Study On The Mechanism Of Icaritin Regulating Apoptosis And Mitophagy In Human Umbilical Cord Mesenchymal Stem Cells

Stem cell therapy is a promising treatment and has been widely used in the treatment of a variety of diseases,but the survival rate of stem cells transplanted into the body is low,which limits its application.Therefore,it is urgent to find a new therapeutic strategy to improve the ability of stem cells to adapt to the harsh microenvironment of the body.Natural medicine has unique advantages.Previous experiments have found that human umbilical cord mesenchymal stem cells(hUC-MSCs)co-cultured with icaritin(ICT)markedly decreased the hepatocytic apoptosis of failing livers in rats,reduced mortality and improved liver function and pathological changes in liver tissue compared to MSCs alone,but the underlying mechanism was still unclear.Pre-experiments found that ICT can increase the cell viability of hUC-MSCs cultured in vitro,regulate the secretion of various cytokines,and reduce the apoptosis of cells.The result of electron microscopy showed that ICT reduced the number of mitochondria incorporated into autophagosomes.The study will explore the mechanism by which icaritin affect apoptosis and mitochondrial autophagy of hUC-MSCs.The paper aimed to provide theoretical basis for traditional Chinese medicine combined with stem cell therapy.ObjectiveFirst,literature review provides theoretical support for experiments.Subsequently,in vitro,serum-free culture was used to simulate the microcirculatory disturbance in vivo,and hUC-MSCs were pretreated with ICT to explore whether it has anti-apoptotic effect.Further experiments were conducted to investigate if it exerts a therapeutic effect through the HGF(hepatocyte growth factor)/c-Met pathway.We also observed whether ICT can affect the mitochondrial autophagy process of hUC-MSCs.Protein mass spectrometry was used to analyze the differentially expressed proteins of hUC-MSCs pretreated by ICT,and bioinformatics analysis such as GO analysis,KEGG analysis and protein interaction analysis were performed to evaluate the biological processes involved.MethodsHuman umbilical cord mesenchymal stem cells were cultured in serum-free medium for 72 hours as a basic modeling method to simulate the internal environment of microcirculatory disturbance.Different concentrations of ICT were used to intervene the modeled cells,and the OD values were measured by the MTS method to select the optimal treatment concentration.The cell proliferation rate of the normal culture group,the serum-free culture group,the icaritin group(ICT group),and the hepatocyte growth factor group(HGF group)was measured using BrdU incorporation assay.The ELISA method was used to detect the effect of ICT on the secretion of HGF by hUC-MSCs.The expression of c-Met receptor in hUC-MSCs was detected by WB and qRT-PCR.The optimal concentration of crizotinib(the c-Met receptor inhibitor)was determined using the CCK8 method.The apoptosis rate of each group was evaluated by Annexin V-FITC/PI apoptosis assay.qRT-PCR and WB were used to detect the expression of apoptosis-related proteins such as Bcl-2,Caspase-3 and Bax.The MTS method was used to detect the effects of different concentrations of 3MA(autophagy inhibitor)on modeled cells.qRT-PCR,WB and immunofluorescence microscope were used to detect the expression of tomm20(mitochondrial outer membrane protein),timm23(mitochondrial inner membrane protein),cytochrome c oxidase subunit II(MT-C02,mitochondrial matrix protein),and LC3B(autophagy key protein).The ultrastructure of each group of cells was observed by transmission electron microscope,and the level of mitochondrial autophagy was observed.The relevant pathway of mitochondrial autophagy induced by ICT was screened by qRT-PCR.The membrane potential was measured using a JC-1 membrane potential detection kit.Bioinformatics analysis of differential proteins in each group was performed using protein mass spectrometry.Results1.Serum-free cultured hUC-MSCs were treated with different concentrations of Icaritin,and the cell viability was increased at each concentration.The increase of the 50ng/ml(0.1uM)group and the 100ng/ml group were statistically significant(P<0.05).The subsequent experiment was performed with 50 ng/ml ICT while 50 ng/ml was selected as the optimal treatment concentration.The result of brdU incorporation test showed that compared with the normal culture group,the proliferation rate of the serum-free culture group decreased(P<0.05),while the use of Icaritin(ICT)or hepatocyte growth factor(HGF)increased the cell proliferation rate(P<0.05).2.Human umbilical cord mesenchymal stem cells can secrete HGF and also express HGF receptor c-Met,while ICT can promote higher levels of HGF and c-Met receptors(P<0.05).After constructing c-Met+ MSCs,WB and qRT-PCR confirmed the successful gene transfection.According to the cell inhibition rate,4 u M of crizotinib(c-Met receptor inhibitor)was selected as the optimal treatment concentration.The apoptosis rate of MSCs was high at 72h serum-free culture,and the apoptosis rate of c-Met+ MSCs was significantly lower(P<0.01).After treatment