| Objective:Silicosis is a systemic disease mainly caused by pulmonary fibrosis after long-term inhalation of dust with high content of free silica in the production process.At present,there is no specific treatment drug for silicosis and it mainly depends on etiological prevention,making it greatly important to explore the pathogenic mechanism of silicosis.The present study further explored the role of PTEN in pulmonary fibrosis induced by silica dust and related signal transduction pathways through the exposure level of dust-exposed workers,changes of methylation level of PTEN specific gene promoter region in pernpheral blood of silicosis patients,and cell experiments,so as to improve the understanding of silicosis fibrosis and provide new evidences for early diagnosis and treatment of silicosis.Methods:According to the main exposure industries of silica dust and the occupational data of silicosis patients in China,a large non-ferrous metal enterprise was selected to detect the concentration of total dust and respirable dust in the main dust-exposed positions by fixed-point sampling method,and the content of free silica dust in the working places was determined at the same time.According to the inclusion criteria of silicosis patients and normal people,the case group and the control group were selected reasonably,and the peripheral blood samples of the two groups were collected followed by DNA extraction and concentration determination.Then,the methylation status of specific promoter fragments of PTEN gene in the two groups was detected by bisulfite sequencing PCR.In cell experiment,SiO2 dust with high purity and uniform particle size was selected to accurately simulate the pathogenesis of pulmonary fibrosis induced by free silica.The dose of SiO2 for stimulation of HELF cells was determined by CCK8 experiment.The effect of SiO2 on cell morphology and the variation trend versus time of exposure was observed by inverted microscope.The Western blot experiment was carried out to observe the effect of SiO2 stimulation on the the expression of PTEN,p-PTEN,FAK,p-FAK,TGF-? and a-SMA as well as the variation trend versus exposure time.Meanwhile,the changes of FAK,p-FAK and a-SMA protein expression were observed when the PTEN phosphatase activity was inhibited by chemical inhibitorsResults:In the process of on-site sampling,the short-term contact concentration of total dust in 34 working places was detected,of which 18 working places were above 2 mg/m3;the short-term contact concentration of respirable dust in 32 working places was detected,of which 13 working places were above 1.4 mg/m3,and the content of free silica in the working places was between 9.7%and 13.2%.In the methylation test of blood sample,50 samples were detected in case group and 35 samples were detected in control group.The average methylation status of PTEN promoter region in case group was 0.059.The average methylation status of PTEN promoter region in control group was 0.067.The methylation level in case group was 0.008 lower than that in control group,and the difference was statistically significant(p<0.05).The ratio between the methylation difference and the average methylation level was 0.130.CCK8 test results in cell experiment showed that the cell viability of HELF cells stimulated by SiO2 dust with concentration of 50 ?g/cm2 was significantly increased compared with that of non-stimulated group(p<0.05).Microscopic observation showed that when the HELF cells were stimulated by SiO2 dust with concentration of 50?g/cm2 for 2-4 hours,some cells were round in shape;when the stimulation time was prolonged to 8-12 hours,the number of cells with round shape increased;when the stimulation time was further prolonged to 24 hours,stretched HELF cells with long spindle shape were observed,which were similar to that of adherent cells after passage.Western blot results showed that the protein expression of PTEN and p-PTEN decreased with the increase of stimulation time.When the stimulation time was 24 hours,the protein expression of PTEN and p-PTEN was significantly lower than that of non-stimulation group.Under the same stimulation conditions,the expression of TGF-? increased,as well as the tyrosine kinase FAK and p-FAK.The expression of a-SMA which was a marker protein of myofibroblasts also increased.The results of inhibitor experiments showed that the cell morphology was less affected when the concentration of inhibitor was 10?M.At this dose,the expression of FAK protein was significantly increased compared with that without inhibitor and the expression of p-FAK and a-SMA protein also increased.However,the expression of FAK,p-FAK and a-SMA protein was not significantly increased when SiO2 dust and PTEN inhibitor were added at the same time,compared to that when Si02 or VO-Ohpic was added alone.Conclusions:(1)The concentration of total dust and respiratory dust in some positions exposed to dust in this occupational place is high,suggesting that it is necessary to adopt relevant dust reduction and protective measures to improve the occupational health of workers.It is also meaningful to explore the pathogenic mechanism of silicosis and provide experimental evidence for diagnosis and treatment.(2)The promoter region of PTEN gene in silicosis patients showed hypomethylation change compared with the normal population,and the difference was statistically significant.(3)The SiO2 stimulation of HELF cells could increase cell viability,accompanied by changes in cell morphology.The expression of PTEN and p-PTEN decreased significantly when SiO2 stimulates HELF cells for 24 hours,suggesting that at that time cells may be in malignant cell proliferation and cell cycle may be disordered.Moreover,the increased expression of FAK,TGF-p and a-SMA also suggests that fibrosis may have occurred in HELF cells.(4)PTEN inhibited can change the expression of FAK,p-FAK and a-SMA proteins.However,the expression of them did not increase significantly when S1O2 and inhibitor were added together,suggesting that the addition of both of them may activate other pathways in HELF cells,thereby affecting the expression of those proteins.Therefore,it is reasonable to speculate that in the process of HELF cell fibrosis induced by S1O2,there may be other signal factors other than PTEN that have a strong regulatory effect on FAK and a-SMA. |
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