Astragaloside ? Regulates TGF-?1/Smad Signaling Pathway Through PPM1A To Reduce Silicosis Injury And Fibrosis

Background and significanceSilicosis,it is a kind of disease controlled by the diffuse nodular fibrosis of lungs result from long-term exposure to the dust containing a large amount of free silica,which finally damages the function of lung,resulting in the respiratory failure and even death.More and more patients with silicosis in China now.Due to the lack of effective occupational protection measures,the incidence of silicosis remains high.In western developed countries,occupational health institutions are also troubled by the same problem.However,the silicosis pathogenesis is not so clear that lack of effective therapeutic drugs in medicine.Therefore,it is necessary to study and explore the pathogenesis of silicosis and new prevention and treatment methods.TGF-?1 is a powerful fibrotic cytokine.There are so many researches indicated that TGF-?1 can take part in the processes of multiple biology,such as cell proliferation,migration,differentiation,extracellular matrix(ECM)transformation,and immune regulation in related fibrotic diseases.As an important regulatory factor,TGF-?1 plays a key role in tissue repair and fibrosis.A large number of studies have confirmed that the effect of TGF-?1 in activating fibrosis is mediated by the signaling pathway of Smads.It is clear that the signaling pathway of TGF-?1/Smad takes an essential part in pulmonary fibrosis.Protein phosphatase magnesium-dependent1A(PPM1A),also known as Protein Phosphatase 2C(Alpha,PP2C?),is a member of the serine/threonine protein phosphatase family,which is It is considered to be a necessary negative regulator in the pressure response pathway of eukaryotic cells.Studies have found that PPM1A(PP2C?)can specifically dephosphorylate activated p-Smad2/Smad3 protein,and PPM1A can significantly reduce the phosphorylation level of Smad2/3 induced by constitutively activated rat T?RI.That is to say,the activiation of TGF-?1/Smads signaling pathway is inhibited by PPM1A.Astragaloside?(ASV)is one of the key substances for Astragalus.Over the past decade,ASV has shown potential cardioprotection and immune enhancement through in vitro and in vivo experiments.Recently,research on the pharmacological effects of ASV has been extensively and intensively conducted.Studies have shown that ASV can put an active effect on focal cerebral ischemia/reperfusion,cardiovascular disease,lung disease,liver cirrhosis and diabetic nephropathy.More and more evidence supports the use of ASV in the treatment of organ fibrosis,inflammation,oxidative stress and apoptosis.Our previous experiments confirmed that ASV has a protective effect on silicosis injury and fibrosis.In view of the above theoretical basis,we believe that the low expression of PPM1A leads to the continuous phosphorylation of Smad3,which in turn causes the high expression of TGF-?1 and promotes the progression of silicosis;ASV regulates the expression of PPM1A to inhibit the continuous phosphorylation of Smad3,thereby delaying the development of silicosis.Rat lung fibrosis.We will further clarify the relevant mechanisms and possible signal pathways of the protective effect of ASV silicosis,and provide new diagnostic markers and therapeutic targets for the biological targeted therapy of silicosis,which may help improve the prognosis.Part ? Protective Effect Of Astragaloside ?(ASV)On Silicosis Injury And FibrosisObjectiveThe aim of this study was to investigate the effect of Astragaloside ? on silicosis injury and fibrosis.Materials and Methods1.Animal experiment:SiO2 treatment method to construct a silicosis rat model,divided into control group,ASV group,silicosis group and experimental group(silicosis+ASV)group.After relevant treatment,the histological changes of each group were determined by HE staining and Masson staining.Next,perform RT-PCR and IHC to detect fibrosis markers type ? collagen,fibronectin 1 and ?-smooth muscle actin.2.Cell experiment:In vitro SiO2 stimulates embryonic lung fibroblasts to construct a silicosis cell model.RT-PCR,Western blot and immunofluorescence were performed to detect the expression of fibrosis markers type ? collagen,?-smooth muscle actin and fibronectin 1.Results1.HE staining and Masson staining confirmed that the silicosis rat model was successfully constructed,and the silicosis injury and fibrosis were reduced after ASV treatment.There are abnormally high expressions of type ? collagen,?-smooth muscle actin,fibronectin 1 in the lung tissue of the silicosis rat model;after treatment with astragaloside ?,the expression of fibrosis markers was significantly lower than that of the silicosis group.2.In vitro cell experiments also confirmed that astragaloside ? can reduce the expression of fibrosis markers type ? collagen,?