| The main component of quartz is free silica(molecular formula SiO2).Long-term inhalation of dust with a high content of free silica can cause an occupational disease––silicosis,which was characterized by progressive pulmonary fibrosis.Silicosis is the occupational disease with the highest incidence in China at present,of which the pathogenic mechanism was not yet fully understood,making it a current research hotspot.Studies have shown that transforming growth factor-β(TGF-β)was the core factor causing fibrosis,for antagonizing its expression level could inhibit the occurrence and development of fibrosis but would also affect its normal function of regulating immunity and cause other immune diseases.Recent studies have shown that IL-11 is an important downstream regulator of TGF-β and plays an extremely important role in the development of fibrosis in various organs such as heart,kidney,and lung.Exogenous supplementation of IL-11 recombinant protein could significantly increase the expression levels of extracellular matrix and collagen in heart and kidney tissues of mouse,which contributed to the formation of tissue fibrosis obviously.While inhibiting the expression of IL-11 could not only independently inhibit the formation of fibrosis,but also effectively block the fibrosis dominated by TGF-β.To explore the role of IL-11 in silica-induced inflammatory reaction and fibrosis in mice lungs,a silicosis model was constructed to observe the changes of IL-11 expression level in the lung tissue of mice and effects on the expression of inflammatory and fiber-related factors after silica exposure,and to evaluate the alleviating effect on inflammatory response and fibrosis progress by inhibiting the expression of IL-11.This study was divided into two parts:(1)to explore the expression of IL-11 after instilling the silica suspension to the mice lungs and perform the dose screening of IL-11 neutralizing antibody;(2)to explore the role of IL-11 in the inflammation and fibrosis progressof mice lung tissue caused by silica.Part Ⅰ.The expression of IL-11 in mice lung exposed to silica and screening IL-11 neutralizing antibody intervention dosesObjective: To assess the expression of IL-11 and the effect of IL-11 neutralizing antibody after silica exposure in mice lung.To screen a reasonable range of intervention doses for IL-11 neutralizing antibody.Methods: Ⅰ.A model of silicosis was established by intracheal instillation of silica dust suspension.90 C57 BL / 6 male mice were randomly divided into 5 groups(18 in each group): blank control group;phosphate buffered saline(PBS)control group;Silica low-dose(0.5 mg/mice)group;Silica medium-dose(1.5 mg/mice)group;Silica high-dose(2.5 mg/mice)group.The blank control group did not receive any treatment;the PBS control group was given 50μL of sterile PBS by intracheal instillation;each Silica group was given 50 μL of Chinese silica of different doses suspended in sterile PBS.In each group,6 mice were sacrificed randomly at 7 days,28 days and 84 days after Silica exposure.Lung tissues were collected,and the protein expression levels of IL-11 in mice lung tissues were detected by western-blotting.Ⅱ.The same method was used to establish a model of silica exposure in mice,an exogenous IL-11 neutralizing antibody was given to establish an IL-11 inhibition model.60 C57 BL / 6 male mice were randomly divided into 5 groups as PBS group;Silica group;Silica + anti-IL-11(5μg / mice);Silica + anti-IL-11(10μg / mice);Silica + anti-IL-11(20μg / mice).The Silica group exposed by 2.5 mg silica andthree anti-IL-11 group treated by 2.5 mg silica and a corresponding dose of IL-11 neutralizing antibody.In each group,6 mice were sacrificed randomly at 7 days and 14 days after exposure.The Broncho alveolar lavage fluids(BALF)were collected and the expression levels of IL-11 in the BALF were detected by enzymelinked immunosorbent assay(ELISA).Results: Ⅰ.After instilling treatment,the expression level of IL-11 protein in the lung tissue of mice increased.With the increase of the dose of Silica,the content of IL-11 in lung tissue gradually increased.Compared with the blank control group and the PBS group,the IL-11 expression level of the silica medium-dose and high-dose groups was significantly increased at 7 days and 28 days after exposure,and the differences were statistically significant(P < 0.05).The expression level of IL-11 in the low-dose group was not significantly increased(P > 0.05).At 84 days after silica exposure,the expression level of IL-11 in the silica high-dose group was significantly higher than that in the blank group and the PBS group,and the differences were statistically significant(P < 0.05),there was no significant increase in IL-11 expression level in the silica low-dose or medium-dose groups(P > 0.05).