Mechanism Of Dahuang Zhechong Pill On Pulmonary And Hepatic Fibrosis Through P38MAPK/TGF—?1 Pathway Was Discussed Based On The Theory Of “Treating Different Diseases With The Same Method”

ObjectiveAt present,pulmonary fibrosis is a common and serious disease worldwide,and its pathogenesis has not been fully elucidated.In this world,there is no effective treatment drugs and means.Liver fibrosis in the process of chronic liver disease,the pathological mechanism is complex,its persistence will gradually form cirrhosis,causing serious complications.Similarly,there has not been a very clear curative effect of drugs for its treatment.Although they are different diseases,they have something in common from the perspective of traditional Chinese medicine and Western medicine.In recent years,traditional Chinese medicine has shown advantages in the treatment of refractory diseases such as organ fibrosis.Therefore,based on the theory of “treating different diseases at the same method”,this study took silicosis fibrosis and liver fibrosis as the research objects,and intervened them with Dahuang Zhechong Pill(DHZCP),a classical prescription in treatise on Febrile Diseases and miscellaneous diseases We hope that we can explain the pathological mechanism of the two from macro and micro perspectives,and reveal the mechanism of DHZCP in the treatment of pulmonary and hepatic fibrosis.MethodsPart 1: Theoretical research1.This study discusses the thought of “treating different diseases with the same method” in “Treatise on Cold Pathogenic and Miscellaneous Diseases(Shanghan Zabing Lun)”,and points out how to treat them according to syndrome differentiation when the prescriptions and syndromes are corresponding,the pulse and syndrome are consistent,and the diseases have different causes,locations and conditions.In addition,some classic prescriptions are summarized and analyzed,and the thought of“treating different diseases with the same method” embodied in them is analyzed respectively.2.Using the method of literature research,in-depth analysis of the pathogenesis of pulmonary fibrosis and liver fibrosis and their common points,to provide a theoretical basis for DHZCP in the treatment of pulmonary fibrosis and liver fibrosis.Part 2: Experimental research1.72 SPF Kunming mice,60 mice were randomly divided into model control group,DHZCP high-dose group(1.560 g/kg),medium-dose group(0.780 g/kg),low-dose group(0.390 g/kg)and positive control group,with 12 mice in each group.The other 12 uninfected mice served as blank control group.At 7 and 28 days after administration,6 mice in each group were killed.Serum was collected to detect the contents of HYP,TNF-?,IL-1? and IL-6.Lung tissue was taken out to observe the organ coefficient and pathological observation.The expressions of p38MAPK/TGF-?1 pathway related proteins and m RNA were detected by Western blot and RT-PCR.2.48 SPF Kunming mice,were randomly divided into 6 groups at the beginning of the experiment: model control group,high-dose Dahuang Zhechong Pill group(1.560 g / kg),medium dose Dahuang Zhechong Pill group(0.780 g / kg),low-dose Dahuang Zhechong Pill group(0.390 g / kg),positive control group and blank control group,8 mice/each group.The mice except for the blank control group were intraperitoneally injected with CCl4(5 ml/kg/time)to establish the mouse model of liver fibrosis.Two months after administration,the serum was collected to detect the contents of TNF-?,IL-1?,IL-6,ALT and AST.The liver tissue was taken out to observe the organ coefficient and pathological observation.The expressions of p38MAPK/TGF-?1 pathway related proteins and m RNA were detected by Western blot and RT-PCR.3.MH-S cell line was used as the model of pulmonary fibrosis in vitro.Si O2suspension(40 ug/ml)was used to establish the model.DHZCP containing serum(15%)and NF-? B inhibitor PDTC(4 mm)were used to intervene.Cell morphology was observed under microscope.Cell activity was detected by CCK8 kit.The contents of TNF-?,IL-6 and IL-1? in cell supernatant were detected by ELISA kit.The expressions of p38 MAPK/TGF-?1 pathway related proteins were detected by Western blot and immunofluorescence staining.4.HSC-T6 cell line was used as the model of liver fibrosis in vitro.TGF-?