The Regulation Mechanism Of Stem Cell Numb Gene On Biliary Hepatic Fibrosis And Its Effect On Anti-biliary Liver Fibrosis Of Huangqi Decoction

Background and objectiveAbnormal bile duct hyperplasia is the key pathological basis for the development of cholestatic liver fibrosis（CLF）,and the Notch signaling pathway plays a key role in this process.Numb gene is a negative regulator of Notch signaling.The previous study of the research team found that the Huangqi Decoction（HQD）can effectively inhibit the progression of bile duct hepatic fibrosis induced by bile duct ligation（BDL）.The mechanism of this action is related to the inhibition of the activation of Notch signaling pathway.Notch signaling was activated in the model rat liver tissue,and the expression level of Numb,a negative regulator of the signaling pathway,was significantly reduced,while HQD significantly increased the expression of Numb.However,whether Numb gene plays a key role in the occurrence and development of biliary fibrosis,and whether it is a key target of HQD against bile-induced liver fibrosis,is still unclear.Therefore,this study aimed to clarify the relationship between Numb gene and CLF,and its therapeutic effect on HQD anti-CLF.Methods（1）Rat bone marrow mesenchymal stem cells（BMSC）were isolated by whole bone marrow adherence method.After purification and culture to third generations,the expression level of CD10,CD14,CD29,CD34,CD45 and CD90 was detected by flow cytometry in order to identify the cell purity.In addition,the cell cycle of BMSC was detected,and its osteogenic and lipid differentiation were induced to determine its biological characteristics.（2）BMSC Numb gene knockdown（Numb-KD）and BMSC Numb gene overexpression（Numb-OE）.The Numb gene of BMSC was knocked down or overexpression by lentivirus transfection technique,and Negative Control（NC-KD,NC-OE）was set up in an empty virus body group,and the cells in each group were transfected to observe the cell morphology and virus transfection rate.RNA was extracted and the knocked down or overexpression of Numb gene was successfully determined by RT-PCR.Successfully transfected BMSCs have Enhanced Green Fluorescent Protein（EGFP）.（3）Establishment of CLF model and BMSC transplantation.The CLF model was prepared by common bile duct ligation（BDL）method.At the same time,BMSC,BMSC^NC-KD,BMSC^Numb-KD,BMSC^NC-OEand BMSC^Numb-OEwere injected into the spleen according to the experimental group.The injected cell volume of each rat was1×10⁶cells.After 4 weeks of modeling,the serum liver function,liver pathology and liver tissue hydroxyproline（Hyp）content were observed.The expressions ofα-SMA,CK19,HNF4αand Alb in liver tissues were observed by immunohistochemical staining.（4）The effect of Numb gene knockdown or overexpression of Numb gene on anti-CLF of HQD.After ligation of the common bile duct and the spleen transplantation of BMSC^Numb-KDor BMSC^Numb-OE,HQD was administered after one week,and DAPT（γ-seacretase inhibitor）was used as a positive control drug for a total of 3 weeks.At the end of the 4th week,the serum liver function,liver histopathology and Hyp content in the liver were observed.The expressions ofα-SMA,CK7 and CK19 in liver tissues were observed by immunohistochemical staining.The protein and m RNA expression levels ofα-SMA,CK19,Numb and Notch signaling pathways in liver tissues were observed by Western blot and RT-PCR.（5）The differentiation orientation of transplanted BMSC^Numb-KDor BMSC^Numb-OEwas observed by Immunofluorescence staining.Results（1）The isolated rat BMSC cells were morphologically characterized by long fusiform and vortex-like growth.The results of flow cytometry identification were CD10（-）,CD14（-）,CD34（-）,CD45（-）,CD29（+）,CD90（+）.the BMSCs showed osteogenic and adipogenic abilities following the differentiation assay,with a large number of calcium deposits noted following osteogenic induction,and a large number of fat droplets noted following adipogenic induction.The BMSC of 80.20%was in the G1 phase by examining the cell cycle via flow cytometry,indicating that the isolated BMSC had better differentiation ability.At MOI=80,the rate of lentiviral transfection cells reached more than 80%.RT-PCR results showed that the Numb expression in the Numb-KD group was significantly lower compared with the NC-KD group（P<0.01）,and Numb expression was significantly increased in the Numb-OE group compared with the NC-OE group（P<0.01）.