| Background and Objective:Smoke inhalation induced acute lung injury(SI-ALI)is a common emergency and severe disease after fire accidents,which can lead to acute respiratory distress syndrome(ARDS)in severe cases.Severe pulmonary fibrosis during the process of ALI/ARDS repair is one of the important reasons for the poor prognosis and high lethality/morbidity of ARDS,which may be related to the activation of Wnt/β-catenin signaling pathway in fibroblasts according to research data.Recent studies have found that hydroxychloroquine(HCQ)can inhibit fibroblast proliferation and alleviate pulmonary fibrosis in models of fibrotic skin disease and bleomycin-induced pulmonary fibrosis.Therefore,the Wnt/β-catenin signaling pathway may also be overactivated in SI-ALI,so whether HCQ alleviates SI-ALI/ARDS pulmonary fibrosis by inhibiting Wnt/β-catenin signaling pathway?Our study intends to explore the effect of HCQ on pulmonary fibrosis by establishing a SI-ALI/ARDS pulmonary fibrosis rat model and take methylprednisolone(MP)as control drug,which is commonly used to inhibit pulmonary fibrosis in clinical,at the same time,explore whether HCQ exerts anti-fibrotic effect by inhibiting the Wnt/β-catenin signaling pathway.Methods:1.Establishment of pulmonary fibrosis model after smoke inhalation induced acute lung injury in ratsMale Sprague-Dawley(SD)rats were randomly divided into smoke(S)group and control(C)group at the age of 8w.The S group was randomly divided into 3 subgroups according to different times of smoke inhalation,which were one time of smoke inhalation(S/1),three times of smoke inhalation(S/3)and seven times of smoke inhalation(S/7).And inhaling smoke stably once a day at the same time,15min each time.A certain amount of smoke was stably inhaled using the smoke generator device designed and made by the research group in the early stage,and the pulmonary fibrosis model after SI-ALI was established.The activity and vital signs of the rats were observed.On the first day and 28d after the prescribed smoke inhalation times,some of the lungs of the rats were taken for hematoxylin-Eosin(HE)staining,Sirius red staining and Masson staining to observe tissue damage and fibrosis changes,and evaluate the degree of alveolar inflammation and fibrosis.Looking for the best way to make the mold.2.The protective effect of HCQ on pulmonary fibrosis after smoke inhalation induced acute lung injury in rats8-week-old healthy adult male SD rats were selected and randomly divided into C group,S group,smoke+phosphate buffered saline(S+PBS)group,smoke+methylprednisolone(S+MP)group,smoke+hydroxychloroquine(S+HCQ)group,and S+HCQ group was divided into 3 groups according to the different doses of HCQ(5mg/kg,l0mg/kg,20mg/kg).One hour before inhaling smoke,the S+MP group was injected with 4 mg/kg/d MP intraperitoneally,the S+HCQ group was injected with 5 mg/kg,10 mg/kg,and 20 mg/kg HCQ,respectively,the S+PBS group was injected with the same volume of PBS,and the C group received no intervention.After one hour,mold was made according to the method determined in Part I.MP and HCQ in each subgroup were continuously intervened for 7 days and reared for 28 days.During the process,the survival rate of the rats was observed,and the wet-dry ratio(W/D)of the lung tissue at 28 days was measured.And western blotting(WB)experiment was used to determineα-smooth muscle actin(SMA),collagen(Col)I/III,and quantitative real-time polymerase chain reaction(qRT-PCR)were performed to detect the mRNA expression of above indexes.The remaining observation indexes and pathological staining were the same as the first part.3.HCQ may play an anti-fibrosis role by inhibiting Wnt/β-catenin signaling pathwayThe grouping and experimental methods were the same as those in the second part.The WB experiment was used to detect β-catenin,p-GSK-3β/GSK-3β,and the qRTPCR experiment was performed to detect the mRNA expression of the above indicators.Results:1.Successful establishment of a rat model of pulmonary fibrosis after smoke inhalation acute lung injury1)Activity and vital signs:During the process of smoke inhalation,the rats were shortness of breath,became restless,and the secretions from the mouth and nose significantly increased.After the smoke inhalation,the vitality of the rats was significantly decreased,and the response was slow.The above performance in group S/7 and S/3 was more obvious than that in group S/1.The rats in each group gradually returned to normal state after 2-3 days after all the times of smoke.The rats in group C had good vitality and no obvious abnormality.2)Survival rate:No rats died in group C;3 rats died in group S/1,and the overall survival rate was 85%;5 rats died in group S/3,and the overall survival rate was 75%;8 rats died in group S/7,and the overall survival rate was 