MiR-708 Induces The Senescence Of Hepatic Stellate Cells Through The Upregulation Of P53 By Targeting ZEB1 And The Effect Of MDP

The main feature of liver fibrosis is the excessive accumulation of extracellular matrix after chronic liver injury.Progressive fibrosis is characterized by the accumulation of myofibroblasts around the portal vein and around the sinus that secrete?-SMA.Persistent inflammation usually leads to a large amount of fibrosis,which eventually leads to cirrhosis,accompanied by structural distortion of hepatic vessels,and lays the foundation for liver decompensation,primary liver cancer and death.After liver injury,hepatic stellate cells(hepatic stellate cells,HSCs),the main collagen synthesis cells in the liver,are activated and transformed into myofibroblast-like cells,which proliferate faster and show stronger chemotaxis,survival rate and collagen production.Hepatic stellate cells also play an important role in the progression of liver inflammation to fibrosis.The activation of HSCs is driven by a variety of mediators,such as chemokines,reactive oxygen species,growth factors,matrix rigidity,stromal cell proteins and injury-related molecular patterns,which are also secreted by neighboring cells and drive HSC scar formation in an autocrine and/or paracrine manner.However,one of the limitations of current anti-fibrosis therapy is that drug candidates cannot specifically target hematopoietic stem cells.More and more studies have found that in addition to inhibiting the proliferation and activation of activated HSCs and promoting the apoptosis of activated HSCs,inducing the senescence of activated HSCs can prevent the process of liver fibrosis.Therefore,understanding the molecular mechanism of activated HSCs aging is of great significance for the development of effective anti-fibrosis therapy.Micro RNAs(mi RNAs)is an endogenous non-coding RNA of about 22 nucleotides in length.Mi RNAs identifies the target genes of mi RNAs by pairing the 5'end of the sequence of about 6-8 nucleotides with the complementary sequences in the target gene messenger RNA,and suppresses the expression of these target genes at the post-transcriptional level through m RNA decay or through RNA-induced silencing complexes containing mi RNA.Mammalian mi RNAs is involved in a variety of biological processes,such as differentiation,proliferation,antioxidant stress and tumor inhibition.In addition,it is known that the expression of mi RNAs is dysfunctional in many diseases,including cancer.Because of its gene regulation ability,mi RNAs has become a very attractive therapeutic target.Previous studies have found that mi RNA plays a regulatory role in the process of many fibrotic diseases.Mi RNA can regulate the expression of a variety of factors related to hepatic fibrosis,which in turn can lead to changes in the expression of a variety of mi RNA.Previously,our group has confirmed that mi R-708 is closely related to the occurrence and development of liver fibrosis.However,most studies have focused on the proliferation and activation of HSCs,but little is known about the mechanism of how mi R-708 participates in the senescence of hepatic stellate cells,which needs further research.Paeoniflorin(Pae)is the main active component of total glucosides of paeony.Due to the slow onset and low bioavailability of Pae,its clinical application is limited.Therefore,monomer derivative of paeoniflorin(MDP)derived from Pae was developed in our laboratory,and its bioavailability and efficacy were better than those of Pae.Our previous studies have shown that MDP can inhibit inflammation.However,the correlation and potential mechanism of MDP in liver fibrosis are still unclear.Objective:1.To study the effects of mi R-708,ZEB1 and p53 in activation of HSCs induced by senescence;2.To clarify the targeting relationship between mi R-708 and ZEB1,ZEB1 and p53promoters;3.To elucidate the therapeutic effect and mechanism of MDP on liver fibrosis;4.It is revealed that MDP further exerts a therapeutic effect on liver fibrosis by promoting the mi R-708/ZEB1/p53 pathway-induced aging by directly acting on Argonaute 2(Ago2).Methods:1.C57 mice were purchased and randomly divided into 6 groups(normal group,CCl4group,25mg/kg MDP+CCl4 group,50mg/kg MDP+CCl4 group,100mg/kg MDP+CCl4 group,40mg/kg colchicine+CCl4 group).Intraperitoneal injection of CCl4 for 8 weeks was used to construct an in vivo model of liver fibrosis.In the second week,MDP and colchicine were given by intragastric administration at different concentrations.After modeling,liver tissues were pathologically detected by HE,Masson and Sirius red staining,and the activities of AST,ALT and TBIL were observed by AST,ALT and TBIL kits.The expressions of?-smooth muscle actin(?