| ObjectiveSilicosis is the world’s most important occupational disease.Although countries continue to update the prevention of silicosis,there are still a large number of new cases every year in developed countries and in low and middle income countries.The deposition of Si O2in lung tissue is the main cause of silicosis.Pulmonary fibrosis caused by Si O2entering the lungs is irreversible.As a result,the patient’s lung function gradually decreases and even fails.So far,there are no effective drugs and measures for silicosis fibrosis at home and abroad.Therefore,it is very important to study the pathogenesis of silicosis and carry out related exploration of traditional chinese medicine to find a new path for treating silicosis.Dahuang Zhechong Pills（DHZCP）is a classic ancient recipe of"Jin Gui Yao Lue",and our preliminary experiments have shown that it can improve silicosis fibrosis in mice.In this study,through animal experiments and cell experiments,starting from the TGF-β1/Smad signaling pathway,we revealed the mechanism of action of DHZCP on silicosis at the overall,cellular,and molecular levels.MethodsPart 1:Determination of a model for mouse silicosis fibrosis modelEighty SPF-grade Kunming mice were randomly divided into a normal control group,an exposed trachea injection group,an endotracheal intubation group,a nasal instillation group,and a static exposure group.There were 5 groups of 16 in each group,of which the normal control group Without any intervention.Groups 1-4 were established on the 40th day after one-time modeling,and Group 5 was continuously modeled for 40 days.The body weight,pulmonary index,survival rate,and lung tissue pathology of each group were compared,and the experimental modeling protocol was comprehensively determined.Part 2:Mechanism of DHZCP on Silicosis Fibrosis in MiceThere were 108 mice in total,of which 90 were randomly divided into model control group,tetrandrine group,DHZCP high,middle and low dose group 5 groups,and each group had 18 mice after 40 days.Eighteen non-exposed mice were the normal control group.The low,middle and high dose groups were administered intragastrically according to the clinical equivalent of DHZCP adult equivalent doses of 1,2 and 3 times,once a day.Tetrandrol was administered orally once a day according to the clinical adult equivalent dose;the normal control group and the model control group were orally administered with an equal amount of normal saline.Six mice were killed in each group at 14,21,and 28 days after administration.The pulmonary index was calculated and the contents of TNF-α,IL-6,IL-1β,and HYP were measured.The pathology was observed with HE and Masson staining,the effect of DHZCP on the expression of TGF-β1/Smad pathway-related proteins（TGF-β1,α-SMA,Smad2,Smad3,Smad7）was detected by Western-Blot method,and the effect of DHZCP on expression of pathway-related genes（TGF-β1m RNA,Smad2m RNA,Smad3m RNA,Smad7m RNA）was detected by RT-PCR.Part 3:Study on the mechanism of DHZCP on Si O2-induced silicosis fibrosis in vitro cell modelSi O2-induced MH-S cells were used to construct a model of silicosis fibrosis cells,and the drug-containing serum of DHZCP rats was extracted.The pharmacological components in the serum were detected by tandem mass spectrometry,and then the drug-containing serum of different concentrations was used for intervention.Cell activity was measured,the contents of TNF-α,IL-6,and IL-1βin the cell supernatant were measured by Elisa method,and the TGF-β1/Smad pathway-related proteins（TGF-β1,α-SMA,Smad2,Smad3,Smad7）expression,immunofluorescence method to detect the fluorescence expression intensity of the above proteins.ResultsPart 1:The static poisoning method was used as the modeling method in this experiment.1 General situation of each groupThe normal control group was generally in good condition.The weight of each model of mice in the later period increased slowly,which was significantly lower than that of the normal control group.The fur was sparse,lacking luster,and foul adhesion.Mice in the static-exposure group had nose scratches and dark purple lips.2 Survival rate and pulmonary index of each groupThe normal control group was 100%,the exposed trachea injection group was62.5%,the tracheal intubation group was 87.5%,the nasal drip group was 93.8%,the static exposure group was 100%,and the static exposure group was significantly higher than the other three group.Compared with the normal