| Objective:The effect of Maimendong decoction on pulmonary fibrosis induced by bleomycin in rats was studied.The changes of lung histopathology and various indexes of rats in each group were observed,and the intervention effect and mechanism of Maimendong decoction on pulmonary fibrosis rats were studied through TLR4/NF-κB signaling pathway.Methods:Ninety male SD rats with SPF grade were purchased and randomly divided into 6 groups with 15 rats in each group,which were blank group,model group,prednisone group,low dose group,medium dose group and high dose group.Except blank group,75 rats in other groups were injected with bleomycin(BLM)(5mg/Kg)to establish pulmonary fibrosis model,and began gavage 24 hours after modeling.The blank group was given 10.0ml/(kg·d)normal saline by gavage,the model group was given 10.0ml/(kg·d)normal saline by gavage,the prednisone group was given 3.0mg/(kg·d)prednisone suspension by gavage,and the low dose group was given Maimendong decoction by gavage as crude drug 0.55g/ml/100 g.The medium dose group was given intragastric administration of gopogon decoction as crude drug1.1g/ml/100 g,and the high dose group was given intragastric administration of gopogon decoction as crude drug 2.2g/ml/100 g,once a day at the same time.14 and 28 days after modeling,lung tissue and serum samples of rats in each group were collected,and the lung histopathological changes were observed by HE staining and Masson staining.The expression of TLR4,NF-κ B,TNF-α,interleukin-6,Cytokeratin and N-cadherin in rat lung tissue was determined by immunohistochemical method.The levels of TNF-α and IL-6 in serum were determined by ELISA,and the m RNA expression levels of Cytokeratin,N-cadherin,TLR4 and NF-κ B in lung tissue were determined by real-time quantitative PCR.Results:1.HE and Masson dyeing:14 days after modeling,compared with blank group,HE and Masson staining of model group showed pathological abnormalities of rat lung tissue,significant damage of lung tissue structure and structural disorder,a large number of inflammatory cells infiltrated,alveolar wall thickened,and blue collagen fibers significantly hyperplasia.After drug intervention,compared with model group,alveolar inflammation and pulmonary fibrosis of rats in each treatment group were reduced.28 days after modeling,compared with the blank group,HE and Masson staining showed that the lung tissue structure of rats in the model group was severely damaged and the structure was seriously disturbed.Compared with the 14 th day,the number of inflammatory cells was more,the number of atrophic alveoli was more,and the damage was more serious.The alveoli were almost covered by fibrils,with large amount of collagen deposition and blue collagen fiber hyperplasia.Lung tissue injury and fibrosis were alleviated in all treatment groups.2.Immunohistochemical test results :TLR4 and NF-κ B protein expression:compared with blank group,the expression of TLR4 and NF-κB in lung tissue of model group increased 14 and 28 days after modeling(P<0.05),with statistical differences;Compared with model group,the expression of TLR4 and NF-κB in lung tissue of rats in prednisone group and ophiopagon decoction groups decreased 14 and 28 days after treatment(P<0.05),with statistical differences.Cytokeratin and N-cadherin protein expression: Compared with blank group,14 and 28 days after modeling,the protein expression of Cytokeratin was decreased in the lung tissue of model group,while the protein expression of N-cadherin was increased(P<0.05),showing statistical differences.At 14 days after treatment,compared with model group,the protein expression of Cytokeratin increased in prednisone group,medium dose group and high dose group,while the protein expression of N-cadherin decreased(P<0.05),showing statistical differences.Compared with model group,the protein expressions of Cytokeratin and N-cadherin in lung tissue of rats in low dose Maimendong decoction group were not different(P>0.05).On day 28 after treatment,compared with model group,the protein expression of Cytokeratin increased in prednisone group and Ophiopindon decoction groups,while the protein expression of N-cadherin decreased(P<0.05),showing statistical differences.Protein levels of TNF-α and IL-6: Compared with blank group,the expressions of TNF-α and IL-6 in lung tissue of model group were increased14 and 28 days