-SMA),Collagen type1(Col.I)and senescence-related proteins(p16 and p21)were detected by IHC and Western blot.To observe the therapeutic effect of MDP on liver fibrosis.2.In vitro,human hepatic stellate cell LX-2 was stimulated by TGF-?1(10 ng/m L)to construct an in vitro model of liver fibrosis,and MDP was given at the same time.The expression changes of?-SMA,Col.I,p16 and p21 were detected by immunofluorescence,q PCR and Western blot.3.Immunofluorescence,q PCR and Western blot were used to detect the expression levels of mi R-708,ZEB1 and p53 in liver fibrosis tissues.In vitro,human hepatic stellate cell LX-2 was stimulated to construct an in vitro model of liver fibrosis by using TGF-?1(10 ng/m L).Immunofluorescence,q PCR and Western blot were used to detect the expression levels of mi R-708,ZEB1 and p53 in liver fibrosis to observe the expression changes of mi R-708,ZEB1 and p53.4.mi R-708 inhibitor,mi R-708 mimics,P-CMV-ZEB1,ZEB1 si RNA,p53 si RNA and PEX-3-p53 were used to silence and overexpression mi R-708,ZEB1 and p53 in vitro.Flow cytometry was used to observe the effects of mi R-708,ZEB1 and p53 on l X-2cell cycle.SA-?-Gal staining kit was used to observe the effects of mi R-708,ZEB1and p53 on SA-?-Gal activity in LX-2 cells.Immunofluorescence and Western blot were used to observe the effects of mi R-708,ZEB1 and p53 on the expression of senescence markers of activated HSCs.5.In vitro co-transfection of mi R-708 mimics and ZEB1 WT-3'-UTR,mi R-708 NC and ZEB1 WT-3'-UTR,mi R-708 mimics and ZEB1 MT-3'-UTR,mi R-708 mimics and ZEB1 MT-3'-UTR,double luciferase reporter assay was used to detect the targeting relationship between mi R-708 and ZEB1.6.ZEB1 si RNA and WT-p53,ZEB1 NC and WT-p53,ZEB1 si RNA and MT-p53,ZEB1 NC and MT-p53 were co-transfected in vitro.Double luciferase reporter gene assay was used to detect the effect of ZEB1 silencing on p53 promoter activity.7.Lib Dock software in Discovery Studio 2017 R2 was used to conduct molecular simulation docking research on MDP.Results:1.Overexpression of mi R-708 can inhibit cell cycle arrest and promote the expression of p16 and p21 in activated LX-2 cells.In order to observe the effect of mi R-708 on senescence of activated HSCs,q PCR results showed that the expression of mi R-708 decreased in the process of liver fibrosis.Then,flow cytometry showed that overexpression of mi R-708 could significantly inhibit the cell cycle progression of activated LX-2,and SA-?-Gal staining showed that overexpression of mi R-708 could increase SA-?-Gal activity.The results of immunofluorescence and Western blot showed that overexpression of mi R-708 could significantly increase the expression of p16 and p21 and inhibit the expression of?-SMA and Col.I.On the contrary,silencing mi R-708 could significantly inhibit the expression of p16 and p21 and promote the expression of?-SMA and Col.I.2.Silencing ZEB1 can inhibit cell cycle arrest and promote the expression of p16 and p21 in activated LX-2 cells.In order to observe the effect of ZEB1 on senescence of activated HSCs,immunofluorescence,q PCR and Western blot results showed that the expression of ZEB1 was increased in activated HSCs.After transfection,flow cytometry showed that silencing ZEB1 could significantly inhibit the cell cycle progression of activated LX-2;SA-?-Gal staining showed that silencing ZEB1 could increase SA-?-Gal activity;immunofluorescence and Western blot results showed that silencing ZEB1 could significantly increase the expression of p16 and p21 and inhibit the expression of?-SMA and Col.I.On the contrary,overexpression of ZEB1 could significantly inhibit the expression of p16 and p21 and promote the expression of?-SMA and Col.I.3.Overexpression of p53 can inhibit cell cycle arrest and promote the expression of p16 and p21 in activated LX-2 cells.In order to observe the effect of p53 on senescence of activated HSCs,immunofluorescence,q PCR and Western blot results showed that the expression of p53 decreased in activated HSCs.After transfection,flow cytometry showed that overexpression of p53 could significantly inhibit the cell cycle progression of activated LX-2,and SA-?-Gal staining showed that overexpression of p53 could increase the activity of SA-?-Gal.The results of immunofluorescence and Western blot showed that overexpression of p53 could significantly increase the expression of p16 and p21 and inhibit the expression of?-SMA and Col.I.On the contrary,silencing p53 could significantly inhibit the expression of p16 and p21 and promote the expression of?