The Role Of Muc5b In Airway Inflammation Induced By Atmospheric PM_2.5 And Its Mechanisms

Objective:To clarify the effect of PM_2.5.5 exposure on mucin Muc5b secretion and related inflammatory factors,to reveal the regulatory role of mucin Muc5b in PM_2.5-induced airway inflammation,and to explore the mechanism of Muc5b in airway inflammation induced by PM_2.5,so as to provide scientific basis for prevention and treatment of respiratory health damage caused by PM_2.5.Method:Twenty-four Wistar rats were randomly divided into control group and low,medium and high concentration PM_2.5.5 exposure group.The rats in the low,medium and high concentration groups were intratracheally dripped with PM_2.5.5 normal saline of equal volume 1.5,7.5 and 37.5 mg/kg body weight respectively,while the rats in the control group were intratracheally dripped with normal saline of equal volume.The pathological changes of airway were observed by HE staining.AB-PAS staining was used to detect airway mucus secretion.The levels of TNF-?and IL-1?in serum and bronchoalveolar lavage fluid and Muc5b protein in lung and trachea tissues were determined by ELISA,and the expression of Muc5b gene in lung and trachea tissues was detected by Q-PCR.A549 cells were cultured in DMEM medium containing 10%fetal bovine serum.siRNA was synthesized and transfected into A549 cells to silence Muc5b gene,and Q-PCR was used to detect the silencing efficiency.Normal and gene silenced A549cells were exposed to 0ug/ml?control group?,100ug/ml?low concentration group?,200ug/ml?medium concentration group?and 400ug/ml?high concentration group?of PM_2.5.5 respectively.There were three parallel samples in each group.Cells and cell culture media were collected after 6,12,24and 48h of exposure.The levels of inflammatory factors TNF-?,IL-1?and mucin Muc5b were detected by ELISA,and the relative expression of Muc5b gene was detected by Q-PCR.IBM SPSS 24.0 software is used to analyze and process data,which is expressed by mean plus minus mark difference.Variance analysis or rank sum test were used to compare the differences of each index between different exposure groups;t test or rank sum test was used to compare the differences of each index between A549 cells and gene silenced A549 cells;linear correlation or rank correlation was used to analyze the correlation between mucin Muc5b and inflammatory factors such as IL-1?and TNF-?.The test level is set at?=0.05.Result:1.Effects of PM_2.5.5 exposure on Muc5b secretion and inflammatory response in rat airway?1?There was no significant difference in body weight of rats before and after PM_2.5.5 exposure?P>0.05?,and there was no significant difference in organ coefficients of liver,trachea and lung of rats in each group?P>0.05?.?2?After PM_2.5.5 exposure,inflammatory cells infiltrated significantly,mucosal morphology changed significantly and mucus secretion increased significantly in the middle concentration group.?3?There was no significant difference in the levels of IL-1?and TNF-?in alveolar lavage fluid and serum of rats exposed to PM_2.5?P>0.05?.?4?After PM_2.5.5 exposure,compared with the control group,the Muc5b mRNA level in lungs of rats in low concentration group decreased significantly?P<0.05?,while there was no significant difference in lung and trachea tissues of rats in other concentration groups?P>0.05?.?5?Muc5b secretion in lung tissue of rats exposed to low and high concentration PM_2.5.5 was significantly lower than that of control group?P<0.05?.Muc5b secretion in lung tissue of rats exposed to medium concentration PM_2.5.5 was significantly higher than that of rats exposed to low and high concentration PM_2.5?P<0.05?.The secretion of Muc5b in trachea tissue of the exposed group with medium concentration of PM_2.5was significantly higher than that of the low and high concentration group?P<0.05?.2.The effect of PM_2.5.5 exposure on the expression of Muc5b and inflammatory factors in A549 cells?1?With the increase of PM_2.5.5 concentration and the prolongation of exposure time,the survival rate of A549 cells decreased significantly?P<0.05?.?2?After 