Artesunate Inhibits Fibroblast Proliferation And Prevents Epidural Prevent Fibrotic Adhesion Through MTOR Mediated Autophagy

Epidural fibrosis is a common complication after laminectomy.The main symptom is pain recurrence,which seriously affects the quality of life in patients after operation.It is one of the problems that perplex orthopedics doctors.At present,although there are many prevention and treatment measures,they can only prevent the occurrence of epidural fibrosis to a certain extent.This problem can not be effectively cured to this day.The mechanism of epidural fibrosis is a complex process.Up to now,it has been found that the activation and proliferation of fibroblasts and the formation of a large number of scar tissue are the main causes of this complication.It can cause extensive adhesion between the dural sac and nerve root,which leads to epidural fibrosis and severe pain.Artesunate(ART)is an antiparasitic drug extracted from artemisinin,which is often used to treat malaria.Recently,it has been found that ART has a good preventive and therapeutic effect on the occurrence and development of fibrotic diseases.m TOR(mammalian rapamycin target protein)signal pathway is an important eukaryotic cell pathway,which can participate in the process of immunosuppression,and play an important role in cell proliferation,apoptosis and autophagy.The purpose of this study is to explore the role of m TOR mediated autophagy pathway in ART prevention of epidural fibrosis,so as to provide potential therapeutic targets for the prevention and treatment of epidural fibrosis in clinical work,improve the surgical effect of patients,and improve their quality of daily life.Chapter Ⅰ Study of artesunate prevents epidural fibrosis in ratsObjective: To explore the effect of ART on the prevention of epidural fibrosis in rats and its possible mechanism.Methods: 72 adult SD rats,male,were randomly divided into four groups: 15,30,60 mg / kg ART group and normal saline group.After 4 weeks,the rats were observed and Rydell’s scorewas measured.Then,the rats were killed,the samples of scar fibrosis tissue in the operation area were taken,the content of hydroxyproline was detected,vimentin and fibroblast surface protein(FSP-1)were detected by immunohistochemistry,the cells in the operation area were identified,and the status of fibroblasts was observed;The degree of epidural fibrosis adhesion was observed by HE staining and the fibroblasts number were counted;Masson staining and Sirius red staining were used to compare the collagen content;The expression of LC3,PCNA,Cyclin D1 and p-m TOR were detected by immunohistochemistry.Results: 1.All SD rats were recovered in the experiment,and there was no death,infection in the operation area,wound nonunion and so on.2.Rydell score found that the degree of epidural fibrosis and adhesion in ART group was reduced.3.Immunohistochemistry was used to detect vimentin and FSP-1 in the operation area.The results showed that there were many positive expression of vimentin and FSP-1,which indicated that there was a large number of fibroblast growth in the operation area.4.HE staining showed that ART could reduce the degree of extradural fibrosis in a concentration dependent manner,and the number of fibroblasts was significantly lower than that of the control group.The number of fibroblasts in 60 mg / kg group was less than that in 15 mg / kg group and 30 mg / kg group,and that in 30 mg / kg group was less than that in 15 mg / kg group.5.The results of hydroxyproline,masson staining and sirius red staining showed that the collagen content and density of the experimental group were significantly lower than that of the control group.6.The expression of PCNA,cyclin D1,p-m TOR and LC3 were detected by immunohistochemistry.It was found that the content of the first three in the experimental group was lower than that in the control group,which was concentration dependent,while LC3 increased with the concentration.Conclusion: 1.There were no death,infection and wound nonunion complications in rats treated with ART of different concentrations,suggesting that ART has a good safety in the prevention of epidural fibrosis.2.In the experiment,fibroblasts participate in the repair process of injury.Different concentrations of ART can prevent epidural fibrosis by inhibiting the proliferation of fibroblasts and reducing the production of collagen tissue,and this effect is concentration dependent.3.ART can increase the expression of LC3 and decrease the expression of p-m TOR,suggesting that autophagy and m TOR signaling pathway areinvolved in the prevention effect of epidural fibrosis by ART.Chapter Ⅱ Artesunate inhibits fibroblast proliferation by inducing autophagyObjective: To explore the effect of artesunate on the prevention of epidural fibrosis in rats and its possible mechanism.Methods: We cultured the fibroblasts in the laboratory.When the density of the cultured cells reached 60%-80%,different concentrations of ART for 24 hours and specific concentrations(10 μM)of ART for different time were used.We detected the viability of fibroblasts by CCK-8,and investigated the expression of PCNA and Cyclin