| Advanced chronic liver disease and acute liver failure are major diseases threatening human health.Approximately 1.7 million people die due to advanced chronic liver disease and acute liver failure annually in the worldwide.Recently,studies have found hepatic stem cells(HSCs)play an important role in liver injury,which can be alleviated by use of drugs that affect HSCs,or intrahepatic transplantation of HSCs.Hepatic oval cells(HOCs)are an important HSCs.After severe liver damage,cells proliferate in large numbers and differentiate into hepatocytes and hepatobiliary cells to assist the repair and regeneration of liver.In this process Notch signalling pathway play an important regulatory role,Abnormal activation of Notch signalling pathway or the deterioration of microenvironment around cells can transform HOCs into liver fibrocytes,or even hepatocellular carcinoma(HCC)cells,thus aggravating liver injury.Drug antagonism of active Notch signalling pathway can promote the transformation of HOCs to normal hepatocytes and inhibit the promotion of liver damage and carcinogenesis.Matrine is an effective alkaloid extracted from Sophora alopecuroides.Many studies have confirmed that matrine can alleviate liver injury through Notch signalling and regulate differentiation of HOCs into hepatocytes.However,matrine-induced differentiation of HOCs has been rarely studied,and its specific mechanism of action is still elucidated.In this study,First of all,to investigate the expression of Notch pathway in HCC tissues and clarify the correlation between Notch pathway and liver function in patients with cirrhosis with HCC,study whether Notch signalling pathways aggravate liver function damage,affect the prognosis of patients,and further explore the role of Notch signalling pathway in the development and progression of HCC.Secondly,HOCs were transfected with si Notch-1 to observe whether HOCs could differentiate into hepatocytes after inhibiting Notch signalling pathway.Thirdly,matrine was used to intervene in HOCs to observe whether it induced differentiation of HOCs into hepatocytes by inhibiting Notch signalling pathway.Finally,the Solt-Farber pre-hepatocellular carcinoma model of rats were made to simulate the liver damage microenvironment in the early stage of the occurrence and development of HCC,and matrine was used for prophylactic treatment to observe whether matrine could inhibit Notch signalling pathway,alleviate liver damage and prevent the occurrence of HCC.Part one Abnormal activation of Notch signalling pathway impaired liver function in patients with cirrhosis with HCC and affected prognosisObjective:To investigate the expression of Notch pathway in HCC tissues and clarify the correlation between Notch pathway and liver function and prognosis in patients with cirrhosis with HCC.Methods:Liver tissue specimens of 58 patients with cirrhosis with HCC were collected,and another 58 cases of paracancerous cirrhosis were taken as control.Expression of Notch-1,Jagged-1,and Hes-1 in liver was detected by immunohistochemistry.The indexes of liver function and coagulation function were collected from 58 patients with HCC before operation.Followed up the survival of the patients,and compared the correlation of between protein expression and prognosis.Result:Immunohistochemistry showed that the expression of Notch-1,Jagged-1,and Hes-1 in HCC tissue was significantly higher than that in cirrhotic tissues.The expression of Notch-1 in HCC tissues was significantly correlated with tumour differentiation and Hbs Ag;Notch-1 expression was stronger in HCC tissue with poor differentiation(OR:13.11,95%CI:1.524-112.77,P=0.019)and Hbs Ag positive(OR:25.35,95%CI:1.429-448.7,P=0.015).The expression of Jagged-1 in HCC tissues was obviously related to HBV-Pres1,and HCC tissues of patients who were Pres1-positive showed a stronger expression of Jagged-1(OR:3.594,95%CI:1.189-10.862,P=0.023).The expression of Hes-1 in HCC tissues was significantly correlated with the BCLC stage and tumour differentiation;the expression of Hes-1 was stronger in patients with later stage(OR:16.07,95%CI:1.36-189.75,P=0.028)and worse differentiation(OR:58.25,95%CI:2.23-1524.11,P=0.015).Kaplan-Meier survival analysis demonstrated that the HCC patients with high Notch-1,Jagged-1,and Hes-1 expression had a significantly worse prognosis