| BackgroundAt present,occupational pneumoconiosis is still the most serious occupational disease in China,and silicosis is the most common type of pneumoconiosis.Silicosis is an occupational disease caused by long-term exposure to large quantity of free silica dust,which is regarded as incurable for the irreversibility of progressing diffuse nodular pulmonary fibrosis,eventually seriously impairs lung function,leading to respiratory failure and even death.The prevalence and incidence of silicosis has been rising,particularly in developing countries.Every year,the direct economic losses caused by silicosis in China amount to more than 8 billion yuan(RMB),and the indirect losses are incalculable.In developed countries,silicosis is also a high-profile occupational health problem.However,the pathogenesis of silicosis is still unclear and none of current therapies can prevent disease progression effectively or reverse lung fibrosis.There is yet a pressing need to advancing novel and efficient approaches.The progress of silicosis is a multifactorial process involving persistent pulmonary inflammation induced by crystalline silica and excessive production of extracellular matrix,which ultimately leads to irreversible destruction of normal lung structure and pulmonary fibrosis.Previous studies have identified many important pathogenic mechanisms that promote silicosis,among which oxidative stress is an important molecular mechanism that regulates a variety of biological processes and signal transduction pathways.When the body inhales silica particles,they are engulfed by alveolar macrophages and persist,which induces oxidative stress through the formation of reactive oxygen species(ROS)and nitrogen species.Oxidative stress can lead to abnormal phenotypic changes of epithelial cells,including increased expression of cytokines,epithelial-mesenchymal transition(EMT)and apoptosis,resulting in the proliferation of fibroblasts and the deposition of extracellular matrix(ECM)in the lungs,and further induces cytotoxicity,oxidative stress,pulmonary inflammation,which eventually leads to silicosis.These pathological events are mainly mediated by various signaling pathways and cytokines.Among these cytokines,transforming growth factor-beta 1(TGF-β1)is considered to be the key fibrogenic factor.TGF-β1 plays an important role in cell proliferation,differentiation,migration,immune regulation and ECM transformation in fibrotic diseases through the classical TGF-β1/Smad signaling pathway.Mountains of researches have shown that the imbalance of the TGF-β1/Smad signaling pathway is an important pathogenesis of tissue fibrosis,which has attracted more and more attention as an effective anti-fibrosis treatment target.Therefore,inhibiting the TGF-β1/Smad signaling pathway may be a treatment strategy to alleviate silicosis fibrosis.In order to maintain an appropriate redox balance,our body has formed a complex oxidative stress response system,including a series of endogenous antioxidant enzymes.Lung injury leads to the elevation of both TGF-β1 and oxidative stress levels.Growing evidence suggests that oxidative stress and ROS production are associated with activation and production of a lot of cytokines and pro-fibrotic growth factors.The activation of the TGF-β1/Smad signaling pathway is the main driving force for the formation of fibrosis,and oxidative stress is an important harmful promoting factor related to the activity of TGF-β1 in promoting fibrosis.In the whole process of fibrogenesis,TGF-β1 has a significant correlation with oxidative stress signals.TGF-β1 can improve the ROS level of lung tissue,which in turn stimulates the activation of TGF-β1-related fibroblasts and myofibroblast differentiation.It was found that the fibrosis events related to TGF-β1 were related to the decrease of ROS scavenging enzyme and/or the increase of ROS producing enzyme.Nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(NOX)is the main source of endogenous ROS.Among them,NOX4 has unique composition activity.As the main enzyme source of extracellular ROS,ROS derived from NOX4 has been recognized as the main source of oxidative stress,NOX4,as a downstream medium of TGF-β1-induced fibrogenic response,induces ROS formation and participates in the pathogenesis of fibrosis by regulating TGF-β1-induced fibroblast activation and myofibroblast differentiation.Studies have shown that NOX4 is up-regulated in fibrotic diseases and plays an important role in the pathogenesis