group,the pulmonary index of each model group was significantly different（P<0.01）.3 Pathological results of lung tissue in each groupLung tissue HE staining results:The alveolar structure of the normal control group was normal.Partial alveolar cavity collapse was seen in all models,local alveolar epithelial cells degenerated and necrotic,and the nucleus was condensed.Among them,the exposed lung trachea injection group,tracheal intubation group and static exposure group had different degrees of thickening of the lung interstitial,with fibrous tissue proliferation and neo-epithelial cell proliferation,inflammatory cell infiltration,mainly round deeply stained lymphocytes.Masson staining showed no significant changes in lung morphology in the normal control group.In the four model groups,the alveolar structure of the mice was destroyed,and the alveolar space was widened.A large number of dense blue collagen fibers were seen in the lung field,which were distributed in a sheet or bundle.4 Conclusions in this sectionIn summary,the three methods of tracheal injection,tracheal intubation,and static exposure can successfully replicate the silicosis model in mice.The relative success rate of nasal instillation is low.Eventually entered the esophagus but did not enter the respiratory tract.Synthesizing the fitting degree,success rate,survival rate and other related factors of the model and the real disease,the static poisoning method was selected as the model.Part 2:DHZCP can inhibit lung fibrosis in silicosis model of mice by regulating TGF-β1/Smad pathway1 Effect of DHZCP on the general situation of silicosis fibrosis miceDuring the modeling,the normal mice were generally in good condition.The mice in the model group were gradually debilitated,with nasal scratching,reduced activity,insignificant weight gain,sparse fur and lack of luster,and dirty adhesions.The nose and lips were darker than the normal group,and purple blood was scattered in some tails.After administration,the DHZCP high-dose group was in a good mental state,and was close to the normal group.The condition of the middle-dose group was improved compared with the model group.The overall situation of the low-dose group was not as good as that of the high and middle groups,but it was better than the model group.2 Effect of DHZCP on pulmonary index of silicosis fibrosis micePulmonary index:The model group was significantly higher than the normal group（P<0.01）;after 14 days of administration,it was seen that the high-dose DHZCP group was lower than the model group and had statistical significance（P<0.05）;The middle-dose group was significantly lower than the model group（P<0.01）.After 28 days,the high,middle and low groups were significantly lower than the model group（P<0.01）.3 Effect of DHZCP on pathological staining of lung tissue in silicosis fibrosis micePathological results of lung tissue:HE staining:normal control group had normal alveolar morphology and structure;model group had congestion,alveolar collapse,fusion,epithelial cell degeneration and necrosis,and nucleus shrinkage.Different degrees of alveolar septal thickening,fibrous tissue proliferation or epithelial cell proliferation;and local lymphocytic infiltration.The morphology and structure of lung tissue in DHZCP high-dose group improved significantly,which was closest to that in normal group.Masson staining:No significant blue-stained collagen deposition was seen in the normal control group;a large number of blue-stained collagen fibers were seen in the model group,and the alveolar wall thickness was different.After DHZCP treatment,collagen fiber deposition in the lung tissue was reduced to varying degrees,the High-dose changes were the most obvious,the blue dye color became significantly lighter,and the area also decreased significantly.Both staining methods showed that the high-dose DHZCP group significantly improved the inflammation and fibrosis of lung tissue.4 Effect of DHZCP on cytokines in silicosis fibrosis miceThe levels of HYP,TNF-α,IL-6 and IL-1βin the serum of the model group mice were significantly higher than those in the normal group（P<0.01）.At 14 days,HYP,TNF-α,IL-6,and IL-1βin the DHZCP high and middle dose groups were significantly lower than those in the model group（P<0.01）,and TNF-αand IL-6 in the low dose group were also significantly lower than those in the model group（P<0.01）.At 21 days,HYP,TNF-α,IL-6,and IL-1βin the high-dose DHZCP group were