than those with a low expression(P<0.01;Fig.2A-C).Cox regression analysis showed that higher Jagged-1 expression was an independent predictor of survival time(HR:2.406,95%CI:1.415,4.092,P=0.007).Combined vascular invasion,the BCLC stage,and Hbe Ab were also independent predictors of survival time.Patients with HCC with positive Jagged-1,combined vascular invasion,late stage,and HBe Ab-negative had worse prognosis.More importantly,our study found that the Notch signalling pathway was significantly associated with liver function in patients with cirrhosis with HCC.Patients who were Notch-1 positive had higher levels of AST,GGT,and PT and lower levels of A/G,and the immunohistochemical score(IHS)of Notch-1 had an obvious linear correlation with AST and GGT.Patients with Jagged-1 positive had higher ALT and AST levels and lower A/G levels,and the IHS of Jagged-1 was significantly linearly correlated with ALT,ALP,and GGT.Patients who were Hes-1 positive had higher ALT,AST,and GGT levels,and the IHS of Hes-1 was significantly linearly correlated with ALT,GGT,and PTA.Summary:The expression of Notch-1,Jagged-1,and Hes-1 in HCC tissue was significantly higher than that in cirrhotic tissues,and the higher the expression of these proteins,the worse the prognosis of patients.More importantly,HCC patients with higher levels of Notch-1,Jagged-1,and Hes-1were associated with higher levels of ALT,AST,and GGT in liver function.Abnormal activity of Notch signalling pathway could promote liver function impairment in HCC patients,promote HBV replication,aggravate the deterioration of hepatic microenvironment,and seriously affect the prognosis of HCC patients.This deterioration of hepatic microenvironment was related to the regulation of the differentiation of HOCs into hepatic fibrosis cells and cancer cells by the abnormally active Notch signalling pathway.Inhibition of Notch signalling pathway may regulate the differentiation of HOCs into normal hepatocytes,improve hepatic microenvironment inflammation,and improve the prognosis of patients.Part two Transfected si Notch-1 inhibited the stem cell characteristics of HOCs and induced a gradual differentiation of HOCs into hepatocytesObjective:To observe whether HOCs could differentiate into hepatocyt-es after the inhibition of Notch signalling pathway by si Notch-1 transfection.Methods:si Notch-1 was transfected into HOCS,and cell viability was determined by CCK-8 assay.Cell cycle and apoptosis were detected by flow cytometry.Immunohistochemical,Western blotting and q RT-PCR were used to detect the expressions of Notch 1,Jagged 1,and Hes-1 in the Notch signalling pathway,HOCS markers AFP,CK-19,and C-Kit,and albumin(ALB)secreted by mature hepatocytes.Results:Immunohistochemistry,western blotting,and QRT-PCR demo-nstrated that the expressions of Notch-1,Jagged-1,and Hes-1 in the si Notch-1were significantly decreased when compared with the control and NC-si RNA groups,which indicates that si Notch-1 successfully inhibited Notch signalling.The expression of AFP and C-Kit of the HOC markers was significantly decreased in the si Notch-1 group compared with the control and the NC-si RNA groups.The expressions of ALB secreted by mature hepatocytes in si Notch-1 group were increased.CCK-8 assays were used to determine cell viability.The si Notch-1 group demonstrated significantly less growth than the other groups did,demonstrating the ability of si Notch-1 to inhibit the viability of HOCs.Flow cytometry demonstrated that compared with the control and NC-si RNA group,the proportion of cells in the S phase and G2 phase were lower in the si Notch-1 group.By contrast,the proportion of cells in G1 phase was increased in si Notch-1 group(P<0.05).Cell apoptosis was detected by flow cytometry and compared with the control and NC-si RNA groups;the proportion of late apoptotic,total apoptosis,and dead cells were increased in the si Notch-1 group(P<0.05).Summary:The aforementioned results showed that after transfection of si Notch-1 into HOCs,the Notch signalling pathway was significantly inhibited.And si Notch-1 inhibited the stem cell characteristics of HOCs and induced a gradual differentiation of HOCs into mature hepatocytes.The Notch signalling pathway plays an important role in maintaining the characteristics of HOCs.Part three Matrine promotes hepatic oval