of liver,kidney,lung and heart fibrosis.NOX4 is considered to be a potential therapeutic target for pulmonary fibrosis associated with enhanced TGF-β1 signaling.Nuclear factor erythroid 2-related factor-2(Nrf2)is a key redox-sensitive transcription factor involved in host defense against oxidative stress.It seems to be a negative regulator of tissue fibrosis.Nrf2 binds to the inhibitor Kelch-like ECH-associated protein 1(Keapl)at rest and is released from Keapl under tissue damage-mediated ROS or electrophilic stimulation,translocating into the nucleus and activating the antioxidant response element(ARE),to induce the expression of downstream antioxidant and detoxifying enzymes,including heme oxygenase-1(HO-1)and NAD(P)H quinone oxidoreductase-1(NQO-1),which is an essential factor to maintain the redox homeostasis of cells.It has been proved that Nrf2 is involved in the dynamic process of fibrosis formation and seems to be a negative regulator of tissue fibrosis.Nrf2 not only balances oxidative stress but also negatively regulates TGF-β1-mediated signal transduction.It seems that Nrf2 can exert cell-autonomous antifibrotic effects.In persistent organ fibrosis,Nrf2 activation is impaired,which changes the redox balance of NOX4-Nrf2 and leads to myofibroblasts to obtain an antiapoptotic phenotype.As the main modulator of many antioxidant,anti-inflammatory,and anti-fibrosis pathways,Nrf2 has potential value as a therapeutic target.The activation of Nrf2 signaling can reduce the progress of lung injury and fibrosis,suggesting that the strategy targeting the Nrf2/ARE signaling pathway has the potential of anti-silicosis fibrosisNatural products play a very important role in the research and development of drugs.Tanshinone ⅡA(Tan ⅡA)is the most important active component of the traditional Chinese herb Salvia miltiorrhiza(Danshen),which possesses superior bio-availability and various pharmacological actions,has been reported to possess anti-inflammatory,antioxidant,and anti-fibrosis properties in various organs.However,there are few studies available on the efficacy of Tan ⅡA in silicosis,and the molecular mechanisms by which Tan ⅡA attenuates silica-induced lung fibrosis remain elusive.Can it effectively alleviate pulmonary injury and fibrosis of silicosis?Objective1.To investigate the therapeutic effect of Tan IIA on silicosis rats by intraperitoneal injection,and the effect of Tan ⅡA on silicosis was observed from the aspects of lung histopathology,airway inflammatory cell recruitment and inflammatory factor content of lung tissue,etc.2.To investigate whether Tan ⅡA can inhibit the TGF-β1/Smad signaling pathway of silicosis rats in vivo and whether Tan IIA can inhibit the activation of EMT and TGF-β1/Smad signaling pathway of A549 and HBE cells induced by silica in vitro,so as to alleviate the silicosis pulmonary fibrosis.3.To investigate whether Tan ⅡA can reduce oxidative stress induced by silica,and play a protective role in silica-induced pulmonary injury and fibrosis by inhibiting the expression of NOX4 and activating Nrf2/ARE signaling pathway in vivo and in vitroMethods1.In vivo experiment1.1 Animal modeling and administration48 SD rats(age,6-8 weeks;weight,200±20 g)were divided into four groups at random(n=12 per group):ⅰ)control group;ⅱ)Tan ⅡA group;ⅲ)silicosis group;ⅳ)silicosis+Tan ⅡA group.The silicosis rat model was established by endotracheal intubation:SD rats were anesthetized by intraperitoneal injection of 2%Pentobarbital Sodium(40 mg/kg),then the rats in silicosis group and silicosis+Tan ⅡA group were implanted with epidural anesthesia catheter,and then 1 ml of 50 mg/ml silica dust suspension and 0.25 ml of air were infused into the trachea.The control group and Tan ⅡA group were perfused with the same amount of normal saline and air by endotracheal intubation.After that,the rats were immediately rotated so that the injection was evenly distributed in the lungs.Two days after modeling,rats in Tan ⅡA group and silicosis+Tan ⅡA group were intraperitoneally injected with 25 mg/kg body weight Tan ⅡA for 40 days.Rats in the control group and silicosis group were intraperitoneally injected with an equal quantity of sterile normal saline at the same time.After 42 days of modeling,the bronchoalveolar lavage fluid(BALF)of the four groups of rats was collected and rats were executed after anesthesia,their lungs were taken for further experiments.1.2 To observe the therapeutic effect of Tan ⅡA on silicosis