-smooth muscle actin,fibronectin 1.SummaryAstragaloside ?(ASV)can alleviate lung injury and fibrosis in silicosis rats;in vivo and in vitro experiments have confirmed that ASV can reduce silicosis-related protein collagen ?(collagen ?),?-smooth muscle actin(?-SMA),fibronectin 1(FN1)expression.Part ? Astragaloside ? Inhibits Inflammation and Oxidative Stress by Regulating The TGF-?1/Smad Signaling Pathway to Play a Protective Role in SilicosisObjectiveTo explore the pathophysiological processes involved in the protective effect of astragaloside ? on silicosis and to study the effects of astragaloside ? on TGF-?1/Smad signaling pathway.Materials and MethodsThe silicosis rat model was constructed,and the experimental animals were divided into control group,ASV group,silicosis group and experimental group(silicosis+ASV).Cell counting method was used to detect the recruitment of inflammatory cells in alveolar lavage fluid,and ELISA method was used to detect oxidative stress and inflammation.Factor situation.RT-PCR and Western blot were used to detect the expression of TGF-?1/Smad pathway related proteins in each group.Results1.Oxidative stress,inflammatory cell recruitment and inflammatory factor levels in silicosis are increased significantly in some extent.ASV can help the oxidative stress,inflammatory cell recruitment and inflammatory factor reduce the levels in lung tissue of silicosis rats;2.TGF-?1/Smad pathway is abnormally activated in silicosis.ASV could not decrease the expression of Smad2 and Smad3 related genes in TGF-?1/Smad signaling pathway,but restrain the behavior of TGF-?1/Smad pathway by affecting the phosphorylation of Smad2 and Smad3.SummaryASV does not reduce the expression of genes related to the TGF-?1/Smad signaling pathway,but dephosphorylates pSmad2 and pSmad3 to partially inhibit the activity of the TGF-?1/Smad pathway in silicosis tissues,reducing oxidative stress and inflammation.Part ? Astragaloside ? Regulates TGF-?1/Smad Signaling Pathway by Regulating the Expression of PPM1AObjectiveTo explore the effect of Astragaloside ? downstream target gene PPM1A on TGF-?1/Smad signaling pathway.Materials and MethodsThe blot in western was took to find the impact of Astragaloside ? for the facial expression of PPM1A in silicosis tissues and cells.The concentration gradient and time gradient of Astragaloside ? were tested to clarify its regulatory effect on PPM1A.The si-PPM1A vector was constructed and divided into si-Control and si-PPM1A groups.The two groups were divided into control,SiO2,SiO2+ASV subgroups,and the expression of PPM1A,lung fibrosis indicators,and TGF-?1/Smad signaling pathway.Results1.In vivo and in vitro experiments confirmed that ASV promotes the expression of PPM1A and reduces the expression of p-Smad2/3 in silicosis.2.In vitro experiments confirmed that the expression of PPM1A in silicosis cell model is concentration-dependent and time-dependent on ASV.3.The expression of p-Smad2/3 was significantly negatively correlated with PPM1 A.4.After silencing PPM1 A,the effects of astragaloside ? on Smad2/3 phosphorylation and downstream fibrotic factors were weakened,indicating that astragaloside ? may regulate Smad2/3 phosphorylation and downstream signal factors through PPM 1 A.SummaryAstragaloside ? may regulate the TGF-?1/Smads signaling pathway through PPM1A-mediated regulation of Smad2/3 phosphorylation to play a protective role in silicosis.ConclusionWe have successfully constructed a silicosis model in vivo and in vitro.We found that Astragaloside ?(ASV)can alleviate lung injury and fibrosis in silicosis rats;in vivo and in vitro experiments have confirmed that ASV can reduce silicosis-related protein type ? collagen(collagen ?),Fibronectin 1(FN1)and?-smooth muscle actin(?-SMA)expression.ASV can reduce oxidative stress,inflammatory cell recruitment and cytokine levels in silicosis tissue,ASV plays an anti-silicotic fibrosis impact by inhibiting the TGF-?1/Smad signaling pathway.Other deep researches have shown that PPM1A is low in silicosis and p-Smad2/3 is high;ASV can promote PPM1A expression and reduce p-Smad2/3 high expression;PPM1A expression is positively correlated with ASV treatment time and concentration;at the same time p-Smad2/3 Expression is negatively correlated with ASV treatment time and concentration;PPM1A expression is significantly negatively correlated with p-Smad expression;Astragaloside? targeted regulation of PPM1A expression affects the phosphorylation of Smad2/3 to regulate the TGF-?1/Smads signaling pathway.Astragaloside ? affects the phosphorylation of Smad2/3 by targeting the expression of PPM1A,regulates the signaling pathway of TGF-?1/Smads.It can results in the oxidative stress and inflammatory response,and then gradually develop a active role in silicosis.