D1 by western blot and use Ed U staining to find the effect of ART on the proliferation of fibroblasts.Meanwhile,the effects of ART on autophagy of fibroblasts were observed by LC3 immunofluorescence staining,western blot of autophagy related proteins(Beclin-1,LC3 II / LC3 I,Atg5 and p62)and transmission electron microscopy.Finally,fibroblasts were treated with autophagy inhibitor 3-MA,and the inhibition of ART on fibroblast proliferation was detected by CCK-8 and western blot.Results: 1.CCK-8 results showed that with the increase of ART concentration and the prolongation of ART action time,the viability of fibroblasts was detected to decrease continuously,showing a significant time and concentration dependence.2.After the fibroblasts were treated with ART at a specific concentration(10 μM),the DNA synthesis in the cells was detected by Ed U staining.The results showed that ART could significantly inhibit the DNA synthesis of fibroblasts.3.ART was used to interfere with fibroblasts and western blot was used to detect proteins related to proliferation.The results showed that PCNA and cyclin D1 decreased with the increase of concentration and time prolongation,which showed a concentration and time dependence.4.LC3 immunofluorescence staining showed that after ART intervention,the number of fluorescent spots in fibroblasts increased,indicating that the number of autophagosome increased.5.ART was used to interfere with fibroblasts,autophagy related proteins were detected by western blot.The results showed that the expression of Beclin-1,LC3 II / LC3 I and Atg5 increased with the increase,while the expression of p62 decreased with the increase of ART concentration and time prolongation,which further indicated that ART induced autophagy was concentration and time dependent.6.The autophagy of fibroblasts after treated with ART was observed by transmission electron microscopy,which fully proved that ART could induce autophagy of fibroblasts.7.After 3-MA treatment,the expression of LC3 by immunofluorescence staining and western blot showed that autophagy induced by ART could be partially reversed.8.After 3-MA treatment,western blot were used to detect the proliferation related proteins.The results showed that the inhibition of ART on the viability and proliferation of fibroblasts decreased after the inhibition of autophagy.Conclusion: 1.ART can effectively inhibit the viability of fibroblasts,and its inhibition ability depends on time and concentration.2.ART can effectively inhibit the proliferation of fibroblasts,and also show a significant concentration and time-dependent,which indicates that ART may inhibit the cell proliferation by inhibiting viability of fibroblasts.3.ART can induce autophagy of fibroblasts in a concentration and time-dependent manner.4.ART can inhibit the proliferation of fibroblasts by inducing autophagy.Chapter Ⅲ: The role of m TOR mediated autophagy pathway in artesunate inhibition of fibroblast proliferationObjective: To investigate the role of m TOR mediated autophagy in the inhibition of fibroblast proliferation by ART.Methods: Fibroblasts were cultured in vitro.When the cell density reached 60%-80%,ART with different concentrations(0,5,10,20 μM)was used for 24 hours.The m TOR signal pathway and the upstream PI3 K / Akt and AMPK signal pathway related proteins were determined by western blot(p-PI3 K,PI3K,p-AKT,AKT,p-AMPK,AMPK,p-m TOR,m TOR,p-4E-BP1 and p-P70S6K).Overexpression the m TOR gene,construction of stable cell lines,western blot were used to detect the overexpression efficiency.The control group and the OE fibroblasts group were treated with or without ART respectively,then LC3 immunofluorescence staining was performed and autophagy related proteins(LC3,Atg5,p62)were detected by western blot.The effect of ART on m TOR mediated autophagy pathway was observed.CCK-8,Ed U staining and western blot were used to determine whether ART affects the proliferation of fibroblasts through m TOR mediated autophagy.Results: 1.After the fibroblasts were treated with ART at different concentrations,the downstream and upstream proteins of m TOR were detected.The results showed that the activation pathway PI3 K / AKT was inhibited,while the inhibition signal pathway AMPK was activated in a concentration dependent manner,and autophagy was enhanced when m TOR was inhibited.2.After m TOR overexpression,LC3 immunofluorescence staining showed that the number of vesicles in fibroblasts decreased,western blot showed that the trend of increasing expression of LC3 II / LC3 I and Atg5 and decreasing expression of p62 were partially reversed.3.After overexpression of m TOR,the inhibition of ART on the viability of fibroblasts was reduced.Ed U staining showed that the inhibition of ART on the synthesis of DNA in fibroblasts was also reduced,while western blot showed that the downtrend of proliferation related proteins(PCNA,cyclin D1)was partially reversed.Conclusion: 1.m TOR mediated signaling pathway is involved in the process of ART on fibroblasts.2.ART affects the autophagy of fibroblasts through m TOR mediated pathway.3.ART inhibits fibroblast proliferation through m TOR mediated autophagy.