(10ng/ml)was used to establish the model.After the intervention with DHZCP containing serum(15%)and NF-?B inhibitor PDTC(4 mm),cell morphology was observed.CCK8 kit was used to detect cell activity.Similarly,ELISA kit was used to detect the contents of TNF-?,IL-6,IL-1?,ALT and AST in cell supernatant.Western blot and immunofluorescence staining were used to detect the protein expression of p38 MAPK/TGF-?1 pathway.ResultsPart 1: Results of theoretical research1.This paper mainly summarizes and discusses the theory and some related prescriptions of “treating different diseases with the same method” in “Shanghan Zabing Lun”.It is pointed out that the treatment is the same when the prescription and syndrome are corresponding,the pulse and syndrome are consistent,and different diseases such as different etiology,disease location and disease trend are faced,because the essence of the disease is identified through the disease appearance,and the essence of the syndrome is the same.Therefore,different diseases can be treated with the same method.2.From the perspective of traditional Chinese medicine,the pathogenesis of pulmonary and hepatic fibrosis has the common characteristics of “deficiency and stasis”,the essence of which is deficiency and blood stasis;According to modern medicine,the common pathological feature of pulmonary and hepatic fibrosis is the process of chronic inflammation and fibrosis.Pathogenic factors lead to cell damage,abnormal signal transduction pathway,abnormal deposition of ECM and changes of tissue structure.Therefore,the two diseases of pulmonary fibrosis and hepatic fibrosis have the same pathogenesis and have the basis of treating different diseases with the same treatment.It is feasible to treat pulmonary and hepatic fibrosis with DHZCP.Part 2: Results of experimental research1.Effect of DHZCP on organ coefficient of mice with pulmonary and hepatic fibrosisFrom the organ coefficient of animal experiment,it can be seen that the organ coefficient of lung and liver fibrosis mice model group is significantly higher than that of blank control group.The high dose(1.560 g/kg)of DHZCP was the most obvious,which indicated that the high dose of DHZCP could significantly reduce the organ coefficient,which might be related to the inhibition of collagen deposition.2.Effect of DHZCP on pathological results of pulmonary and hepatic fibrosis in miceIn the experiment of pulmonary fibrosis,inflammatory cell infiltration,degeneration and necrosis,nuclear pyknosis,alveolar septum thickening and fibrous tissue hyperplasia were observed in the lung tissue of the model group.The degree of fibrosis was more serious at 28 days than at 7 days.After treatment,the improvement was most obvious in the DHZCP high-dose group,but there was still a small amount of inflammatory cell infiltration and alveolar structure recovered to a great extent,followed by the middle dose group,then the low-dose group and the positive control group.In the liver fibrosis experiment,the liver tissue cells in the model group swelled significantly,inflammatory cells infiltrated and gathered,there was also degeneration or necrosis,nuclear pyknosis,fragmentation or dissolution disappeared,capsule thickening was obvious,and fibrous tissue proliferation was visible.The three dose groups of DHZCP and positive control group had different degrees of improvement.The liver tissue structure of high dose group of DHZCP was complete,no obvious fibrosis,no obvious thickening of capsule,which was significantly improved compared with the model group;the degeneration and necrosis of cells,infiltration of inflammatory cells and swelling of liver parenchymal cells were still found in the middle dose group,which were the most obvious improvement in the two groups,without obvious fibrosis Change.The results showed that the high(1.560 g/kg)and medium(0.780 g/kg)dose groups of DHZCP had a good inhibitory effect on liver fibrosis,and showed a certain degree of anti fibrosis effect.3.Effect of DHZCP on cytokines of pulmonary and hepatic fibrosisIn vivo and in vitro experiments of pulmonary fibrosis,HYP,TNF-?,IL-1? and IL-6 in the model control group were significantly higher than those in the blank control group,while the high,medium and low dose groups of DHZCP had a decreasing effect on these indexes,but only the high dose group of DHZCP showed a stable decreasing effect,followed by the medium dose group.In the experiment of liver fibrosis in vivo and in vitro,DHZCP not only significantly reduced TNF-?,IL-1? and IL-6,but also inhibited ALT and AST.These results indicate that DHZCP has a good anti-inflammatory effect on pulmonary and hepatic fibrosis,can delay the process of fibrosis to a certain extent,and can improve liver function indexes.4.Effect of DHZCP on cell viability of MH-S and HSC-T6The cell viability of the two cell lines in the model control group was significantly lower than that in the blank control group.After the intervention of DHZCP containing serum and PDTC,the cell viability was improved in varying degrees.The combination of