cells differentiation into hepatocytes by suppression of Notch signalling pathwayObjective:To observe whether matrine can induce differentiation of HOCs into hepatocytes by inhibiting Notch signalling pathway.Methods:Different concentrations of matrine were used to intervene HOCs,and the optimal concentration of matrine was selected.Cell viability was determined by CCK-8 assay,and cell cycle and apoptosis were detected by flow cytometry.The matrine was used to interfere with HOCs.Immunohistochemical,Western blotting and q RT-PCR were used to detect the expressions of Notch 1,Jagged 1,and Hes-1 in the Notch signalling pathway,HOCS markers AFP,CK-19,and C-Kit,and ALB secreted by mature hepatocytes.Results:CCK-8 method was used to screen the optimal intervention concentration of matrine for HOCs.Matrine inhibited the viability of HOCs in a concentration-dependent manner.The IC50 of treatment with matrine for 72h was 1.47 mg/ml.The next experiment used matrine at a concentration of1.47 mg/ml for the high dose group and 0.5 mg/ml for the low dose group.The cell cycle showed that compared with control group,the proportion of cells in the G2 phase in the matrine group was decreased,whereas readouts such as G1,S,and SPF were increased(P<0.05).Flow cytometry demonstrated that compared with the other groups,the proportion of cells in the S phase and G2phase were lower in the matrine+si Notch-1 group,The proportion of cells in the G1 phase were more significantly increased in the matrine+si Notch-1group.Other readouts such as PI and SPF were decreased(P<0.05).Cell apoptosis detected by flow cytometry showed that early apoptotic and total apoptosis increased in the matrine group when compared with the control group(P<0.05).Cell apoptosis was detected and compared with the other groups,the proportion of early apoptotic cells,late apoptotic cells,and dead cells were more significantly increased in the high matrine+si Notch-1group(P<0.05).The immunohistochemistry,QRT-PCR,and western blotting results demonstrated that compared with the control group the expression of Notch-1,Jagged-1,and Hes-1 were reduced after treatment with matrine.Moreover,the effect of Notch inhibition in the high-matrine group was more obvious than that the low-matrine group,showing a dose-dependent effect.Immunohistochemistry,Western blotting and QRT-PCR demonstrated that compared with the si Notch-1 group and high matrine alone the expressions of Notch-1,Jagged-1,and Hes-1 in HOCs were significantly inhibited in the high matrine+si Notch-1 group.The immunohistochemistry,QRT-PCR,and western blotting results demonstrated that treatment with matrine,the levels of AFP and C-Kit in HOCs were significantly lower than those in the control group,whereas matrine increased ALB expression.Moreover,the effect of high matrine was more obvious than that of low matrine,showing a dose dependence.The expression of AFP and C-Kit in the high matrine+si Notch-1group was significantly decreased in HOCs,whereas the expression of ALB was increased ompared with control group,the si Notch-1 group and high matrine.Summary:The aforementioned results showed that matrine inhibited the proliferative viability of HOCs and induced their apoptosis,inhibiting the stem cell properties of HOCs.Matrine could inhibit the expression of Notch-1,Jagged-1,and Hes-1 in the Notch signalling pathway in a dose-dependent manner.Moreover,after treatment with matrines,induced a gradual differentiation of HOCs into mature hepatocytes.Matrine and si Notch-1 can synergically enhance the inhibition of Notch signalling pathway and induce the differentiation of HOCs into liver cells.Part four Matrine alleviates liver damage and inhibits Notch signalling pathway in the rat Solt-Farber model of precancer lesionsObjective:The Solt-Farber pre-hepatocellular carcinoma model of rats were made to simulate the liver damage microenvironment in the early stage of the occurrence and development of HCC,and matrine was used for prophylactic treatment to observe whether matrine could inhibit Notch signalling pathway,alleviate liver damage.Methods:The rats Solt-Farber pre-hepatocellular carcinoma model was prepared.Sprague Dawley(SD)rats were randomly divided into 4 groups(control group,model group,low-matrine group,and high-matrine group).The model group,low-matrine group,and high-matrine