6h exposure to PM_2.5,the expression of Muc5b gene in A549 cells in medium and high concentration group was significantly lower than that in control group?P<0.05?;after 12h exposure,there was no significant difference in the expression of Muc5b gene between different concentration groups?P>0.05?;after 24h exposure,the expression of Muc5b gene in low concentration group was significantly higher than that in control group?P<0.05?;after 48h exposure,the expression of Muc5b gene in medium concentration group was significantly higher than that in control group?P<0.05?.It was lower than that of control group and low concentration group?P<0.05?.The expression of Muc5b gene in PM_2.5-exposed 24h A549 cells in low concentration group was significantly higher than that in 6h and 12h?P<0.05?,while that in medium concentration group was significantly higher than that in 6h and12h?P<0.05?,and that in 48h was significantly lower than that in 6h,12h and 24h?P<0.05?.?3?After 6h exposure to PM_2.5,the secretion of Muc5b in A549 cells in medium and high concentration groups was significantly higher than that in control group?P<0.05?,and that in high concentration groups was significantly higher than that in low and medium concentration groups?P<0.05?,and that in high concentration groups was significantly higher than that in control,low and medium concentration groups?P<0.05?at 12 and 48h after exposure.Muc5b secretion of A549 cells exposed to PM_2.5.5 for 24h and 48h in control group,low and medium concentration group was significantly higher than that of 6h and 12h?P<0.05?,and that of 48h was significantly higher than that of 24h?P<0.05?.?4?After 6h exposure to PM_2.5,the secretion of IL-1?in A549 cells in low concentration group was significantly higher than that in control group?P<0.05?,and the levels of IL-1?in medium and high concentration group were significantly lower than those in low concentration group?P<0.05?;after 12h exposure,the levels of IL-1?in medium and high concentration group were significantly lower than those in control group?P<0.05?,and the levels of IL-1?in high concentration group were significantly lower than those in low concentration group?P<0.05?;after 24h exposure,there was no Change?P>0.05?.After 48h exposure,the expression of IL-1?in low and high concentration groups was significantly lower than that in control group?P<0.05?.In the control group,the secretion of IL-1?in PM_2.5-exposed A549cells at 12h was significantly higher than that in 6h-exposed group?P<0.05?,and the levels at 24h and 48h were significantly lower than those in 12h-exposed group?P<0.05?;the levels at 24h and 48h in low concentration group were significantly lower than those at 6h,and at 24h and 48h were significantly lower than those in12h-exposed group?P<0.05?.In the medium concentration group,the 24h exposure was significantly lower than the 6h exposure?P<0.05?,and the 24h and 48h exposure were significantly lower than the 12h exposure group?P<0.05?.?5?After 6h exposure to PM_2.5,the secretion of TNF-a in A549 cells in low concentration group was significantly higher than that in control group?P<0.05?,while that in high concentration group was significantly lower than that in low and medium concentration group?P<0.05?.There was no significant change in each concentration group?P>0.05?at 12h and 48h,but at 24h,the levels in the medium and high concentration groups were significantly lower than those in the control group?P<0.05?.In the control group,the secretion of TNF-?in A549 cells was significantly lower at 48h after PM_2.5.5 exposure than at 6h and 24h after exposure?P<0.05?;in the low concentration group,the exposure time was significantly lower than that at 6h?P<0.05?;in the medium concentration group,the exposure time was significantly lower at 24h and 48h than that at 6h?P<0.05?;in the high concentration group,the exposure time was significantly higher at 12h than that at 6h?P<0.05?,and the infection time was significantly lower at 48h than that at 12h?P<0.05?.48h was significantly lower than 6h,12h and 24h?P<0.05?,and 24 h was significantly lower