Study On The Pharmacological Effect And Mechanism Of Xueshuantong Injection Against Bleomycin Induced Pulmonary Fibrosis In Rats

ObjectiveThe study used bleomycin induced pulmonary fibrosis rat model to observe the pharmacodynamic effect of Xueshuantong injection on the rat model,and based on the whole animal and cell experiments to conduct Exploratory research on its pharmacodynamic mechanism.The research of this topic provides theoretical and experimental basis for the clinical application of Xueshuantong Injection and the development of drugs for treating pulmonary fibrosis.Method1 Pharmacodynamic study of Xueshuantong injection on bleomycin induced pulmonary fibrosis in ratsWistar rats were induced by intratracheal injection of bleomycin(5 mg·kg-1).According to body mass,they were randomly divided into sham operation group,model group,pirfenidone group(50 mg·kg-1),Xueshuantong injection low,medium and high dose groups(27,54,81 mg·kg-1).Both sham operation group and model group were given the same volume of normal saline.Once a day,continuously administered for 7 days,14 days and 28 days,the general survival status and body mass of rats were observed,and the following indicators were detected:①lung coefficient,② lung function indicators,③lung imaging,④hematoxylin eosin staining(HE)and Masson staining(Masson staining,Masson)were used to observe the lung histopathology,⑤Enzyme linked immunosorbent assay(ELISA)was used to determine the expression levels of type I collagen(COL-I)and fibronectin(FN)in rat lung tissue.⑥ the expression levels of α-smooth muscle actin(alpha smooth muscle action,α-SMA)and COL-I were measured by immunofluorescence,⑦Western blot(WB)was used to determine the expression levels of α-SMA,COL-I,Vimentin(Vim)and E-cadherin(E-cad)in lung tissue of rats in each group.ELISA was used to determine the content of coagulation related factors thrombin antithrombin complex(TAT),soluble fibrin monomer complex(SFMC),and plasma prothrombin fragment 1+2(F1+2)in rat lung tissue.2 Exploring the Pharmacodynamic Mechanism of Xueshuantong InjectionMRC-5 cells were co cultured with thrombin(THR)and different concentrations of Panax Notoginseng saponins(PNS).The appropriate concentration and administration time were selected using the cell counting kit 8(CCK-8)method,and their effects on cell numbers were detected using high content analysis techniques.The levels of α-SMA,Vimentin,and E-Cad changes in cells were measured using RTPCR and Western Blot methods;The expression level of PAR-1 in cells was measured using RT-PCR and Western Blot methods;Using small interfering RNA(siRNA)interference technology,PAR-1 was silenced,and the effective component of Xueshuantong Injection,Panax notoginseng saponins,was used for intervention.The levels of α-SMA,Vimentin,and E-Cad in cells were measured using RT-PCR and Western Blot methods,and the changes in protein levels were observed to verify the results obtained.Result1 Pharmacodynamic study of Xueshuantong injection on bleomycin induced pulmonary fibrosis in ratsGeneral condition:The rats in the sham operation group were in a good mental state,active and active,and their body mass continued to increase normally.The rats in the model group were depressed and their hair color was dim.The overall survival status of rats in the low,medium and high dose groups of Xueshuantong Injection was better than that in the model group.Comparing the body mass of each group,it was found that compared with the model group,the body mass of rats in the pirfenidone group and the low,medium,and high dose groups of Xueshuantong injection were significantly higher than those in the model group at 7,14,21,and 28 days.Lung coefficient:Compared with the sham operation group,the lung coefficient of rats in the model group significantly increased.Compared with the model group,in the results on days 7,14,and 28,the lung coefficient of rats in the pirfenidone group and the low,medium,and high dose groups of Xueshuantong injection significantly decreased.Pulmonary function:Compared with the sham operation group,various pulmonary function indicators in the model group have undergone significant changes,mainly manifested by a significant decrease in tidal volume,a significant increase in airway resistance,and a significant decrease in dynamic lung compliance.Compared with the model group,in the results of 7,14,and 28 days,both the pirfenidone group,the Xueshuantong injection,and the high-dose group can increase tidal volume,reduce airway resistance,and increase dynamic lung compliance,Comparing the three-time points at the same time,the results at 7 days were significantly better than the results at 14 and 28 days.Lung Imaging:In the sham operation group,there was no abnormality in the lung CT,with clear lung markings,uniform transmittance,and no abnormal lines and patchy shadows.In the model group,the lung texture of rats was more disordered,with uneven transmittance,dense shadows,and blurred boundaries.Compared with the model group,the pulmonary texture clarity in the pirfenidone group and the Xueshuantong injection group was significantly improved,with uniform transmittance,fewer