group were used to generate a Solt-Farber model(Partial hepatectomy[HP]+diethylnitrosamine[DEN]+2-acetylaminofluorene[AAF])for preneoplastic lesions.The low-or high-matrine groups,from the day of DEN injection,received intragastric administration of matrine at a dosage of 1.25 mg/kg and 12.5 mg/kg,respectively,twice per day,which lasted from the intraperitoneal injection of DEN to the killing of the rats.The rats were sacrificed at 2,4,and 7 weeks after hepatectomy(4 rats per group,per time);next,we isolated the liver immediately and fixed the liver in 10%formaldehyde.Liver tissue was collected for HE and immunohistochemical detection.Results:In the model group,the hepatic lobule structure and the structure of the liver cell cord were significantly injured compared with those of the control group,Moreover,a large number of oedema and inflammatory cell infiltration in the hepatic portal area 2 and 4 weeks after partial hepatectomy(PH)were observed.Seven weeks after PH,a small proportion of cellular cord structures was detected.In the low-matrine group,a small proportion of hepatocyte cord structure was restored 4 weeks after PH.Seven weeks after PH,hepatocyte oedema degeneration and inflammation were significantly reduced,and hepatocyte nodule structures were recovered.In the high-matrine group,significantly higher protective effects were observed at week 4 and 7 weeks after PH than in the model group and low-matrine group.No obvious expression of Notch-1,Jagged-1,Hes-1,AFP,CK-19,and C-Kit were detected in the control group.By contrast,in the model group,these proteins'expression was significantly upregulated 2,4,and 7 weeks after PH.The expression of Notch-1,Jagged-1,and Hes-1 in the matrine group was weaker than in the model group 4 weeks after PH.Moreover,the high-matrine group had a more obvious inhibitory effect on the Notch signalling pathway.The expression of AFP and C-Kit were significantly decreased in the matrine group 2 and 4 weeks after PH.ALB was highly expressed in the control group,but ALB was significantly downregulated in the model group.The expression of ALB was significantly increased after matrine prophylactic treatment,which was concentration-dependent.The higher the matrine concentration,the higher the ALB expression.Summary:Prophylactic application of matrine in the rat Solt-Farber pre-cancerous model could protect liver,which may be related to matrine-induced differentiation of HOCs into mature hepatocytes by inhibition of the Notch signalling pathway.Conclusion:1.Notch-1,Jagged-1,and Hes-1 of the Notch signalling pathway were strongly expressed in HCC tissues,and the higher the expression of these proteins,the worse the prognosis of patients.More importantly,HCC patients with higher levels of Notch-1,Jagged-1,and Hes-1 had worser liver function.Abnormal activity of Notch signalling pathway could promote liver function impairment in HCC patients,promote HBV replication,aggravate the deterioration of hepatic microenvironment,and seriously affect the prognosis of HCC patients.This deterioration of hepatic microenvironment was related to the regulation of the differentiation of HOCs into hepatic fibrosis cells and cancer cells by the abnormally active Notch signalling pathway.Inhibition of Notch signalling pathway may regulate the differentiation of HOCs into normal hepatocytes,improve hepatic microenvironment inflammation,and improve the prognosis of patients.2.The Notch signalling pathway plays an important role in maintaining the stem cell characteristics of HOCs.After transfection of HOCs with si Notch-1 technology,Notch signalling pathway can be inhibited and HOCs can be induced to differentiate into hepatocytes.3.Matrine can inhibit Notch signalling pathway in a concentration-dependent manner,and induce HOCs to differentiate into mature hepatocytes rather than into hepatic fibrosis cells and cancer cells,helping to improve the liver microenvironment.4.In this study,a classic rat Solt-Farber pre-hepatocellular carcinoma model model was established to simulate the liver damage microenvironment before the occurrence of HCC,and different concentrations of matrine were used for prophylactic treatment.It was observed that matrine could inhibit Notch signalling pathway,and had alleviate liver damage. |
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