| Objective: Carbon black particles not only exist in workplaces such as rubber,automobile tires and ink manufacturing industries,but also derive from agricultural production emissions,road traffic exhaust,fuel and biomass combustion in our living environment.Prolonged inhalation of carbon black particles is associated with the occurrence or development of various respiratory diseases such as chronic bronchitis,asthma,chronic obstructive pulmonary disease.Pulmonary inflammation is an important common response in the pathogenesis of these diseases.Histone deacetylase 6(HDAC6)and its inhibitors have been proved to play a certain regulatory role in the development of some chronic inflammation.But it is still unclear whether HDAC6 participates in the pulmonary inflammation caused by carbon black particles.Therefore,the purpose of this study is to explore the role and mechanism of HDAC6 in lung inflammation caused by carbon black particles in mice.Methods: C57BL/6 male mice were randomly divided into carbon black particle exposure group(n=30),normal saline group(n=30)and blank control group(n=30).Carbon black granule group and normal saline group were administered with single intratracheal instillation of carbon black particles(Printex90,250?g / 50?l)or normal saline(50?l),respectively.The blank control group was given no treatment.Mice were sacrificed on days 1,7,28,56 and 84 after carbon black particles exposure,and then lung tissues,bronchoalveolar lavage fluid(BALF)and peripheral blood samples were collected.Hematoxylin-eosin staining and immunohistochemical staining were used to observe the degree of inflammation and the localization of HDAC6 and intraflagellar transport proteins(IFT20,IFT88)in the lung tissue of mice.The expressions of IL-8 in BALF and peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA).Western blotting(WB)was used to detect the expressions of HDAC6,SHh(an important marker protein of sonic hedgehog signal pathway),IFT20,IFT88,and LC3-II/ LC3-?(autophagy related protein).In addition,HDAC6 specific inhibitor(tubastatin a,TSA,1 mg / ml,injection volume: 2 mg / kg)was given by intraperitoneal injection to mice when exposed to carbon black particles on days 0,7 and 28.The reagent control group was given intraperitoneal injection with 0.2% DMSO(n=30).The mice with TSA treatment were divided into three groups including TSA treatment before carbon black particles exposure(day 0)group(n=30),TSA treatment on day 7 exposure(day 7)group(n=24),TSA treatment on day 28 exposure(day 28)group(n=18).Then mice were sacrificed on days 1,7,28,56 and 84 with carbon black particles exposure.We compared the changes of pulmonary inflammatory response of mice treated with TSA at different exposure times to explore the role of HDAC6 specific inhibitors in the process of pulmonary diseases caused by carbon black particles.Results:(1)On days 1,7,28,56 and 84 after carbon black particles exposure,there was obvious inflammation in the lung tissue of mice,especially on the day 56.On day 84,the alveoli wall became thinner,broken and fused into sacs,and blood capillary of alveolar wall was decreased,showing similar to the pathological changes of emphysema.The expression of IL-8 in BALF and serum increased continuously on days 1,7,28 and 56 exposure(P < 0.05),and decreased to normal level on day 84.At the same time,HDAC6 and LC3-?/LC3-? in the lung tissue of mice exposed to carbon black particles showed an overall increasing trend,and reached the peak value on day 56.Compared with the blank group,the difference was statistically significant(P<0.05),while the expression on day 84 was slightly lower than that on day 56.However,the expression of IFT88 protein showed a downward trend,and reached the lowest level on the day 56,significantly lower than the blank group(P<0.05),then recovered to the initial level on the day 84.In addition,the expression of IFT20 increased gradually with the prolongation of the exposure time,and reached the highest level on the day 84.Compared with the blank control group,the difference was statistically significant(P< 0.05).The expressions level of SHh protein on the day 28 and 56 after exposure was significantly higher than that in the blank control group(P < 0.05).(2)Compared with the carbon black exposure group,TSA given before exposure could not reduce the degree of carbon black particles induced lung inflammation in mice,and the expression of IL-8 in serum did not decrease significantly.TSA given on on day 7 exposure reduced lung inflammation on the day 56 and 84.At these two exposure time points,the alveolar structure was basically restored,the expression of IL-8 in serum was significantly reduced,the difference was statistically significant(P<0.05);TSA treatment on day 28 exposure could reduce inflammation on day 84,the structure of alveolar wall and alveolar septum was basically restored,and the expression level of IL-8 in serum was significantly reduced on day 56.The difference was statistically significant(P <0.05).The expression of HDAC6 protein in lung tissue of each group treated with TSA decreased,especially on the day 28 after exposure,whether it was given on day 0 exposure or on day 7,the expression of HDAC6 in lung tissue was significantly lower than the pure carbon black particles exposure group(P <0.05).In addition,on day 56 after exposure,when TSA given before,on day 7 or on day 28 exposure,the expression of IFT88 was significantly increased,compared with the pure carbon black exposure group(P < 0.05).In addition,compared with the carbon black particles exposure group at each time point,although the expression of autophagy-related proteins LC3-?/LC3-? in the lung tissue decreased after TSA administration,but difference was not statistically significant(P> 0.05).And no matter when TSA is administered,there is no significant effect on the expression of IFT20 and SHh protein in the lungs of mice(P> 0.05).Conclusion:(1)Carbon black particles could induce inflammation in the lungs of mice,and with the prolongation of the exposure time,the inflammation of the lungs was the most serious on day 56,and even developed into emphysema on day 84.The expression levels of inflammatory cytokine IL-8 in serum and BALF also gradually increased,basically consistent with the degree of lung inflammation.HDAC6 specific inhibitor was administered on day 7 exposure,which significantly reduced the level of pulmonary inflammatory response and the expression of IL-8.(2)Carbon black particles could induce the specific expression of HDAC6 in the lung tissue of mice,and the expression level of autophagy related protein,IFT20 and SHh were also increased,while the expression level of IFT88 protein was decreased.After HDAC6 specific inhibitor was given,the expression level of IFT88 in the lung was increased,and the autophagy level wass decreased,suggesting that HDAC6 and primary ciliary autophagy pathway may be involved in the inflammatory reaction induced by carbon black particles.(3)Carbon black particles could significantly increase the expression level of IFT20 protein and SHh protein(an upstream protein in SHh signaling pathway),However,HDAC6 specific inhibitors had no significant effect on the expression of IFT20 protein and SHh protein,suggesting that the pulmonary inflammatory response induced by carbon black particles may be involved by IFT20 and other signaling pathways like SHh signaling pathway.(4)In the process of lung inflammation induced by carbon black particles,there is no significant effect on lung inflammatory reactants if HDAC6 specific inhibitor was given before the inflammation.However,in the early stage of lung inflammation,HDAC6 specific inhibitor could effectively inhibit the middle and late stage of lung inflammation caused by carbon black particles,suggesting that the best time point of HDAC6 specific inhibitor may be the early stage of lung inflammation caused by carbon black particles. |
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