Mechannism Of Triptolide Inhibiting Myofibroblasts Activation In Radiation-induced Pulmonary Fibrosis

Objective:Myofibroblast(MFBs)activation plays a key role in the progression of radiation-induced pulmonary fibrosis(RIPF).In this study,in vivo and in vitro model were applied to observe the regulating effect and the related mechanism of Triptolide(TPL)on the two central factors which promote the activation of MFBs in RIPF,Transforming Growth Factor-1(TGF-β1)and matrix stiffness,to provide a basic support for the anti-RIPF application of TPL.Methods:(1)The RIPF model of C57BL/6 mice was established by whole lung irradiation(15Gy).TPL was administered(0.25mg/kg,iv,qod)for 1 month;(2)The levels of TGF-β1 and matrix stiffness-related factors including type I Collagen,Col I,Lysyl oxidase(LOX),Lysyl oxidase-like-1(LOXL-1)and Lysyl oxidase-like-2(LOXL-2)were detected by ELISA;(3)Fibroblasts were stimulated by TGF-β1,and the levels ofα-smooth muscle actin(α-SMA),ERK,Smad3(ser208)and Smad3(ser423)were detected by Western blot.The role of ERK and Smad3 in the activation of MFBs promoted by TGF-β1 was determined by RNA interference.(4)The hardness of Col I gel and lung tissue was detected by using rotational rheometer.High throughput immunofluorescence imaging system was used to analyze the different effects of soft matrix and stiff matrix on the activation of MFBs(α-SMA~+).Western blot and immunofluorescence were used to detect the expression levels of integrin 1,FAK and MYPT1.(5)To investigate the effects of TPL on TGF-β1 and matrix stiffness-induced MFBs activation in vivo and in vitro,as well as the respective regulation on corresponding signaling pathways.Results:(1)The anti-radioactive pulmonary fibrosis effect of TPL was related to the reduction of MFBs activation through inhibiting TGF-β1/ERK/Smad3 pathway.TGF-β1stimulation upregulated levels of p-ERK,p-smad3(ser208)and p-smad3(ser423),and resulted in MFB activation(increasedα-SMA expression).ERK siRNA down-regulated the level of p-Smad3(ser208),but had no significant effect on p-Smad3(ser423),suggesting that ERK is involved in TGF-β1-related MFBs activation through phosphorylation of Smad3(ser208).TPL significantly down-regulates levels of p-ERK,p-Smad3(ser208)and p-Smad3(ser423)in vivo and in vitro,confirming that TPL potently inhibits TGF-β1-related MFBs activation by inhibiting classical activation of Smad3 downstream of TGF-β1(phosphorylation at ser423)and bypass activation of Smad3(ser208 phosphorylation by ERK).(2)TPL alleviates the hardening of the lung matrix in RIPF and reduces MFB activation by inhibiting the FAK/ROCK signaling pathway.Excessive Col I,LOX,and LOXL-2 in lung tissues after irradiation were major contributors to matrix stiffness.Stiff matrix upregulated the levels of p-FAK and p-MYPT1 and promoted the activation of MFBs.TPL could reduce the secretion of Col I and LOXL-2,and down-regulate the hardness of lung matrix in RIPF;Furthemore,TPL reduced the levels of p-FAK/FAK and p-MYPT1/MYPT1 which indicating FAK/ROCK signaling pathway inhibition,and prevent the activation of MFBs related to matrix stiffness.Conclusion:The potent anti-RIPF effect of TPL is related to its multi-target inhibition of MFBs.TPL inhibits both TGF-β1 and matrix stiffness,two major factors that promote MFBs activation,by suppressing TGF-β1/ERK/Smad3 pathway,alleviating matrix hardening and inhibiting FAK/ROCK pathway.The results of this study reveal the key mechanism of TPL against RIPF,and provide a theoretical basis for the practical application of TPL.