（2）The results of liver Hyp assay showed that the Hyp content of liver tissue in the BDL group was significantly increased compared with the Sham group（P<0.01）.Compared with the BDL group,the Hyp content was significantly decreased in the BMSC group（P<0.05）;but compared with the BMSC^NC-KDgroup,the Hyp content was significantly increased in the BMSC^Numb-KDgroup（P<0.01）,and compared with the BMSC^NC-OEgroup,the Hyp content was significantly reduced in the BMSC^Numb-OEgroup（P<0.01）.（3）HE staining results showed that extensive bile duct hyperplasia was observed in the BDL group,the inflammatory cell infiltration and tissue necrosis were less.The hepatic bile duct hyperplasia and inflammatory infiltration was significantly less in BMSC group than the BDL group.Compared with BMSC^NC-KDgroup,BMSC^Numb-KDgroup aggravated bile duct hyperplasia and inflammatory infiltration.BMSC^Numb-OEgroup had significantly reduced hepatic bile duct hyperplasia and inflammatory infiltration compared with BMSC^NC-OEgroup.Sirius red staining results showed that there was a large amount of collagen deposition around the neonatal bile duct in the BDL group,and it extended to the liver parenchyma as the center of the portal area.Compared with the BDL group,the collagen deposition in the liver tissue of the BMSC group was significantly alleviated.The liver collagen deposition in the BMSC^Numb-KDgroup was aggravated compared with the BMSC^NC-KDgroup,and the liver collagen deposition in the BMSC^Numb-OEgroup was significantly reduced compared with the BMSC^NC-OEgroup.（4）Immunohistochemical staining showed that the expression ofα-SMA,CK7and CK19 were significantly decreased and the expression of HNF4αand Alb were significantly increased in the BMSC group compared with the BDL group.Compared with the BMSC^NC-KDgroup,the expression ofα-SMA,CK7 and CK19 in BMSC^Numb-KDgroup were significantly increased.In addition,compared with BMSC^Numb-KDgroup,the expression ofα-SMA,CK7 and CK19 in HQD+BMSC^Numb-KDgroup were no significantly change but were significantly increased compared with BMSC^Numb-KDgroup.Compared with the BMSC^NC-OEgroup,the expression ofα-SMA,CK7 and CK19 in BMSC^Numb-OEgroup were significantly decreased,and the expression of HNF4αand Alb were significantly increased.Compared with HQD group,the expression ofα-SMA,CK7 and CK19 in HQD+BMSC^Numb-OEgroup was significantly decreased,but no significant change compared with BMSC^Numb-OEgroup.The protein and m RNA results ofα-SMA and CK19showed similar trends.（5）The results of immuno-staining showed that EGFP/CK7 and EGFP/CK19were co-stained in BMSC^Numb-KDgroup,suggesting that Numb gene knockdown can promote BMSC differentiation into biliary epithelial cells;HQD+BMSC^Numb-KDgroup also showed a large number of co-staining of EGFP/CK7 and EGFP/CK19which indicated that HQD could not inhibit the differentiation of BMSC^Numb-KDinto biliary epithelial cells.EGFP/HNF4αand EGFP/Alb were co-stained in BMSC^Numb-OEtransplantation group,suggesting that overexpression of Numb gene can promote BMSC differentiation into hepatocytes.（6）Notch signal pathway RT-PCR results showed that the expression of Numb m RNA in HQD group was significantly higher than that in BDL group（P<0.05）,and the RBPjκand Hes1 m RNA were significantly decreased（P<0.01）.Compared with BMSC^NC-KDgroup,the expression of Numb m RNA in the BMSC^Numb-KDgroup was significantly decreased（P<0.01）,and the RBPjκand Hes1 m RNA were significantly increased（P<0.01）.Compared with the BMSC^Numb-KDgroup,the expression of the RBPjκand Hes1 m RNA were no significantly change in the HQD+BMSC^Numb-KDgroup.Compared with BMSC^NC-OEgroup,the expression of Numb m RNA in BMSC^Numb-OEgroup was increased（P<0.05）,RBPjκand Hes1 m RNA were significantly decreased（P<0.05）;compared with BMSC^Numb-OEgroup,the Numb,RBPjκand Hes1 m RNA in HQD+BMSC^Numb-OEgroup were no significant change.Related protein expression presents a similar trend.Conclusion（1）Numb gene knockdown can promote the progression of CLF,while Numb gene overexpression can inhibit the progression of CLF,indicating that Numb gene plays an important role in the development of CLF;（2）BMSCs Numb gene knockdown transplantation can significantly reduce the effect of HQD on anti-CLF,which may be related to the weakening effect of HQD on inhibiting Notch signaling after Numb knockdown,indicating that the Numb gene may be one of the effective targets of HQD against CLF.