| Objective: 1.To observe the effective of pirfenidone(PFD)on paraquat(PQ)toxic lung injury in mice.2.To observe the difference of arterial blood gas between paraquat poisoning rats and pirfenidone treatment rats at the same time node,the changes of arterial blood gas and other inflammatory factors in paraquat poisoning rats at different time nodes.3.To investigate the efficacy and mechanism of pirfenidone in the treatment of paraquat-induced pulmonary fibrosis in rats.Methods: 1.Thirty-two male C57 mice(SPF)(18-22g)were divided into four groups according to the random number table method,with eight mice in each group.They were divided into control group(NS group),PQ poisoning group(PQ group),low-dose PFD intervention group(PQ + 100 mg / kg PFD,PFD100group)and high-dose PFD intervention group(PQ + 200 mg / kg PFD,PFD200group).The PQ group and PFD intervention group were treated with a single intraperitoneal injection of 30 mg / kg PQ to establish a lung injury model,and the saline of the same dose was intraperitoneally injected into mice of NS group.1 hour later,the PFD intervention group was given different doses of PFD,while the PQ group and NS group were given the same amount of saline.Observe the general condition of the mice.After 24 hour,anesthesia was obtained.Collect the venous blood of the eyeballs,keep the supernatant after centrifugation,and measure the content of SAA1 by ELISA.Collect the alveolar lavage fluid,leave the supernatant after centrifugation,and measure the content of IL-17 by opening the chest cavity.Removing the lung tissue and leaving the left lung the upper lobe was stained with HE,Masson,and TUNEL.The lower lobe of the left lung was removed and the wet / dry weight ratio(W / D ratio)of the lung tissue was calculated.The right lung tissue was used to detect and α-Relative expression levels of TGF-β1,cTGF,EGF,SMA,RORγt mRNA and TGF-β1,α-SMA,IL-1β protein.2.Observe the difference in arterial blood gas between paraquat poisoned rats and pirfenidone-treated rats at the same time point,and the arterial blood gas situation of paraquat poisoned rats at different time points.Fifty-six male SD rats(SPF)(180-220g)were divided into 7 groups according to the random number table method,with 8 in each group.They were divided into control group(NS group),7d PQ poisoning group(7d PQ group),7d low-dose PFD intervention group(PQ + 100 mg / kg PFD,7d PFD100 group),and 7d high-dose PFD intervention group(PQ + 200 mg / kg PFD,7d PFD200 group),14 d PQ poisoning group(14d PQ group),14 d low-dose PFD intervention group(PQ + 100 mg / kg PFD,14 d PFD100 group),and 14 d high-dose PFD intervention group(PQ + 200 mg / kg PFD,14 d PFD200 group).PQ group and PFD intervention group were treated with a single intraperitoneal injection of 20 mg / kg PQ to establish a pulmonary fibrosis model,and the saline of the same dose was intraperitoneally injected into mice of NS group.After 1 hour,the PFD intervention group was orally administered with different doses of PFD,while the PQ group and the NS group were respectively orally administered with an equal amount of normal saline for once every 24 hours.Observing the general condition of rats.The materials were anesthetized after 7d and 14 d.Arterial blood was collected by puncture of the abdominal aorta,of which 1 ml of arterial blood was used for blood gas detection,and the supernatant was collected by centrifugation to detect the contents of COL1A1.The left upper lobe was subjected to HE staining,Masson staining,and TUNEL detection.The left lower lobe was removed and the wet / dry weight ratio(W / D ratio)of the lung tissue was calculated;the right lung tissue was used to detect protein expression levels of TGF-β1,Smad3,NF-κB,MMP2,MMP9,α-SMA and IL-1β.3.Thirty-two male SD rats(SPF)(180-220g)were divided into four groups according to the random number table method,with eight rats in each group.They were divided into control group(NS group),PQ poisoning group(PQ group),low-dose PFD intervention group(PQ + 100 mg / kg PFD,PFD100group)and high-dose PFD intervention group(PQ + 200 mg / kg PFD,PFD200group).).PQ group and PFD intervention group were treated with a single intraperitoneal injection of 20 mg / kg PQ to establish a pulmonary fibrosis model,and NS group was intraperitoneally injected with the same amount of normal saline.After 1 hour,the PFD intervention group was given different doses of PFD,while the PQ group,and the saline of the same dose was intraperitoneally injected into mice of NS group,and then once every 24 hours.Observe the general condition of rats.After 7 days,anesthesia was obtained.Arterial blood was collected by puncture of the abdominal aorta,and the supernatant was collected by centrifugation,and the contents of SAA1,TNF-α,and COL1A1 were detected.The lung tissue was opened to remove the lung tissue,and the left upper lobe was subjected to HE staining,Masson staining,and TUNEL detection.In the left lower lung tissue,the wet / dry weight ratio(W/ D ratio)of the lung tissue was calculated.The right lung tissue was used to detect the expression levels of TGF-β1,Smad3,Wnt1,β-catenin,α-SMA mRNA and protein.Results: 1.There were no deaths in each group.In the experiment of PFD treatment of PQ poisoned mice,compared with the NS