significantly lower than the model group（P<0.01）;HYP,IL-6,and IL-1βin the middle-dose group were significantly lower than the model group（P<0.01）;the content of IL-1βin the low-dose group was significantly lower than that in the model group（P<0.01）.At 28 days,the contents of HYP,TNF-α,IL-6,and IL-1βin the high-dose DHZCP group were significantly lower than those in the model group（P<0.01）,and IL-1βin the middle-dose group was significantly lower than the model group at this stage（P<0.01）.5 Effect of DHZCP on TGF-β1/Smad Pathway Protein Expression in Silicosis Fibrosis MiceCompared with the normal group,the content of TGF-β1,α-SMA,Smad2,and Smad3 in the model group increased significantly（P<0.01）,and Smad7 decreased significantly（P<0.01）.At 14 days,the protein content of TGF-β1,α-SMA,Smad2,and Smad3 in the high dose group of DHZCP was significantly lower than that in the model group（P<0.01）,and Smad7 was significantly increased（P<0.01）.In the DHZCP medium dose group,Smad2 was significantly lower than the model group（P<0.01）,α-SMA and Smad3 were significantly lower than the model group（P<0.05）;Smad7 was significantly increased（P<0.01）.The content of Smad2 protein in the low-dose group was significantly lower than that in the model group（P<0.01）,and Smad7 was significantly increased（P<0.01）.At 21 days,except the Smad2 in the low-dose group was lower than the model group,which was statistically significant,the TGF-β1,α-SMA,Smad2,and Smad3 protein levels in the other DHZCP high,middle,and low dose groups were significantly lower than those in the model group（P<0.01）,Smad7 increased significantly（P<0.01）.At 28 days,the protein content of TGF-β1,α-SMA,Smad2,and Smad3 in the high-dose group was significantly lower than that in the model group,and Smad7 was significantly increased.The difference between TGF-β1 and Smad2 and the model group was statistically significant（P<0.05）,The other three indicators have significant differences（P<0.01）.The protein content of Smad3 in the middle and low dose groups was significantly lower than that in the model group（P<0.01）,and Smad7 was significantly increased（P<0.01）.6 Effect of DHZCP on TGF-β1/Smad Pathway Gene Expression in Silicosis Fibrosis MiceThe expression levels of Smad2 m RNA in the model group at 14 days and 21days were higher than those in the normal group at 14 days,which was statistically significant（P<0.05）.The expression levels of Smad7m RNA at 21 days were significantly lower than those in the normal group（P<0.05）.The expression levels of TGF-β1m RNA,Smad2m RNA,and Smad3m RNA were significantly higher in the other periods than in the normal group（P<0.01）,and the expression levels of Smad7m RNA were significantly lower than those in the normal group（P<0.01）.At 14days,Smad2 m RNA and Smad3m RNA were reduced（P<0.05）,TGF-β1 m RNA was decreased（P<0.01）,and Smad7m RNA was increased（P<0.01）in the high-dose group of DHZCP compared with the model group.The TGF-β1 m RNA in the middle and low dose groups was significantly lower than that in the model group（P<0.01）,and Smad7m RNA was significantly increased（P<0.01）.The middle-dose Smad2m RNA was significantly lower than that in the model group（P<0.05）.At 21 days,the expression levels of TGF-β1 m RNA and Smad3m RNA in the high-dose group were significantly lower than those in the model group（P<0.01）,Smad2m RNA expression was lower than that in the model group（P<0.05）,and Smad7m RNA expression was higher than in the model group（P<0.05）.The TGF-β1m RNA and Smad3 m RNA in the middle and low dose groups were significantly lower than those in the model group（P<0.01）.At 28 days,TGF-β1 m RNA,Smad2 m RNA,and Smad3 m RNA in the high,medium,and low dose groups of DHZCP were significantly reduced,which were significantly different from the model group（P<0.01）;the expression level of Smad7m RNA was also significantly increased,which was also similar to There was a significant difference between the model groups（P<0.01）.Part 3:DHZCP-containing serum can inhibit cell fibrosis in vitro by regulating the TGF-β1/Smad pathway1 Effect of DHZCP drug-containing serum on cell viability of MH-S cell line induced by Si O2After the cells were stimulated by Si O2suspension,the cell viability decreased significantly（P<0.05）.After intervention with different concentrations of DHZCP,the cell viability was higher than that of the model control group,and it was concentration-dependent.2 Effect of DHZCP drug-containing serum on Si O2-induced