The Effect And Mechanism Of Pirfenidone On Fibroblast Proliferation And Preventing Epidural Adhesion

Objective:1.To observe the preventive effect of pirafenidone on epidural scar adhesion after laminectomy in rats.2.To investigate the effect of pirfenidone on proliferation and apoptosis of fibroblasts.3.To elucidate the molecular mechanism of pirfenidone in the prevention of epidural scar adhesion.Methods:1.We establish a epidural adhesion rat model.All 60 rats were divided into four groups(10 mg/ml pirfenidone group,40 mg/ml pirfenidone group,80 mg/ml pirfenidone group and control group).After 4 weeks,gross observation,hydroxyproline content determination,histological observation,fibroblast count and collagen density observation were performed to evaluate the effect of pirfenidone on prevention of epidural scar adhesion.2.The human epidural scar tissue fibroblasts were treated with different concentrations of pirfenidone.Cell viability was measured using cck-8 kit.Cell proliferation was detected by Ed U staining.Apoptosis effect was detected by AV/PI double staining follow by flow cytometry,and cytotoxicity was detected using LDH release release assay.3.Pretreatment of fibroblasts with TGF-?1 to induce fibroblast proliferation,and then treatment with different concentrations of pirfenidone.Cell viability wasmeasured using cck-8 kit.Cell proliferation was detected by Ed U staining.q PCR was used to detect the transcription of related m RNA,and western blot was used to detect the expression of related proteins.Result:1.Gross dissection showed that the epidural scar tissue in the 40 mg/ml and 80mg/ml pirfenidone groups was looser than the control group,and the hydroxyproline content in the epidural scar tissue of the pirfenidone treated group was significantly lower than that of the control group.The content of fibroblasts in the scar area of the pirfenidone treated group was significantly lower than that of the control group(P<0.05).The density of epidural collagen in the pirfenidone group was lower in Masson staining.The area of collagen was significantly lower than that of the control group(P<0.05).2.It was found that TGF-?1 can induce proliferation of fibroblasts.Pirfenidone inhibits TGF-?1-induced fibroblast proliferation.No apoptosis of fibroblasts was observed by Annexin V/PI double-stained.The use of the LDH release kit did not reveal that pirfenidone increased the release of LDH.3.TGF-?1 can up-regulate ?-SMA and type I collagen levels at the transcriptional and translational levels,while pirfenidone can inhibit TGF-?1-induced?-SMA and type I collagen.TGF-?1 activates Smad 2,Smad 3,p38 and Akt to increase the expression of phosphorylated Smad 2,Smad 3,p38 and Akt,and pirfenidone inhibits phosphorylated Smad 2,Smad 3,p38 and Akt expression.Conclusion:1.Pirfenidone can reduce the formation of epidural scar tissue after laminectomy in rats,and effectively prevent epidural adhesion.2.Pirfenidone reduces epidural scar tissue formation by inhibiting fibroblast proliferation,reducing hydroxyproline content,and reducing collagen tissue density.3.Pirfenidone can inhibit the proliferation of fibroblasts without increasing the apoptosis of fibroblasts and showing no cytotoxic effects.4.Pirfenidone can inhibit the expression of ?-SMA and type I collagen induced by TGF-?1 suggesting that pirfenidone can inhibit the activation of fibroblasts,and pirfenidone can also inhibit the induction of TGF-?1.Phosphorylation of Smad 2,Smad 3,p38 and Akt suggests that pirfenidone can inhibit fibroblast proliferation by inhibiting TGF-?1-induced Smad-dependent and non-dependent pathways.