increased significantly(P<0.01).Compared with the Baf A1 inhibitor group,the mRNA expression of LC3-Ⅱ in the Qizhu Granules group and the Baf A1+Qizhu Granules group was significantly reduced,with a statistically significant difference(P<0.01).Compared with the blank group,the model group showed a significant increase in p62,with a statistically significant difference(p<0.01);Compared with the model group,the mRNA expression of p62 in the Qizhu Granule group decreased significantly,with a statistically significant difference(P<0.01).The expression of p62 in the Baf A1 inhibitor group increased significantly,with a statistically significant difference(P<0.01).Compared with the Baf A1 inhibitor group,the mRNA expression of p62 in the Qishu Granules group and the Baf A1+Qishu Granules group was significantly reduced,with a statistically significant difference(P<0.01).②WB detection Col-I,LC3-Ⅰ,LC3-Ⅱ Expression of α-SMA,Atg3,p62,and Beclinl proteins:Compared with the blank group,the model group showed a significant increase in Col-Ⅰ,with a statistically significant difference(p<0.05);Compared with the model group,the Col-Ⅰ in the Qizhu Granule group decreased with statistical significance(P<0.05).Compared with the Baf A1 inhibitor group,the Col-Ⅰ levels in the Qizhu Granules group and the Baf A1+Qizhu Granules group decreased significantly(P<0.01).Compared with the blank group,the model group showed a significant increase in LC3-Ⅱ,with a statistically significant difference(p<0.05).Compared with the model group,the Compound Biejia Ruangan tablet group and Qishu granules group showed a decrease in LC3-II,with a statistically significant difference(P<0.05).The Baf A1 inhibitor group showed a significant increase in LC3-Ⅱ,with a statistically significant difference(P<0.01).Compared with the Baf A1 inhibitor group,the LC3-Ⅱ levels in the Qizhu Granules group and the Baf A1-Qizhu Granules group decreased significantly(P<0.01).The changes in LC3-Ⅰ groups were not significant.Compared with the blank group,the model group α-SMA significantly increased,with a statistically significant difference(p<0.01);Compared with the model group,the Compound Biejia Ruangan tablets group and the Qizhu granules group α-SMA decreased with statistically significant differences(P<0.05);Baf A1 inhibitor group α-SMA increased with statistically significant differences(P<0.05).Compared with the Baf A1 inhibitor group,the Qizhu Granules group and the Baf A1+Qizhu Granules group α-SMA decreased with statistically significant differences(P<0.01).Compared with the model group,the Atg3 levels in the Qishu Granule group and the normal serum control group were significantly increased,with a statistically significant difference(P<0.05).Compared with the model group,the Baf A1 inhibitor group showed a significant increase in p62(P<0.01).Compared with the Baf A1 inhibitor group,the Baf Al+Qishu granules group showed a statistically significant decrease in p62(P<0.01).There was no significant change in the levels of Beclinl in each group.Conclusions1.Qizhu Granules can improve liver function,four items of liver fibrosis,liver elasticity,and TCM syndromes in patients with liver fibrosis due to Qi deficiency and blood stasis.2.Animal experiments have shown that Qizhu granules have therapeutic effects on rat liver fibrosis models,which can reduce the degree of liver fibrosis,decrease the activation of hepatic stellate cells,and affect hepatic cell autophagy..3.Cell experiments have shown that Qizhu Granules can reduce the activation of hepatic stellate cells(HSCs),and this mechanism is related to its effect on HSCs’autophagy. |
PDF Full Text Request