Therapeutic Effect And Mechanism Of MG132 On Bleomycin-induced Pulmonary Fibrosis In Rats

Objective:The rat pulmonary fibrosis model was constructed by bleomycin,and the therapeutic effect of MG132 on pulmonary fibrosis was observed.the anti-fibrotic mechanism of MG132 and its regulation mechanism on TGF-?1/Smad signaling pathway were also studied.Methods:1.Establishment of pulmonary fibrosis model and experimental grouping:120 SD rats were randomly divided into pulmonary fibrosis model group(90 rats)and saline control group(NS group,30 rats).The model group was given intratracheal injection of bleomycin(0.3 ml,5 mg/kg),and the control group was intratracheal injected with 0.3 ml of normal saline.On the first day after modeling,the model group was randomly divided into three groups: the bleomycin group(BLM group),the dimethyl sulfoxide group(DMSO group),and the MG132 group,with 30 rats in each group.MG132 group was intraperitoneally injected with MG132(0.3ml,0.1mg/kg.d)daily,DMSO group was intraperitoneally injected with 0.3ml dimethyl sulfoxide solution daily,BLM group and NS group were intraperitoneally injected with normal saline 0.3ml.2.Observe and record the general condition of rats at each time point.Ten rats in each group were randomly sacrificed on the 7th,14 th and 28 th day,and lung tissues were taken to observe the gross structural changes and inflammatory response of lung tissue.3.Pathological examination of lung tissue: The lung tissue removed was embedded in paraffin,sectioned,Masson stained,HE stained,and assessed by Ashcroft score.4.Molecular mechanism study: Western blot and immunohistochemistry were used to detect the expression of CD31,?-SMA,TGF-?1,Smad3,Smurf2 and SnoN.The mRNA expression of Smurf2 and SnoN was detected by PCR.Results:1.General condition of the rats: On days 7,14,and 28 of the NS group,therats were in good general condition,the skin was smooth,the breathing was stable,the activity was sensitive,the food intake was normal,and the body weight gradually increased.In the BLM group,the state of the rats gradually deteriorated,the spirits were sluggish,the activity was slow,the breathing was accelerated,the food intake was reduced,and the body weight was decreased.The DMSO group and the BLM group performed similarly.In the MG132 group,the general state,activity,feeding,respiration,and body weight of the rats were between the NS group and the BLM group.(On the sixth day,one rat died in the BLM group and one rat died in the MG132 group.The rats were found to have red and swollen skin in the neck and yellowish white purulent fluid exudation.The reason is that when the lung fibrosis is molded,the wound is not strictly disinfected after the neck is opened,causing infection to cause death.On the 18 th day,1 rat died in the DMSO group.The rats were dissected and found to have severe abdominal adhesions.The death of the rats was considered in consideration of peritonitis.).2.Lung tissue: The NS group is pink at each time point,smooth,full and elastic.BLM group,there is scattered bleeding point in the early stage,the elasticity is slightly poor,the texture of late lung tissue is hard,the color is dark red,and the volume is reduced.In the DMSO group,lung tissue was generally similar to the BLM group.In the MG132 group,the early stage was pink with a small amount of bleeding points.The late color became darker,the volume was reduced,and the elasticity decreased,but the degree was lighter than that of the BLM group.3.Pathology:(1)HE staining: the alveolar structure was normal in NS group at 7,14 and 28 days.BLM group: As time progresses,the alveolar structure is filled with collagen fibers,the alveolar septum is thickened,congestion,bleeding range is increased,inflammatory cell infiltration.The performance of DMSO group was similar to that of BLM group.In the MG132 group,on the 7th day,the alveolar structure was slightly damaged,and a small amount of inflammatory cells infiltrated.On the 14 th and 28 th,the alveolar structure was destroyed,the alveolar septum was thickened,and a large number of inflammatory cells were infiltrated,but the degree was lighter than that of the BLM group.(2)Masson staining: On days 7,14,and 28 of the NS group,the alveolar structure was normal,and no obvious blue collagen deposition was observed(P>0.05).In the BLM group,the alveolar structure was destroyed with time,a large amount of blue collagen fibers were deposited,and the alveolar septal thickening was obvious.The difference was statistically significant(P<0.05).The performance of DMSO group was similar to that of BLM group(P>0.05).In the MG132 group,the alveolar structure was destroyed,the blue collagen fibers were deposited,and the alveolar septum was thickened,but the degree was between the NS group and the BLM group.The difference between the groups was statistically significant(P<0.05).4.Ashcroft score: The Ashcroft score was similar at each time point in the NS group(P>0.05).In the BLM group,the Ashcroft score increased significantly on the 7th,14 th,and 28 th days compared with the NS group(P<0.05).In the DMSO group,the Ashcroft score was similar to that of the BLM group,and the difference was not statistically significant(P>0.05).In the MG132 group,the Ashcroft score was lower than that in the BLM group(P<0.05),which was higher than that in the NS group(P<0.05).5.Molecular mechanisms:(1)immunohistochemistry(CD31,?-SMA,TGF-?1,Smad3,Smurf2,SnoN expression),Compared with NS group,the expression of ?-SMA,TGF-?1,Smad3 and Smurf2 in BLM group increased gradually(P<0.05),and the expression of CD31 and SnoN decreased gradually(P<0.05).The positive expression area of each antibody in the DMSO group was similar to that of the BLM group(P>0.05).The positive expression area of each antibody in the MG132 group was between the BLM group and the NS group(P<0.05).(2)Western blot(CD31,?-SMA,TGF-?1,Smad3,Smurf2,SnoN xpression),On days 7,14,and 28,compared with the NS group,the expression of?-SMA,TGF-?1,Smad3,and Smurf2 in the BLM group gradually increased,and the expression of CD31 and SnoN decreased gradually.The expression levels of each antibody in the DMSO group were substantially similar to those in the BLMgroup.The expression level of each antibody in the MG132 group was between the BLM group and the NS group.(3)qRT-PCR(Smurf2,SnoN): There was no significant difference in mRNA expression of SnoN between each group and each time period(P>0.05).Compared with the NS group,the mRNA expression of Smurf2 in the BLM group was significantly up-regulated(P<0.05);The mRNA expression of Smurf2 in DMSO group was similar to that in BLM group(P>0.05).In the MG132 group,the mRNA expression of Smurf2 was similar to that of the BLM group(P>0.05).Conclusion1.Pulmonary fibrosis rat model can be successfully established by intratracheal injection of bleomycin.2.MG132 can reduce inflammation and collagen deposition and has a therapeutic effect on rats with pulmonary fibrosis.3.MG132 may delay pulmonary fibrosis by regulating Smurf2-SnoN signaling,affecting the TGF-?1/Smad pathway.