cytokine in MH-S cell lineThe contents of TNF-α,IL-6,and IL-1βin the cell supernatant of the model control group were significantly higher than those in the normal control group（P<0.01）.The content of-6 was significantly lower than the normal control group（P<0.01）,and the content of TNF-αand IL-1βin the 15%serum-containing group was lower than that in the model group,which was statistically significant（P<0.05）.The content of TNF-αin the medicated serum 10%group was lower than that in the model group,which was statistically significant（P<0.05）.3 Effect of DHZCP drug-containing serum on Si O2-induced TGF-β1/Smad pathway protein expression in MH-S cell line（1）Western-Blot results:The expression of TGF-β1,α-SMA,Smad2,and Smad3in the model control group was significantly higher than that in the normal control group（P<0.01）,and the expression of Smad7 was opposite.The model control group was lower than the normal group and had Statistical significance（P<0.05）.The expression of TGF-β1,α-SMA,Smad2,and Smad3 in the DHZCP drug-containing serum 15%group was significantly lower than that in the model control group（P<0.01）,and the expression of Smad7 protein was opposite and higher than that in the model group,which was statistically significant（P<0.05）.The expression of TGF-β1,α-SMA,and Smad2 protein in the medicated serum 10%group was significantly lower than that in the model control group（P<0.01）.The expression ofα-SMA protein in the 5%group of medicated serum was significantly lower than that in the model control group（P<0.01）,and the protein expression of TGF-β1 and Smad2 was lower than that in the model group,which was statistically significant（P<0.05）.（2）It can be seen from the results of cellular immunofluorescence that the average optical expression of TGF-β1,α-SMA,Smad2,and Smad3 in the model control group was significantly higher than that in the normal control group（P<0.01）,and Smad7 expressed AO value was lower than that of the normal control group（P<0.01）.After adding DHZCP drug-containing serum,the AO value of TGF-β1,α-SMA,Smad2,and Smad3 in the 15%drug-containing serum group was significantly lower than the model control group（P<0.01）,and the AO value of Smad7 was significantly higher than the model control group.（P<0.01）;the AO value of TGF-β1,α-SMA,Smad2,and Smad3 in the 10%drug-containing serum group was significantly lower than that in the model control group（P<0.01）,and the AO value of Smad7 expression was higher than that in the model control group.Statistical significance（P<0.05）;the AO values ofα-SMA and Smad2 in the 5%drug-containing serum group were significantly lower than those in the model control group（P<0.01）,and the AO values of TGF-β1 and Smad3 were lower than those in the model control group,statistical significance（P<0.05）,the AO value of Smad7 expression was significantly higher than the model control group（P<0.01）.This shows that DHZCP can significantly inhibit the AO value of TGF-β1,α-SMA,Smad2,and Smad3,and enhance the AO value of Smad7,especially the inhibitory effect of the 15%drug-containing serum group is more obvious.The conclusion is consistent with Western-Blot.Conclusion1.Through modeling experiments,it has been shown that three methods of tracheal injection,tracheal intubation and static poisoning can successfully replicate the silicosis model of mice,but in order to reduce the impact of trauma during modeling,and the mortality of mice is more suitable and close to the conditions of real silicosis.Finally,the static poisoning method was selected as the modeling method for this subject.2.Pulmonary index and pathology from animal experiments;serum HYP content,serum TNF-α,IL-6,IL-1βcontent;expression ofα-SMA,TGF-β1,Smad2,Smad3,and Smad7 protein,TGF-β1m RNA,Smad2m RNA,Smad3m RNA,and Smad7m RNA expression in lung tissue confirmed that DHZCP can inhibit the pulmonary fibrosis of silicosis model in mice through the TGF-β1/Smad pathway,and there is a certain dose and time dependence.3.In vitro cell experiment:DHZCP-containing serum was used to interfere with Si O2-induced alveolar macrophages MH-S cell line,CCK8 was used to test cell viability,and the relevant inflammatory factor contents were also observed.The expression of Smad7 protein is consistent with animal experiments,which proves that DHZCP can also inhibit lung fibrosis through TGF-β1/Smad in vitro,and there is a concentration dependence. |
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