rats1)The wet/dry weight ratio of the lung tissue of rats in each group was measured.2)The paraffin-embedded sections of lung tissues of rats in each group were stained with H&E and Masson.3)The expression of Collagen Ⅰ,fibronectin 1(FN1)and α-smooth muscle actin(α-SMA)in lung tissue of rats in each group were detected by immunohistochemistry and RT-PCR.4)Giemsa staining method was used to count the total cells,neutrophils,macrophages,and lymphocytes in BALF.5)The contents of TNF-α,IL-6,and IL-1β in the lung tissue of rats in each group were determined by ELISA kits.1.3 Detection of oxidative stress level in lung tissue of rats in each groupThe oxidative stress level in the lung tissue of rats in each group was detected using commercial test kits of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px).1.4 Determination of key factors of TGF-β1/Smad signaling pathway1)The mRNA expression of Tgfb1 and its downstream genes Smad2,Smad3 and Smad7 in lung tissues of rats in each group were detected using quantitative RT-PCR.2)The protein expression of TGF-β1,p-Smad2/3,Smad2/3,p-Smad3,Smad3,and Smad7 in lung tissues of rats in each group were detected using Western Blot.1.5 Determination of the expression of NOX4 and key factors of Nrf2/ARE signaling pathway1)The mRNA expression of Nox4,Nrf2,Keapl and their downstream genes Ho1 and Nqo1 in lung tissues of rats in each group were detected using quantitative RT-PCR.2)The protein expression of NOX4,cytoplasmic Nrf2,nuclear Nrf2,Keap1 and their downstream HO-1 and NQO-1 in lung tissues of rats in each group were detected using Western Blot.2.In vitro experiment2.1 Silica(Si02)and Tan IIA treatment concentration screeningA549 and HBE cells were cultured in vitro.The effects of different concentrations of silica and Tan IIA on cell viability of A549 and HBE cells were measured by CCK-8 kit.Cells were treated with different concentrations of silica(0,25,50,100,200,400,800 μg/ml)or Tan ⅡA(0,2.5,5,10,20,40,80 μM)for 24 h,and then the cell viability was detected by CCK-8 kit.Appropriate silica and Tan ⅡA treatment concentrations were selected for subsequent experiments.2.2 Cell grouping and treatmentA549 and HBE cells were cultured in vitro.After changing the serum-free medium,A549 cells and HBE cells were divided into the following groups:1)Blank control group;2)SiO2 group:cells were treated with single SiO2(100 μg/ml)for 24 h;3)SiO2+Tan ⅡA group:cells were treated with Si02(100 μg/ml)combined with different doses of Tan ⅡA(5,10,or 20 μM)for 24 h.2.3 Determination of EMT markers and key factors of TGF-β1/Smad signaling pathway1)The protein expression of E-cadherin,Vimentin,FN1,Collagen Ⅰ and α-SMA in each group of cells were detected using Western Blot.2)The protein expression of TGF-β1,p-Smad3,Smad3 and Smad7 in each group of cells were detected using Western Blot.3)The expression of TGF-β1 in each group of cells was detected by immunofluorescence.2.4 Detection of ROS level in cells of each groupThe level of ROS in each group of cells was detected by ROS detection kit.2.5 Determination of the expression of NOX4 and key factors of Nrf2/ARE signaling pathway1)The protein expression of NOX4,Nrf2,Keap1 and their downstream HO-1 and NQO-1 in each group of cells were detected using Western Blot.2)The expression of Nrf2 in each group of cells was detected by immunofluorescence.3.Statistical analysisIBM SPSS statistics 19.0 software was used for data processing.If the measurement data conform to the normal distribution,the mean ± SD was used.One-way ANOVA or Kruskal Wallis nonparametric test was used for comparison between groups.LSD or Dunn Bonferroni method was used for comparison between groups.The difference was statistically significant when P<0.05.Results1.The therapeutic effect of Tan ⅡA on silicosis rats1.1 Tan ⅡA alleviated pulmonary injury and fibrosis in silicosis ratsCompared with the control group,the wet/dry weight of rats in silicosis group increased significantly,and decreased significantly after Tan ⅡA treatment.We made paraffin embedded sections and histopathological staining on the lung tissues of the rats in each group.H&E staining showed that the lung tissues of the rats in silicosis group lost normal alveolar structure,the alveolar walls were thickened,accompanied by a large number of inflammatory cells infiltration,and characteristic silicosis nodules were formed around the bronchial tree and vascular bed,showing diffuse pulmonary fibrosis.After treatment with Tan