| Objective:To investigate the effect and mechanism of Artemisinin-Hydroxychloroquine（AH）in the treatment of bleomycin-induced pulmonary fibrosis（PF）in Sprague-Dawley（SD）rats through in vivo experiments,in an attempt to provide new ideas for the development of new drugs for the treatment of PF.Methods:1.Replication animal model of PF:Bleomycin hydrochloride（BLM）was prepared into 4.5mg·mL-1 solution with normal saline,and the solution was kept away from light for later use.After deep anesthesia of SD rats by intraperitoneal injection,the skin in front of the cervical part was prepared and disinfected.A 1-cm longitudinal incision was made along the anterior medium of the rat’s cervical region to expose the trachea,and a 1mL syringe was used to puncture the trachea under the cricoid cartilage and inject BLM solution(4.5 mg·kg-1)slowly to replicate the PF animal model.2.Grouping and administration:Eighty SPF male SD rats were randomly pre-divided into 8 groups,with 10 rats in each group.Except for the sham group,the remaining 70 rats were induced PF by one-time intratracheal instillation of BLM.After 1week,the dead and low body weight rats were excluded,and the rats were formally grouped by random number method:The sham Group（SHAM）,model group（BLM）,AH Low dose group(AH-L,38.2 mg·kg-1·d-1),AH Medium dose group(AH-M,76.4 mg·kg-1·d-1),AH High dose group(AH-H,152.8 mg·kg-1·d-1),Artemisinin group(ART,33.3mg·kg-1·d-1),Hydroxychloroquine group(HCQ,43.1 mg·kg-1·d-1),and Prednisolone group(PDN,3 mg·kg-1·d-1),with 8 rats in each group.The drug was administered by daily gavage after randomization grouping and continued for 21 days.The SHAM and BLM group were administered with equivalent volume of physiological saline.3.The effect of AH in the treatment of bleomycin-induced pulmonary fibrosis in rats:the changes of body weight,general state and appearance behavior score were observed every week after treatment.Pulmonary function was tested at the end of the experiment.The lung index,liver index and kidney index were calculated.Blood routine and serum biochemistry were tested.Pathological sections of lung tissue were prepared and stained with HE to evaluate alveolitis（Szapiel method）,and masson staining pathological slides were used to evaluate pulmonary fibrosis（Ashcroft method）.Combined with the above indicators,the efficacy and safety of AH in the treatment of pulmonary fibrosis were verified.4.Study on the mechanism of AH in the treatment of bleomycin-induced pulmonary fibrosis in rats:Elisa was used to detect the contents of tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）,vascular endothelial growth factor（VEGF）,E-cadherin（E-Cad）,hydroxyproline（Hyp）,and Krebs von den Lungen-6（KL-6）in serum;Western Blot and q PCR were used to detect the protein and mRNA expression levels of molecules（transforming growth factor-β1（TGF-β1）,Smad2/3,P-Smad2/3,andα-actin（α-SMA））in the TGF-β1/Smad2/3 signaling pathway in lung tissue,and these indicators were used to preliminarily explore the possible mechanism of AH in the treatment of pulmonary fibrosis.Results:1.Compared with the SHAM group,the rats in the BLM group showed a decrease in body weight and an increase in appearance behavior scores within 1 week after modeling,and then gradually recovered from 2 weeks.Pulmonary function tests showed a decrease in dynamic compliance,response compliance,inspiratory capacity,tidal volume,and peak expiratory flow rate（P<0.05）,increased respiratory rate and functional residual capacity（P<0.05）,and the lung index was significantly increased（P<0.01）,liver and kidney index did not change significantly（P>0.05）;Routine blood test showed a slight increase in white blood cells,and serum biochemical tests showed normal levels of alanine aminotransferase,aspartate aminotransferase,creatinine,and urea（P>0.05）.The pathological examination of lung tissue showed large area of alveolar fusion and fibrosis changes,and the Szapiel score of alveolitis and Ashcroft score of pulmonary fibrosis were significantly increased（P<0.01）.The above indicators together suggested that the model was successfully replicated.Compared with the BLM group,the body weight of the AH group increased faster from week 2 to week 3,and the main effect of time on body weight change was statistically significant（P<0.01）;The appearance behavior score of AH group was significantly decreased,and the main effects of treatment and time on appearance behavior score were statistically significant（P<0.05）;Pulmonary function tests showed that AH had a slight improvement in dynamic compliance,response compliance,inspiratory capacity,tidal volume,peak expiratory flow rate,respiratory rate,and functional residual capacity（P>0.05）;The lung index decreased slightly,and the degree of decrease of lung index was positively correlated with AH dose（P<0.05）,liver and kidney index did not change significantly（P>0.05）;Blood routine test showed that AH could reduce the level of white blood cells.The changes of alanine aminotransferase,aspartate aminotransferase,creatinine and urea in serum biochemistry did not have clinical significance.The pathological examination of lung tissue showed that AH reduced the severity of lung fibrosis in a dose-dependent manner.More complete alveolar structures were observed in the lung tissue of the AH high-dose group,and the Szapiel score of alveolitis in AH-H group was significantly decreased（P<0.05）,fibrosis Ashcroft score decreased（P>0.05）.The results of these studies suggest that high-dose AH can safely and effectively delay the disease process of BLM-induced PF animal models by inhibiting specific inflammation and extracellular matrix deposition.2.Compared with the SHAM group,the serum levels of TNF-α,IL-6,KL-6,VEGF and Hyp in the BLM group were increased to varying degrees,while E-Cad was slightly lower than that in the SHAM group.The increase of VEGF was significantly different（P<0.01）;Western Blot showed that the expressions of TGF-β1,Smad2/3 and P-Smad2/3in lung tissue were increased,and q-PCR results also showed that the expression of Smad3 mRNA was increased,suggesting that the pathogenesis of this model involves TGF-β1/Smad2/3 signaling pathway.Compared with the BLM group,the levels of TNF-α,IL-6,KL-6,VEGF and Hyp were decreased in a dose-dependent manner in the AH group,and the levels of VEGF decrease significantly（P<0.01）,while the level of E-Cad was increased.Western Blot showed that the protein levels of TGF-β1,Smad2/3 and P-Smad2/3 in the lung tissue of each dose group of AH were decreased,and the protein expression ofα-SMA in the AH-H group was decreased.The results of q-PCR also showed that the expression of Smad3 mRNA was increased.AH can regulate the levels of pro-inflammatory and pro-fibrotic cytokines in the serum of rats with pulmonary fibrosis in a dose-dependent manner,and down-regulate the levels of key molecules in the TGF-β1/Smad2/3 signaling pathway,suggesting that the anti-pulmonary fibrosis effect of AH is related to the inhibition of the activation of TGF-β1/Smad2/3 signaling pathway.Conclusion1.AH can inhibit bleomycin-induced lung inflammation and extracellular matrix deposition,and safely and effectively delay the progression of pulmonary fibrosis.2.The anti-pulmonary fibrosis effect of AH is related to the inhibition of TGF-β1/Smad2/3 signaling pathway. |
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