dense shadows,and no abnormal lines and shadows.Histopathology:Observe the lung tissue status of rats in each group.Compared with the sham operation group,the pathological changes in the model group were relatively severe,with the compensatory expansion of the alveoli,destruction of the bronchial mucosa,thickening of the alveolar septa in the surrounding lung tissue,infiltration of inflammatory cells,the proliferation of fibrous tissue,partial destruction of local structures in the lung tissue,and affected lung function.The effects of pirfenidone and Xueshuantong injection can significantly improve the severity of pulmonary fibrosis in model group rats,specifically manifested by reducing inflammatory cell infiltration,reducing pulmonary interstitial thickening,reducing alveolar wall widening,reducing fibrous tissue proliferation to varying degrees,and reducing histopathological scores.Expression of extracellular matrix:Western Blot results showed that the expression of α-SMA,Vimentin and COL-I increased and the expression of E-cad decreased in the model group compared with the sham operation group 7,14 and 28 days after modeling.The expression of α-SMA,Vimentin and COL-I decreased and the expression of E-cad increased in the pirfenidone group and Xueshuantong injection group.Collagen deposition:ELISA results showed that after 7,14,and 28 days of modeling,compared with the sham surgery group,the levels of COL-I and FN in the lung tissue of the model group rats were significantly increased.Compared with the model group,the levels of COL-I and FN in the lung tissue of rats in the pirfenidone group and the high dose Xueshuantong injection group were significantly reduced in the results of 7 days,14 days and 28 days.At the same time,the results of 7 days were significantly better than those of 14 days and 28 days when compared with three time points.The results of immunofluorescence showed that after 7,14,and 28 days of modeling,the expression of α-SMA and COL-I in the model group rats was significantly increased compared to the sham operated group.The expression of αSMA and COL-I in lung tissue of rats in pirfenidone group and Xueshuantong injection group decreased significantly.Coagulation factors:ELISA results showed that after 7,14,and 28 days of modeling,compared with the sham surgery group,the levels of TAT,F1+2,and SFMC in the lung tissue of the model group rats were significantly increased.Compared with the model group,there was no significant difference in the results of 7 days,14 days and 28 days in the pirfenidone group,and the relative content of SFMC in the lung tissue of the high dose Xueshuantong injection group decreased significantly at 7 days;At 14 days,the relative content of SFMC in the lung tissue of rats in the low,medium,and high dose groups of Xueshuantong injection significantly decreased;At 28 days,there was no significant difference between the groups.There was no significant difference in the relative content of TAT,F1+2 in the lung tissue of rats in each treatment group at days 7,14,and 28.2 Exploring the Pharmacodynamic Mechanism of Xueshuantong InjectionPNS inhibits THR induced proliferation of MRC-5 cells:Thrombin was used to stimulate MRC-5 cells,and safe concentrations of total saponins of Panax notoginseng were selected for intervention.Thrombin modeling can significantly induce MRC-5 cell proliferation.Compared with the thrombin group,total saponins of Panax notoginseng can inhibit the proliferation of MRC-5 cells induced by thrombin.Effect of PNS on THR induced in vitro PF model:An in vitro pulmonary fibrosis model based on THR stimulated fibroblasts MRC-5 cells:THR can promote abnormal proliferation of MRC-5 cells and increase α-SMA and Vimentin levels,while E-cad levels decrease.PNS can reduce the relative expression level of α-SMA and Vimentin and increase the relative expression level of E-cad.PNS regulates the anti PF effect of PAR-1:The experimental results show that the expression of PAR-1 increases after thrombin stimulation,while PNS can reduce the expression of PAR-1.By using siRNA silencing technology,the expression of PAR-1 was knocked down.After thrombin stimulation,the expression of α-SMA and Vimentin in the model group was significantly increased,while the expression of ECad was significantly reduced.The improvement effect of PNS on α-SMA,Vimentin,and E-Cad was not significant,indicating that Xueshuantong Injection regulates the expression of related proteins by regulating the target of PAR-1,thereby improving pulmonary fibrosis.Conclusion1 Xueshuantong Injection can improve the overall survival status of pulmonary fibrosis rats,improve lung coefficient and function,alleviate inflammatory cell infiltration and fibrous tissue proliferation,improve collagen deposition,and inhibit epithelial mesenchymal transformation,with anti pulmonary fibrosis effects.2 The anti pulmonary fibrosis effect of Xueshuantong Injection is related to the improvement of coagulation system abnormalities.3 The effective component of Xueshuantong Injection,total saponins of Panax notoginseng,can regulate the key coagulation target PAR-1 and exert anti pulmonary fibrosis effects.