than 12h?P<0.05?.?6?After 48h exposure to PM_2.5,the Muc5b secretion of A549 cells in low concentration group was negatively correlated with the level of inflammatory factor TNF-??P<0.05?.3.Effect of PM_2.5.5 on Muc5b gene silencing and expression of inflammatory factors in A549 cells?1?After 6h exposure to PM_2.5,the expression of Muc5b in A549 cells in low and medium concentration groups was significantly higher than that in control group?P<0.05?;after 12h exposure,the expression of Muc5b in high concentration group was significantly higher than that in control group and low concentration group?P<0.05?;after 24h exposure,the expression of Muc5b in medium concentration group was significantly lower than that in control group?P<0.05?;after 48h exposure,the expression of Muc5b in medium concentration group was significantly lower than that in control group?P<0.05?.The expression level of Muc5b mRNA in low concentration group?P<0.05?,medium concentration group?P<0.05?and high concentration group?P<0.05?was significantly lower than that in 6h,12h and 24h?P<0.05?and 24h?P<0.05?respectively,while that in high concentration group?P<0.05?was significantly lower than that in 12h?P<0.05?.?2?At 6h and 48h of PM_2.5.5 exposure,the Muc5b secretion level of A549 cells in each concentration group was significantly higher than that of control group?P<0.05?;at 6h,the Muc5b secretion level in high concentration group was significantly lower than that in low and medium concentration group?P<0.05?;at 24h,the Muc5b secretion level in medium and high concentration group was significantly higher than that in control group?P<0.05?.Muc5b secretion level of Muc5b gene silenced A549cells in low concentration group was significantly higher than that in 12h?P<0.05?,24h and 48h in medium concentration group,6h and 12h in Muc5b secretion level?P<0.05?,24h and 48h in high concentration group,and 6h in Muc5b secretion level?P<0.05?.?3?After 6 and 24h of PM_2.5.5 exposure,the secretion of IL-1?in A549 cells in medium and high concentration groups was significantly higher than that in control and low concentration groups?P<0.05?,and 48h after PM_2.5.5 exposure,the secretion of IL-1?in medium and low concentration groups was significantly lower than that in control and low concentration groups?P<0.05?.PM_2.5.5 in medium concentration group was significantly lower than 6h?P<0.05?and 24h was significantly higher than 12h?P<0.05?;PM_2.5.5 in high concentration group was significantly lower than 6h?P<0.05?and 48h were significantly lower than 24h?P<0.05?.?4?After 6h exposure to PM_2.5,the level of TNF-?in Muc5b gene silenced A549cells in high concentration group was significantly higher than that in control group and medium concentration group?P<0.05?.PM_2.5.5 was exposed to PM_2.5.5 for 6h in low concentration group and medium concentration group,the TNF-?level was the lowest?P<0.05?.?5?Compared with normal A549 cells,the levels of IL-1?secretion of Muc5b gene silencing A549 cells increased significantly after 6h and 24h of moderate and high concentration PM_2.5.5 exposure and 48h of low concentration PM_2.5.5 exposure?P<0.05?;the levels of TNF-?silencing A549 cells by Muc5b gene decreased significantly after 6h of PM_2.5.5 exposure,and increased significantly in high concentration group?P<0.05?;and the levels of TNF-?silencing A549 cells by Muc5b gene silencing increased significantly after 12h,24h and 48h of PM_2.5exposure?P<0.05?.Muc5b gene silencing of inflammatory factor TNF-?in A549cells increased significantly in all concentration groups?P<0.05?.Conclusion:1.PM_2.5.5 exposure can affect the morphology and mucus secretion of trachea in rats.2.High concentration of PM_2.5.5 is cytotoxic and can affect the survival rate of A549 cells.3.Overall,the secretion of inflammatory factors IL-1?and TNF-?increased after Muc5b gene silencing in A549 cells compared with before gene silencing,suggesting that the inhibition of Muc5b may lead to an increase in the level of inflammatory factors.4.With the increase of Muc5b concentration,the inflammatory reaction was alleviated to some extent.