after modeling(P<0.05),with statistical differences.At 14 days after treatment,compared with model group,the expressions of TNF-α and IL-6 protein in lung tissues of rats in prednisone group,Maimendong decoction medium dose group and Maimendong decoction high dose group were decreased(P<0.05),with statistical differences.Compared with model group,the expression of TNF-α and IL-6 protein in lung tissue of rats in low dose Maimendong decoction group had no difference(P>0.05).At 28 days after treatment,compared with model group,the expressions of TNF-α and IL-6protein in lung tissues of rats in prednisone group and each dose Maimendong decoction groups were decreased(P<0.05),with statistical differences.3.Quantitative real-time PCR results: TLR4 and NF-κB m RNA expression: 14 and 28 days after modeling,compared with blank group,TLR4 and NF-κB m RNA expression in lung tissue of model group increased(P<0.05),with statistical differences;14 days after drug intervention,TLR4 m RNA expression in lung tissue of rats in prednisone group and medium and high dose groups of ophiopagon decoction decreased(P<0.05),while NF-κB m RNA expression in lung tissue of rats in prednisone group and Maimendong decoction groups decreased(P<0.05),with statistical differences.Compared with model group,TLR4 m RNA expression was not significantly different in lung tissues of Maimendong decoction low-dose group(P>0.05).After 28 days of drug intervention,the m RNA expressions of TLR4 and NF-κB in lung tissues of rats in model group,prednisone group and Maimendong decoction groups were decreased(P<0.05),with statistical differences.m RNA expression of Cytokeratin and N-cadherin: At 14 and 28 days after modeling,compared with blank group,the m RNA expression of Cytokeratin was decreased and the m RNA expression of N-cadherin was increased in the lung tissue of model group(P<0.05),showing statistical differences.At 14 and 28 days after drug intervention,compared with the model group,The m RNA expression of Cytokeratin was increased in prednisone group and Maimendong decoction groups,while the m RNA expression of N-cadherin was decreased(P<0.05),which were statistically significant differences.4.ELISA test results: 14 and 28 days after modeling,compared with blank group,serum TNF-α level in model group was increased(P<0.05),with statistical differences;At 14 days after treatment,compared with model group,serum TNF-α level in prednisone group and Maimendong decoction high-dose group decreased(P<0.05),with statistical difference;Compared with model group,there was no significant difference in serum TNF-α level between low dose group and medium dose group(P>0.05).At 28 days after treatment,the serum TNF-α level in prednisone group was decreased compared with model group(P<0.05),and the difference was statistically significant.Compared with model group,there was no significant difference in the serum TNF-α level of rats in different dosage groups of Maimendong decoction(P>0.05).Compared with blank group,serum IL-6 level in model group was increased(P<0.05),with statistical difference.14 days after treatment,compared with model group,serum IL-6 level in prednisone group and Maimendong decoction groups decreased(P<0.05),with statistical differences;At 28 days after treatment,compared with model group,serum IL-6 level of rats in prednisone group and Maimendong decoction high-dose group was significantly decreased(P<0.05),with statistical difference,while serum IL-6 level of rats in Maimendong decoction low-dose and medium-dose groups was not significantly different in comparison model group(P>0.05).Conclusion(s):1.In the bleomycin-induced rat model of pulmonary fibrosis,Maimendong decoction can reduce the expression level of N-cadherin and increase the expression level of Cytokeratin,inhibit the EMT process,and improve the pulmonary fibrosis.2.In the bleomycin-induced rat model of pulmonary fibrosis,Maimendong decoction can reduce the levels of TNF-αand IL-6 in the lung tissue and serum of rats,thereby reducing lung inflammation and delaying the progression of pulmonary fibrosis.3.By detecting the gene and protein of TLR4 and NF-κ B in bleomycin-induced pulmonary fibrosis model,it is speculated that Maimendong decoction may improve pulmonary fibrosis in rats by regulating TLR4-NF-κ B signaling pathway. |
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