DHZCP containing serum and PDTC had the best effect,followed by DHZCP containing serum group and PDTC group.5.Effect of DHZCP on apoptosis of MH-S and HSC-T6 cellsThe apoptosis of the two cell lines in the model control group was significantly higher than that in the blank control group.After the intervention of DHZCP containing serum and PDTC,the apoptosis was reduced in varying degrees.The combination of DHZCP containing serum and PDTC had the best effect,followed by DHZCP containing serum group and PDTC group.The effect was consistent with the trend of cell viability.6.Effect of DHZCP on p38MAPK/TGF-?1 pathway protein and m RNAIn vivo,the protein and m RNA expression levels of p38 MAPK,NF-?B p65,TGF-?1,?-SMA,Smad2 and Smad3 were significantly increased in the lung fibrosis mice stimulated by Si O2 and liver fibrosis mice stimulated by CCL4.The expression level of Smad7 decreased,indicating that stimulation successfully activates p38MAPK/TGF-?1 signaling pathway,induces inflammatory response,starts fibrosis and activates Smad signal downstream.After treatment with DHZCP,the protein and m RNA levels of p38 MAPK,NF-?B p65,TGF-?1,?-SMA,Smad2 and Smad3 were significantly reduced,while Smad7 increased significantly.It suggested that DHZCP could down regulate p38 MAPK,NF-?B p65,TGF-?1,?-SMA,which could inhibit inflammation and reduce collagen deposition in the upstream.It also down regulated the expression of TGF-?1,Smad2 and Smad3,and up regulated Smad7,suggesting that DHZCP could inhibit TGF-?1,and regulate the signal pathway of Smad in the downstream,and effectively inhibit the occurrence of fibrosis.In vitro,PDTC had no significant effect on p38 MAPK.However,PDTC pretreatment could down regulate the expression of NF-?B p65 by reducing the activation of NF-?B,making the end of the pathway unable to receive stimulation signals,thus inhibiting the release of TNF-?,IL-6,IL-1? and the occurrence of inflammation.When treated with DHZCP containing serum,in addition to down regulating the expression of NF-?B p65 and blocking the TGF-?1/Smad signaling pathway,the expression of p38 MAPK was also significantly down regulated.It is suggested that DHZCP can inhibit inflammation and downstream TGF-?1/Smad signaling pathway by blocking the activation of NF-?B,and can also inhibit p38 MAPK in the upstream of NF-?B.DHZCP can control the upstream inflammatory reaction with multiple targets,which is more effective than PDTC alone in inhibiting NF-?B,which is also in line with the rule of multi-target treatment of diseases in traditional Chinese medicine.The inhibitory effect of DHZCP on silicosis fibrosis and liver fibrosis is related to the regulation of p38 MAPK/TGF-?1 signaling pathway.Conclusion1.This study discusses the idea of “treating different diseases with the same method” in “Shanghan Zabing Lun”,and concludes that whether there are corresponding prescriptions and syndromes for different diseases,corresponding pulse and syndrome,or different etiologies,disease location,disease situation,disease nature and symptoms,if the pathogenesis is the same,then “treating different diseases with the same method” can be achieved.2.The basic pathogenesis of pulmonary and hepatic fibrosis is deficiency and blood stasis.DHZCP has the effect of breaking blood,removing blood stasis and dredging collaterals,nourishing Yin,nourishing blood and moistening dryness,which is exactly the common pathogenesis of the two.Although they are “different diseases”,they have the same pathogenesis,which is the theoretical basis of treating pulmonary and hepatic fibrosis with DHZCP.3.DHZCP can reduce the organ coefficient of mice with pulmonary and hepatic fibrosis.4.DHZCP can inhibit TNF-?,IL-1?,IL-6 in serum of mice with pulmonary and hepatic fibrosis and supernatant of MH-S cells and HSC-T6 cells.It can inhibit inflammation.5.DHZCP can reduce the content of HYP in serum of mice with pulmonary fibrosis,and reduce the degree of pulmonary fibrosis.6.DHZCP can reduce the content of ALT and AST in serum of liver fibrosis mice and improve the liver function.7.DHZCP can improve the cell viability of MH-S cells and HSC-T6 cells.8.DHZCP can inhibit the protein and m RNA expression of p38 MAPK,NF-?B p65,?-SMA,TGF-?1,Smad2,Smad3,Smad7 in lung and liver tissues and MH-S and HSC-T6 cells,but increase Smad7,which indicates that the inhibitory effect of DHZCP on lung and liver fibrosis is related to the regulation of p38 MAPK/TGF-?1signaling pathway.It was dose-dependent in the range of 0.390-1.560 g/kg.Therefore,from the micro level,the treatment basis of DHZCP on pulmonary and hepatic fibrosis is related to p38MAPK/TGF-?1 signaling pathway.