ⅡA,these destructive effects were relieved.Masson staining showed that Tan ⅡA treatment could significantly reduce the Collagen deposition in the lung tissue of silicosis rats.Immunohistochemical staining and RT-PCR results showed that Tan ⅡA treatment could significantly reduce the expression levels of Collagen Ⅰ,α-SMA and FN1 in the lung tissue of silicosis rats.These results indicate that Tan ⅡA treatment can alleviate pulmonary injury and fibrosis in silicosis rats1.2 Tan ⅡA reduced the inflammatory level of silicosis ratsCompared with the control group,the number of total cells,neutrophils,macrophages,and lymphocytes in the BALF of the silicosis group increased significantly,but decreased significantly after treatment with Tan IIA;the levels of inflammatory factors TNF-α,IL-6,IL-1β in the lung tissue of the silicosis group increased significantly but decreased significantly after treatment with Tan IIA.2.Tan ⅡA could inhibit EMT and activation of the TGF-β1/Smad signaling pathway induced by silica.2.1 Tan ⅡA inhibited TGF-β1/Smad signaling pathway activation in silicosis ratsThe activity of TGF-β1 and its downstream signaling Smad2,Smad3 and Smad7 were determined by RT-PCR and Western Blot analysis.RT-PCR result showed that the mRNA and protein expression levels of TGF-β1 in the lung tissue of silicosis rats increased significantly,but decreased significantly after treatment with Tan ⅡA;There was no significant change in the mRNA and protein levels of Smad2 and Smad3 in the lung tissue of silicosis rats,while the protein expression levels of p-Smad3 and p-Smad2/3 increased significantly,but decreased significantly after treatment with TanⅡA.As a negative regulator of TGF-β1/Smad pathway,the mRNA and protein levels of Smad7 decreased significantly in the lung tissue of silicosis rats but increased significantly after treatment with Tan IIA.These results indicate that the inhibition of the TGF-β1/Smad signaling pathway is related to the protective effect of Tan IIA on silicosis fibrosis.2.2 Tan ⅡA inhibited EMT of A549 and HBE cells induced by silicaWestern Blot was used to detect the inhibitory effect of Tan ⅡA on EMT of A549 and HBE cells induced by silica exposure.The results showed that after incubated with silica(100 μg/ml)for 24 h,the protein expression of epithelial marker E-cadherin was downregulated,accompanied by the upregulation of the mesenchymal marker Vimentin,FN1,Collagen Ⅰ and α-SMA.Tan ⅡA treatment can upregulate the expression of E-cadherin,downregulate the expression of Vimentin,FN1,Collagen Ⅰand α-SMA with a dose-dependent manner in A549 and HBE cells.These results indicate that Tan ⅡA can inhibit EMT of A549 and HBE cells induced by silica exposure.2.3 Tan ⅡA inhibited the activation of TGF-β1/Smad signaling pathway induced by silica in A549 and HBE cellsWestern Blot was used to detect the inhibitory effect of Tan ⅡA on the activation of the TGF-β1/Smad signaling pathway induced by silica in A549 and HBE cells.The results showed that the expression of TGF-β1 and its downstream p-Smad3 in A549 and HBE cells was significantly up-regulated after silica exposure,and the expression of Smad7 was significantly down-regulated,while the expression of Smad3 had no significant change,indicating the activation of TGF-β1/Smad signaling pathway in A549 and HBE cells induced by silica.The expression of TGF-β1 and p-Smad3 was significantly down-regulated,while Smad7 is significantly up-regulated by Tan ⅡA in a dose-dependent manner.The inhibition of Tan IIA on the activation of TGF-β1 in A549 and HBE cells induced by silica was also confirmed by immunofluorescence analysis.The above results showed that Tan IIA inhibited silica-induced pulmonary fibrosis in vitro partly by inhibiting the activation of the TGF-β1/Smad signaling pathway.3.Tan IIA could reduce oxidative stress injury induced by silica,inhibit the expression of NOX4 and activate the Nrf2/ARE signaling pathway3.1 Tan IIA reduced oxidative stress injury,inhibited the expression of NOX4 and activated the Nrf2/ARE signaling pathway in silicosis ratsThe oxidative stress level in lung tissue of rats in each group was detected using commercial test kits of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px).The results showed that the content of ROS and MDA in silicosis group increased with the decrease of SOD and GSH-Px activity,while the content of ROS and MDA in Tan ⅡA treatment group decreased with the increase of SOD and GSH-Px activity compared with