Ⅱ.After treatment,the content of IL-11 in mice alveolar lavage fluid was significantly higher than that in PBS group,the difference was statistically significant(P < 0.05).IL-11 neutralizing antibody could inhibit the expression level of IL-11 at 7 days after silica exposure.Compared with silica group,IL-11 expression levels in silica + anti-IL-11(10μg / mice),Silica + anti-IL-11(20μg / mice)groupwere significantly reduced,the differences were statistically significant(P < 0.05).After 14 days of silica exposure,the inhibitory effect of IL-11 neutralizing antibody was reduced.Compared with the silica group,only the silica + anti-IL-11(20μg / mice)group had a significant difference in IL-11 expression(P < 0.05).Conclusions: After silica exposure,the expression level of IL-11 in the lung tissue of mice increased;IL-11 neutralizing antibody could effectively inhibit the expression level of IL-11 in BALF of mice,and the effect of highest intervention dose(20μg)was the most obvious.The neutralization effect decreased from 14 days after silica exposure,suggesting that IL-11 neutralizing antibody should be supplemented every 14 days to maintain its inhibitory effect.Part Ⅱ.The effect of IL-11 in silica-induced inflammatory reaction and fibrosis in mice lungObjective: Ⅰ.To explore the role of IL-11 in silica-induced pulmonary inflammation;Ⅱ.To investigate the role of IL-11 in silica-induced lung fibrosis.Methods: 144 C57 BL / 6 male mice were randomly divided into 6 groups(24 in each group): blank control group,PBS group,silica low-dose(0.5 mg/mice)group,silica mediumdose(1.5 mg/mice)group,silica high-dose(2.5 mg/mice)group,and silica(2.5 mg/mice)+anti-IL-11(20 μg/mice)group.The treatment method was the same as the Part I.In the Silica + anti-IL-11 group,IL-11 neutralizing antibody was injected intraperitoneally every 14 days(20 μg/mice).Eight mice were sacrificed randomly at 7 days,28 days,and 84 days after silica exposure,BALFs and lung tissues were collected.Western-bloting was used to assess the expression of IL-11 protein in lung tissue;ELISA was used to detect the expression levels of IL-6 and TNF-α in BALF;RT-q PCR was used to detect the relative m RNA expression levels of IL-6,IL-1β,COL1A1,and fibronectin in lung tissue;HE,Masson,and Sirius red staining were used to measure the degree of inflammation and fibrosis in lung tissue.Results: Ⅰ.The effect of IL-11 on the inflammatory reaction of the lung caused by silica: H&E staining results showed that compared with the blank control group and the PBS group,the mice in each silica exposure group showed more obvious inflammatory infiltration and granulomatous inflammation.With the increase of the dose of silica,the inflammatory response tended to increase.Compared with the silica high-dose group,the IL-11 neutralizing antibody group’s lung tissue inflammation degree was alleviated;compared with the blank control group and PBS group,the relative expression levels of IL-6 and IL-1-β m RNA in the lung tissue of the Silica high-dose group were significantly increased at 7 days and 28 days after exposure,and the differences were statistically significant(P < 0.05);The expression levels of IL-6 and TNF-α in mice BALF increased significantly,the differences were statistically significant(P < 0.05).IL-11 neutralizing antibody could significantly reduce the relative expression levels of IL-6 and IL-1β m RNA in mice lung tissue and the expression levels of IL-6 and TNF-α in alveolar lavage fluid(P <0.05),but TNF-α had no statistically significant difference in the 84 days group(P > 0.05).Ⅱ.The effect of IL-11 on silica-induced pulmonary fibrosis: The results of Masson staining and Sirius red staining showed that compared with the control group,mice in the high-dose silica group had different levels of collagen fiber deposition and fiber clumps at each time point,and the degrees of fibrosis gradually increased with time after exposure.The above-mentioned lesions of mice lungs of IL-11 neutralizing antibody group at all time points were significantly alleviated.At each time point,the relative expression levels of COL1A1 and Fibronectin m RNA in the lung tissue of the silica high-dose group were significantly increased compared with the blank control group and PBS groups,and the differences were statistically significant(P < 0.05).At 28 days and 84 days,the relative expression levels of COL1A1 and Fibronectin m RNA in the IL-11 neutralizing antibody group were significantly lower than those in the high-dose group(P < 0.05).At 7 days,COL1A1 protein level showed no significant difference between each two groups,,but was higher in the Silica high-dose group than that of the control group at 28 days and 84 days(P < 0.05),with lower in the IL-11 neutralizing antibody group than the corresponding Silica group.The above-mentioned indicators in the low and medium dose Silica group showed an upward trend,but there were no statistically significance(P > 0.05).Conclusions: Silica caused inflammation and fibrosis in lung tissue of mice;IL-11 neutralizing antibody could effectively reduce the inflammation and fibrosis degree in mice lung tissue. |