group,HE,TUNEL,and Masson staining in the PQ group showed that the lung tissue in the PQ group was more severely damaged,apoptotic cells and collagen fibers in the lung tissue were significantly increased.The wet/dry weight ratio was significantly increased,and the relative expression levels of TGF-β1,cTGF,EGF,α-SMA,and RORγt mRNA in lung tissue were significantly increased,and TGF-β1,α-SMA,IL-1β protein was significantly increased in lung tissue.The levels of IL-17 in BALF and SAA1 in serum were significantly increased(all p < 0.05).After PFD treatment,lung tissue damage was reduced,apoptotic cells and collagen fiber in lung tissues were significantly reduced,wet/dry weight ratio of lung tissues was significantly reduced.The expression levels of TGF-β1,cTGF,EGF,α-SMA,and RORγt mRNA and TGF-β1,α-SMA,and IL-1β protein in lung tissue were significantly reduced.The level of SAA1 in serum and IL-17 in BALF were decreased(all p < 0.05).In addition,the efficacy in PFD200 is better than in PFD100(p < 0.05).2.There were 3 rats dead in the 14 d PQ group,and no deaths were found in the other groups.In the experiment of observing the changes of blood gas in different groups of rats,the pathological results showed that the lung tissue of the PQ group was more severely damaged,the inflammatory cells and red blood cells exuded significantly increased,the lung interval was significantly thickened,and the lung tissue was apoptotic.The formation of cells and collagen fibers increased significantly,the wet-to-dry weight ratio of lung tissue increased significantly.The PH,SaO2 and PaO2 were decreased,and the Pa CO2 was increased(all p < 0.05).And the expression level of TGF-β1,Smad3,NF-κB,MMP2,MMP9,α-SMA,and of IL-1β protein was significantly increased(all p < 0.05).The level of COL1A1 in serum increased significantly(p < 0.05).After PFD treatment,pathological results showed that lung tissue damage was reduced.Inflammatory cells and red blood cells exuded were significantly reduced,and lung space was significantly narrower.The apoptotic cells and collagen fibers in the lung tissue were significantly reduced.The ratio of wet-to-dry weight of the lung tissue was reduced(p < 0.05).The levels of COL1A1 in the serum and TGF-β1,Smad3,NF-κB,MMP2,MMP9,α-SMA and IL-1β protein in the lung tissue were significantly reduced(all p <0.05).The PH,SaO2 and PaO2 were increased,but the Pa CO2 was decreased(all p < 0.05);the PFD200 group had better effective than the PFD100 group.In the PQ group of different batches of rats,contrast with the 7d PQ group,the HE staining results of the 14 d PQ group showed that the lung tissue was more severely damaged,the inflammatory cells and red blood cells exuded significantly,the lung interval was significantly thicker.The apoptotic cells and collagen fibers were significantly increased showed by TUNEL and Masson staining.The W/D of lung tissue was significantly increased(p < 0.05).The level of COL1A1 in serum and TGF-β1,Smad3,NF-κB,MMP2,MMP9,α-SMA and IL-1β protein in lung tissue were significantly increased(all p < 0.05).The PH,SaO2 and PaO2 were decreased while the Pa CO2 was increased(all p <0.05).3.There were no deaths in each group.In experiments to study the mechanism of PFD for paraquat-induced pulmonary fibrosis,compared with the NS group,the lung tissue in the PQ group was more severely damaged,inflammatory cells and red blood cells exuded significantly,the lung interval was significantly thickened,and the lung tissues were withered.The formation of dead cells and collagen fibers increased significantly,the wet-to-dry weight ratio of lung tissue increased significantly.The relative expression of TGF-β1,Smad3,Wnt1,β-catenin,and α-SMA mRNA and protein and the levels of SAA1,TNF-α,and COL1A1 in serum were significantly increased(all p < 0.05).After PFD treatment,the degree of lung tissue damage was reduced,inflammatory cells and red blood cells exuded decreased.Lung interval was narrower.Apoptotic cells and collagen fibers formed in lung tissues were significantly reduced.Wet-dry weight ratio of lung tissues was reduced(p < 0.05),and the expression levels TGF-β1,Smad3,Wnt1,β-catenin,α-SMA mRNA and protein were reduced of lung tissue(all p < 0.05).the levels of SAA1,TNF-α and COL1A1 in serum were reduced,too(all p < 0.05).Moreover,compared with PFD100,the degree of lung tissue destruction,inflammatory cells and red cells exudation,the thickness of the lung interval were reduced.The number of apoptotic cells and collagen fibers was reduced.The ratio of wet/dry weight of lung tissue was smaller.The expression levels of TGF-β1,Smad3,Wnt1,β-catenin,α-SMA mRNA and protein and the contents of SAA1,TNF-α and COL1A1 in the lung tissue were significantly decreased(all p < 0.05).The PFD200 group was more effective than the PFD100 group.Conclusion: 1.PFD can reduce lung injury in mice induced by PQ poisoning.2.PQ poisoned rats,with time,the higher the degree of pulmonary fibrosis,the worse the ability to use oxygen;PQ poisoned rats after PFD treatment,the ability to use oxygen was increased and the pulmonary fibrosis was increased.3.PFD may reduce PQ-induced pulmonary fibrosis in rats via TGF-β1 / Smad3 and Wnt1 / β-catenin signaling pathways. |
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