Effect Of Proteasome Inhibitor MG132 On End MT Of Rats With Pulmonary Fibrosis And Its Mechanism

Background:Pulmonary fibrosis is a chronic and fatal pulmonary disease.It is caused by a variety of body and external factors,which lead to the damage of the integrity of the basement membrane.The regeneration ability of the epithelial cells and endothelial cells are weakened,and the fibrotic tissue is proliferated and eventually forms the fibrosis focus.The pathogenesis of PF is not fully understood.It also lacks an effective treatment and has a very poor prognosis.Therefore,it is necessary to find further exploration of effective strategies to improve PF.Recent studies have shown that EndMT is an important mechanism of pulmonary fibrosis.TGF-?1 regulates the key cytokines on EndMT of fibrosis,and Smad protein is an important signal transduction factor in TGF-? signaling.Smad7 of receptor-inhibitory Smad protein plays an important role in TGF-? signaling pathway.The close combination of Smad7 and T?RI produce a competitive antagonism,which plays a negative regulatory role in the TGF-?1 signaling.Ubiquitin proteasome pathway(UPP),an important protein regulation system in eukaryotic cells,which can specifically degrade intracellular proteins.Smad ubiquitin regulatory factor 2(Smurf2)is a kind of E3 ubiquitin ligases,which can regulate TGF-?1signaling through ubiquitination of Smad7,and may play an important role in the development of PF.The ubiquitin proteasome inhibitor MG132 is an aldehyde peptide proteasome inhibitor that binds to the proteasome and reversibly inhibits proteasome activity,thereby inhibits UPP.It has been widely used in experimental research.Studies have shown that ubiquitin-proteasome inhibitor MG132 can reduce the occurrence and development of liver,kidney,heart,skin and other organ fibrosis.However,rare research has been done on pulmonary fibrosis and the mechanism is still not clear.Objective:By detecting the expression of endothelial cell(CD31)and interstitial cell phenotype(?-SMA)at different time points of PF,the role of EndMT in PF and its changes characteristics were understood,rat models of PF were established with Proteasomal inhibitor MG132 intervention by,detecting the expression of Smurf2,TGF-?1 and Smad7,to investigate the effect of proteasome inhibitor MG132 on EndMT of pulmonary fibrosis and the regulatory mechanism of Smurf2-Smad7 signaling on EndMT of PF.Methods:96 healthy male SD rats were randomly divided into four groups(the control group,the BLM group,the DMSO group and the MG132 group).The BLM group,the DMSO group and the MG132 group rats were intratracheally injected with bleomycin solution 0.3ml(5mg/kg)to establish animal model of PF.In the same way,the rats in the control group were given the same amount of saline.After model establishment,the MG132 group was intraperitoneally injected with0.3ml of proteasome inhibitor MG132(0.1mg/kg/d)everyday,the BLM group was intraperitoneally injected with the same amount of saline everyday,the DMSO group was intraperitoneally injected the same amount of solvent reagents everyday,and the control group was injected with equal amount of saline everyday.Eight rats were sacrificed randomly on the 7th,14 th,and 28 th day in each group.The lung tissues were taken out,HE staining and Masson staining were performed.Besides,Hydroxyproline(HYP)assay and Ashcroft score were used to evaluate the degree of lung fibrosis in rats,the expression of Smurf2,TGF-?1,Smad7,CD31 and?-SMA were detected by immunohistochemistry and the expression of Smurf2,TGF-?1and Smad7 mRNA were detected by RT-PCR at different time points in the rats.Results:(1)General condition of rat:On the 7th,14 th,and 28 th day,the rats in the control group were sensitive reaction,normal feeding,good activity,and stable breathing.But inthe BLM group,the reaction is slow,the feeding is gradually reduced,the activity decreases,and the breathing is accelerated.The DMSO group was similar to the BLM group;the response,feeding,activity and respiration of rats in the MG132 group were between the control group and the BLM group.On the 16 th day,one rat died in the MG132 group,and it was considered that there was a high possibility of death due to the occurrence of mid-peritoneal peritonitis.(2)Pulmonary fibrosis index test:The lung tissue changes,HE Masson staining,hydroxyproline(HYP)content detection,Ashcroft score are all suggested that On the 7th,14 th and 28 th day,the indexes of lung fibrosis in BLM group,compared with the control group,were significantly increased(P<0.05),and the rate of early stage change was significantly higher than that of the late stage;The indexes of lung fibrosis in the DMSO group were similar to the BLM group(P>0.05),The index of pulmonary fibrosis in the MG132 group was lower than the BLM group(P<0.05),but it was higher than the control group(P<0.05).(3)Immunohistochemical detection of Smurf2,TGF-?1,Smad7,CD31 and ?-SMA in lung tissue:On the 7th,14 th and 28 th day,the degree of pulmonary fibrosis in BLM group,compared with control group,was significantly aggravated,the positive area of CD31 expression gradually decreased,the positive area of ?-SMA expression gradually increased,and the positive area of Smurf2 and TGF-?1 expression gradually increased,but the Smad7 expression positive area gradually decreased(P<0.05);The results of immunohistochemistry in the DMSO group were similar to those in the BLM group(P>0.05).The immunohistochemistry results in the MG132 group were between the BLM group and the control group(P<0.05).(4)RT-PCR detection of Smurf2,TGF-?1,Smad7 mRNA expression:On day7 th,14th and 28 th day,the expression of Smurf2 and TGF-?1 mRNA of lung tissue in the BLM group compared with the control group,were significantly increased(P<0.05)and Smad7 mRNA was down-regulated(P<0.05).The DMSO group were similar to the BLMgroup(P>0.05).The expression of Smurf2 and TGF-?1 mRNA in lung tissue of MG132 group was up-regulated(P<0.05),while the expression of Smad7 was down-regulated(P<0.05).The expression of Smurf2,TGF-?1,Smad7 mRNA in lung tissue of MG132 rats was between the BLM group and the control group(P<0.05).The expression of Smurf2 mRNA was positively correlated with the degree of fibrosis and the expression of TGF-?1 mRNA in rats,and negatively correlated with the expression of Smad7 mRNA.Conclusion:1.EndMT is involved in the formation of pulmonary fibrosis,with an EndMT peak occurring on 14 th day.2.The proteasome inhibitor MG132 participates in the regulation of EndMT in rats with pulmonary fibrosis,which inhibits the development and development of pulmonary fibrosis.3.The proteasome inhibitor MG132 can regulate pulmonary fibrosis in rats by regulating Smurf2/Smad7 signal transduction,thereby inhibiting the occurrence and development of pulmonary fibrosis.4.Smurf2 may become a new target for clinical prevention and development of pulmonary fibrosis.