60%.Compared with the C group,the survival rate of the rats in the S/3 and S/7 groups was significantly decreased,and the difference was statistically significant(P<0.05).3)W/D of lung tissue:the W/D of the S group was significantly increased on the first day after smoke inhalation,and the W/D of S/7 and S/3 was significantly higher than that of S/1,and the difference was statistically significant(P<0.05),repeated smoke inhalation increased lung injury;W/D in each S group on the 28th day was significantly lower than that on the 1st day,and the difference was statistically significant(P<0.05).4)Lung tissue pathology:Compared with the C group,the rats in the S groups had different degrees of lung tissue damage and fibrosis,and with the increase of the number of smoke inhalation,the lung damage and fibrosis gradually increased,and the degree of pulmonary fibrosis in S/3 and S/7 group are more obvious.5)Lung injury score and fibrosis grade:Compared with C group,with the increase of smoke inhalation times,the lung tissue injury score of rats in S groups increased significantly(P<0.05),and the pulmonary fibrosis grade gradually increased.2.HCQ attenuates pulmonary fibrosis in rats after smoke inhalation1)Activity and vital signs:The performance during smoke inhalation and after modeling was roughly the same as in the first part,and there was no significant difference between all smoking groups;And there was no obvious abnormality in group C.2)Survival rate:Compared with the C group,the survival rate of the S group was significantly decreased(70%,P<0.05);compared with the S+PBS group,the survival rate of the rats in the S+MP group and the S+HCQ group were improved,among which 20mg/kg HCQ and 4mg/kg MP had more obvious effects on the survival rate of the rats(90%,P>0.05 and 95%,P<0.05),there was no significant difference between 20mg/kg HCQ and 4mg/kg MP(P>0.05).3)W/D of lung tissue:Compared with the C group,the W/D of the S group was significantly increased(P<0.05);compared with the S+PBS group,the W/D of the S+MP group and the S+HCQ group was significantly decreased(P<0.05).The effects of 20mg/kg HCQ and 4mg/kg MP on rat W/D were more obvious,and there was no significant difference between them(P>0.05).4)Lung tissue homogenate fibrosis index:Compared with group C,the levels of Col Ⅰ,Col Ⅲ and α-SMA in lung tissue homogenate in group S were significantly increased(P<0.05);compared with group S+PBS,the levels of Col Ⅰ,Col Ⅲ and αSMA in group S+MP and group S+HCQ were significantly decreased(P<0.05),among which 20mg/kg HCQ and 4mg/kg MP had obvious effects on fibrosis indexes in rats,and the difference was statistically significant(P<0.05)..The mRNA levels of Col I,Col Ⅲ and α-SMA in the qRT-PCR experiment were roughly the same as those in the WB experiment.5)Lung tissue pathology:The pathological changes of lung tissue in the S group and S+PBS group were the same as part I.Compared with the S+PBS group,inflammation and fibrosis such as alveolar congestion,inflammatory cell infiltration and Sirius red staining were significantly reduced.The effects of 20mg/kg HCQ and 4mg/kg MP on pulmonary fibrosis in rats were more obvious.6)Lung injury score and fibrosis grade:Compared with group C,the scores of lung tissue damage in group S and group S+PBS were significantly increased(P<0.05),and the grade of pulmonary fibrosis was increased.Compared with the S+PBS group,the lung injury scores of the S+MP group and S+HCQ group decreased(P<0.05),and 20mg/kg HCQ and 4mg/kg MP had more significant effects on the lung injury scores of the rats.No significant difference was found(P>0.05).3.HCQ may play an anti-fibrosis role by inhibiting Wnt/β-catenin signaling pathwayCompared with the C group,the levels of β-catenin and p-GSK-3β/GSK-3βsignaling pathway proteins in lung tissue homogenate in the S group were significantly increased(P<0.05);Compared with S+PBS group,the levels of β-catenin and p-GSK3 β/GSK-3β signaling pathway protein in S+MP group and S+HCQ group were significantly decreased(P<0.05),and the effects of 20mg/kg HCQ and 4mg/kg MP on signaling pathway were more obvious.And the two had no significant difference(P<0.05).20mg/kg HCQ had slightly weaker effect on signaling pathway than 4mg/kg MP.The mRNA levels of β-catenin in the qRT-PCR experiment were roughly the same as those in the WB experiment.Conclusion:1.Lung fibrosis occurred in the late stage of smoke inhalation lung injury in rats,and the HCQ could alleviate the pulmonary fibrosis after smoke inhalation in rats.2.The Wnt/β-catenin signaling pathway is overactivated during the pulmonary fibrosis in the advanced stage of smoke inhalation injury in rats,and the HCQ may alleviate the pulmonary fibrosis by inhibiting the Wnt/β-catenin signaling pathway. |
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