-SMA and Col.I.4.ZEB1 was a candidate target for mi R-708In order to observe the relationship between mi R-708 and ZEB1,it is found that ZEB1is a candidate target for mi R-708 by Target Scan prediction,and the complementary site of mi R-708 is found in ZEB1.The results of double luciferase assay showed that overexpression of mi R-708 could significantly inhibit the luciferase activity of WT3'-UTR of ZEB1.However,overexpression of mi R-708 did not affect the luciferase activity of ZEB1 MT 3'-UTR reporter vector.The results of q PCR and Western blot showed that overexpression of mi R-708 could inhibit the expression of ZEB1,while silencing mi R-708 could promote the expression of ZEB1.5.ZEB1 can bind to p53 promoter regionIn order to observe the relationship between ZEB1 and p53,double luciferase report test data show that silencing ZEB1 can increase the luciferase activity in p53 promoter compared with empty carriers.However,overexpression of ZEB1 can inhibit the luciferase activity of p53 promoter.Immunofluorescence analysis,q PCR and Western blot results showed that overexpression of ZEB1 could significantly inhibit the expression of p53,while silencing ZEB1 could significantly promote the expression of p53.6.MDP can reduce CCl4-induced liver fibrosis in vivo.In order to observe the therapeutic effect of MDP on hepatic fibrosis,the results of HE,Masson and Sirius red staining showed that the liver tissue of mice had more fatty degeneration,necrosis and fibrosis interval than the normal group.At the same time,CCl4 could significantly increase the activities of ALT,AST and TBIL in serum of mice.In addition,?-SMA and Col.I are two proteins that are usually considered to be markers of fibrosis.The results of immunohistochemistry and Western blot showed that the expression of?-SMA and Col.I in mouse liver fibrosis was significantly increased.We further measured whether aging markers changed with the progression of liver fibrosis.The results of immunohistochemistry and Western blot showed that CCl4 inhibited the expression of p16 and p21.More importantly,we observed the effects of MDP(25mg/kg,50mg/kg,100mg/kg)on CCl4-induced liver fibrosis in mice.The results showed that MDP could significantly inhibit hepatic steatosis,necrosis and fibrosis interval.After the addition of MDP,the increase of ALT,AST and TBIL activity was reversed.In addition,with the increase of the expression of aging markers(p16 and p21),the expression of?-SMA and Col.I decreased in a dose-dependent manner.These results show that MDP can reduce CCl4-induced liver fibrosis in vivo.7.MDP can promote activated HSCs senescence in vitro.MTT results showed that the higher the concentration of MDP,the higher the toxicity,and reached the critical value at 10^-5mol/L.In addition,Western blot results show that TGF-?1 decreased the expression of p16 and p21 and up-regulated the expression of?-SMA and Col.I compared with the normal group in LX-2 cells.However,MDP(10^-8,10^-7,10^-6,10^-5mol/L,24 h)significantly decreased the expression of?-SMA and Col.I,and increased the expression of p16 and p21 in LX-2 cells.More importantly,high concentration(10^-5mol/L)of MDP significantly inhibited the viability of LX-2 cells induced by TGF-?1,so 10^-5mol/L was selected for further study.Then,the results of SA-?-Gal staining showed that a large number of SA-?-Gal positive cells could be found in activated LX-2 cells treated by MDP.The results of immunofluorescence,q PCR and Western blot showed that TGF-?1 promoted the expression of?-SMA and Col.I,and inhibited the expression of p16 and p21.However,MDP could inhibit the expression of?-SMA and Col.I,and up-regulate the expression of p16 and p21.It is suggested that MDP can promote the senescence of activated HSCs in vitro.8.MDP targeting Ago2 indirectly regulates mi R-708 to reverse liver fibrosisIn order to observe the relationship between mi R-708 and MDP,q PCR results showed that MDP could significantly promote the expression of mi R-708.In addition,immunofluorescence and Western blot results showed that the expression of Ago2 was inhibited in CCl4-induced hepatic fibrosis and activated LX-2 cells,and the decrease of Ago2 expression was reversed after the addition of MDP to liver fibrosis and activated LX-2 cells.q PCR results showed that the inhibition of Ago2 decreased the expression of mi R-708.On the contrary,overexpression of Ago2 can promote the expression of mi R-708.In addition,Discovery Studio2017 R2 software analysis showed that MDP was bound to the active pocket of the target protein by folding.Western blot results showed that silencing Ago2 gene could increase the expression of?-SMA and Col.I,which could be inhibited by MDP.On the contrary,overexpression of Ago2 inhibited the expression of?-SMA and Col.I,while MDP further inhibited the expression of?-SMA and Col.I,indicating that the effect of MDP was similar to that of plasmid.Conclusion:1.mi R-708 significantly promote the senescence of HSCs cells via trageting ZEB1 by p53 in liver fibrosis,and thus inhibited the progression of liver fibrosis.2.MDP can inhibit liver fibrosis by inducing activated HSCs senescence.3.MDP can further upregulate the expression of mi R-708 by directly combining with Ago2 and thus inhibit liver fibrosis.