with crizotinib,the apoptosis rate was significantly increased(P<0.01).The HGF/c-Met pathway had an anti-apoptotic effect on MSCs.The result of WB showed that,compared with MSCs group,the expression of Bcl-2 was up-regulated and the expression of Cleaved caspase-3 and Bax was down-regulated in the c-Met+MSCs group(P<0.05),while crizotinib decreased Bcl-2 protein level and increased the expression of Cleaved caspase-3 and Bax.We confirmed the above conclusion using qRT-PCR.The above experiments suggested that the HGF/c-Met pathway exerts an anti-apoptotic effect by regulating the expression of apoptosis-related proteins.3.The apoptosis rate of MSCs was high in serum-free group for 72h.After ICT or HGF treatment,the apoptosis rate of serum-free MSCs was significantly decreased(P<0.05).When the c-Met receptor inhibitor(crizotinib)was used,the anti-apoptotic effect of ICT was weakened,and the difference was statistically significant(P<0.01).It indicates that crizotinib can inhibit the protective effect of ICT on MSCs,and the anti-apoptotic effect of ICT on MSCs is closely related to HGF/c-Met pathway.The result of WB showed that Bcl-2 was up-regulated,Cleaved caspase-3 and Bax were down-regulated in ICT or HGF treated MSCs(P<0.05).Compared with ICT group,crizotinib group(ICT+crizotinib)reduced Bcl-2 protein levels and increased the expression of Cleaved caspase-3 and Bax.The above conclusion was confirmed by qRT-PCR.It is suggested that ICT can play an anti-apoptotic role by regulating the expression of apoptosis-related proteins through the HGF/c-Met pathway.4.Autophagy inhibitor(3-MA)of low concentration promoted the cell viability of serum-free cultured MSCs(P<0.05),while autophagy inhibitor(3-MA)of high concentration inhibited cell viability(P<0.01).It was suggested that under serum-free culture conditions,moderate inhibition of autophagy increased cell survival rate,while excessive inhibition of autophagy reduces cell viability.5.Compared with the control group,the mitochondrial outer membrane protein tomm20,the mitochondrial inner membrane protein timm23 and the mitochondrial matrix protein cytochrome c oxidase subunit ?(MT-C02)were reduced and the expression of LC3I and LC3II were increased,the ratio of LC3-?/LC3-? were raised,mitochondrial membrane potential was decreased in the serum-free group,which indicated the upregulation of mitochondrial autophagy levels in the serum-free group.The result of electron microscopy showed that "mitochondrial autophagosomes"(mitochondria incorporated into autophagosomes)was increased significantly,damaged mitochondrial was increased,and cell morphology was poor,confirming that serum-free culture upregulated mitochondrial autophagy.After pretreatment with ICT or HGF,the expression of mitochondrial protein mentioned above was increased,the expression of LC3I and LC3II were decreased,the ratio of LC3-?/LC3-? were lowered,and mitochondrial membrane potential was increased,indicating that mitochondrial autophagy was down-regulated.Electron microscopy showed a decrease in "mitochondrial autophagosomes" and impaired mitochondria,and improved cell morphology,confirming that ICT or HGF reduced mitochondrial autophagy.6.The Pinkl/Parkin pathway,FUNDC1 pathway and Bnip3/Nix pathway weredetected by qRT-PCR.The results were as follows:The change of mRNA of Pinkl and Bnip3 was consistent with the previous mitochondrial autophagy trend,and the mRNA of Nix and FUNDC1 showed contradictory results.7.Protein mass spectrometry:the differential protein between the normalculture group and the serum-free culture group,between the ICT group and the serum-free culture group,between the HGF group and the serum-free culture group were analyzed.It was found that all of them were involved in apoptosis,autophagy,and mitochondrial autophagy-related biological process(P<0.05).The differential proteins of ICT group and serum-free culture group were analyzed.AKT1 was located at the center of protein interaction chart.The PI3K-AKT pathway was statistically significant in KEGG analysis and could be used as an important pathway.The differential proteins between the HGF group and the serum-free culture group were analyzed,and the NF-? pathway was screened as the research focus.Conclusion1.Icaritin can reduce the apoptosis rate of human umbilical cord mesenchymal stem cells under serum-free culture conditions through HGF/c-Met pathway,which is closely related to the protein expression of Cleaved Caspase-3,Bax and Bcl-2.2.Icaritin or hepatocyte growth factor can reduce the mitochondrial autophagy level of human umbilical cord mesenchymal stem cells under serum-free culture conditions,reduce the number of damaged mitochondria and improve cell morphology,increase mitochondrial protein expression of tomm20?timm23?MTC02 and raise mitochondrial membrane potential.3.Protein mass spectrometry revealed that icaritin or hepatocyte growth factor-mediated differential proteins are involved in the biological processes involved in apoptosis,autophagy,and mitochondrial autophagy.For ICT,the PI3K-AKT pathway can be used as a research focus.For HGF,the NF-?B pathway can be used as a research focus.