silicosis group.The above results showed that Tan IIA treatment reduced the oxidative stress injury in the lungs of silicosis rats.We used RT-PCR and Western Blot to detect whether Tan IIA can inhibit NOX4 expression and activate Nrf2/ARE signaling pathway in silicosis rats,to protect the oxidative injury of silicosis rats.The results showed that the mRNAs and protein levels of NOX4 were significantly upregulated in silicosis group,and down-regulated after treatment with Tan IIA.The mRNAs of Nrf2 and its downstream HO-1 and NQO-1 were significantly increased in silicosis group,and further increased after treatment with Tan IIA.Since Nrf2 nuclear translocation is a necessary step for Nrf2 activation,we evaluated the nuclear accumulation of Nrf2 and the expression of Nrf2 in the cytoplasm.The results showed that Nrf2 expression in the nucleus of silicosis group was up-regulated,while Nrf2 expression in the cytoplasm was down-regulated.These effects were further enhanced after treatment with Tan ⅡA.In accordance with Nrf2,the expression levels of HO-1 and NQO-1 were also significantly increased in silicosis group,and were further increased in Tan IIA treatment group.In contrast to Nrf2,the mRNA and protein expressions of its inhibitor Keap1 were down-regulated in silicosis group,while Tan IIA treatment further down-regulated its expression.The above results indicate that Tan ⅡA partially protects against oxidative stress injury of silicosis by inhibiting NOX4 expression and activating Nrf2/ARE signaling pathway.3.2 Tan ⅡA reduced oxidative stress induced by silica,inhibited the expression of NOX4 and activated Nrf2/ARE signaling pathway in A549 and HBE cellsThe inhibition of Tan ⅡA on the oxidative stress level of A549 and HBE cells induced by silica was detected by the ROS test kit.After treatment of A549 and HBE cells with silica(100 μg/ml)for 24 h,ROS detection with DCFH-DA showed that the green fluorescence intensity of A549 and HBE cells was significantly enhanced,while the green fluorescence intensity of A549 and HBE cells was decreased by co-treatment with Tan ⅡA.In order to further elucidate the anti-oxidation mechanism of Tan ⅡA,we used Western Blot to detect the NOX4 expression and activity of the Nrf2/ARE signaling pathway in A549 and HBE cells.The results showed that silica exposure significantly increased NOX4 protein expression,while Tan ⅡA treatment decreased NOX4 expression.Silica exposure increased Nrf2 protein expression,while Tan ⅡA treatment further enhanced Nrf2 expression.Immunofluorescence analysis showed that Tan ⅡA activated Nrf2 and promoted its nuclear aggregation in A549 and HBE cells.Accordingly,silica exposure resulted in the up-regulation of HO-1 and NQO-1 expression,which was further up-regulated by Tan ⅡA treatment.In contrast to Nrf2,the protein expression of Keap1,an inhibitor of Nrf2,was down-regulated in the silica exposed group,and further down-regulated in Tan ⅡA treated group.These data indicated that Tan ⅡA can reduce oxidative stress induced by silica exposure in vitro by activating the Nrf2/ARE signaling pathway.Conclusion1.Tan ⅡA could effectively alleviate the damage of lung tissue structure andpulmonary fibrosis,as well as reduce the inflammatory response of silicosis rats.2.Tan ⅡA could inhibit epithelial-mesenchymal transition(EMT)and TGF-β1/Smad signaling pathway activation induced by silica.3.Tan ⅡA could alleviate oxidative stress induced by silica by inhibiting the expression of NOX4 and activating the Nrf2/ARE signaling pathway.SignificanceIn this study,we used silicosis rat model and cell experiment in vitro to clarify the anti-inflammatory,anti-oxidation and anti-fibrosis effects of Tan ⅡA,a monomer of traditional Chinese Medicine,on silicosis and further explored its molecular mechanism of efficacy,which proved that Tan IIA can reduce silicosis fibrosis by inhibiting epithelial mesenchymal transformation(EMT)and TGF-β1/Smad signaling pathway induced by silica,and reduce oxidative stress injury induced by silica by inhibiting the expression of NOX4 and activating Nrf2/ARE signaling pathway,so as to play a therapeutic and protective role in pulmonary injury and fibrosis of silicosis This study showed that Tan IIA can be used as a potential drug for the treatment of silicosis,providing theoretical support for the prevention and treatment of silicosis,and providing a theoretical basis for the development of new drugs. |
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