| Objective:Public health problems caused by arsenic exposure in drinking water have attracted worldwide attention.As one of the main target organs,lung tissue is particularly sensitive to arsenic exposure.The inflammatory response and oxidative stress caused by arsenic exposure are currently widely accepted and studied mechanisms of arsenic toxicity,and autophagy dysfunction is also closely related to arsenic exposure.Curcumin has a variety of pharmacological activities such as anti-inflammatory,anti-tumor and antioxidant.In order to further explore the possible antagonistic effect of curcumin on chronic lung arsenic exposure induced by drinking water,this study took NaAsO2 and curcumin as treatment factor and intervention factor,respectively,to observe the curcumin intervention on the lung weight of mice with chronic inorganic arsenic exposure.At the same time related factors such as organ coefficients,lung tissue pathological changes,serum cytokine content,oxidative stress（GSH and ROS）,and inflammatory response（MPO and BALF total protein）;and explore the nuclear transcription factors NF-κB and The changes of MAPK pathway,Nrf2 pathway and autophagy-related pathways were attempted to explore the antagonism and possible mechanism of curcumin on inflammatory response,oxidative damage and autophagy in lung tissue of mice induced by arsenic exposure to drinking water.Methods:1.Experimental animals and grouping:Experimental animals were selected.SPF-grade female Kunming and Babl/c mice,54 each of them were adaptively reared for one week,and then divided into 6 groups according to the random number table according to body weight.The grouping is as follows:control group,arsenic-exposed group（10 mg/L As,25 mg/L As）,curcumin-only intervention group（200 mg/kg Cur）,and curcumin-intervention group（10 mg/L As+200 mg/kg Cur,25 mg/L As+200mg/kg Cur）.NaAsO2 was used for free drinking water and curcumin was given at the same time.Gavage was given twice a week for 6 weeks and 12 weeks.After the exposure,the mice were sacrificed after CO2 anesthesia,and the whole blood,serum,urine and complete lung tissue samples of mice was collected and tested.2.Detection indicators and methods:（1）Determination of total arsenic content in mouse urine and lung tissue in vivo:atomic fluorescence absorption assay;（2）pathological changes in mouse lung tissue:HE staining;（3）serum cells Factor level detection:Elisa kit detection;（4）Whole blood GSH activity measurement:DTNB method;（5）Mouse lung tissue ROS measurement:flow cytometry method;（6）MPO activity measurement in lung tissue:kit method;（7）Determination of protein content in mouse bronchoalveolar lavage fluid（BALF）:BCA kit method;（8）Expression of transcription factor NF-κB and MAPK pathway proteins in lungs:western blot analysis;（9）Nrf2 And downstream protein expression:western blot analysis and immunofluorescence analysis;（10）protein levels of Akt-mTOR signaling pathway:western blot analysis and immunofluorescence analysis.Results:1.Effect of curcumin intervention on organ weight,organ coefficient and total arsenic content in lung tissue of mice exposed to arsenic in drinking water.There was no significant difference in lung weight and organ coefficient between groups after 6 and 12 weeks of arsenic exposure and curcumin intervention.The total arsenic content in urine of arsenic exposed mice increased（P<0.05）;after curcumin intervention,compared with the corresponding simple exposure group,the total arsenic content in urine increased（P<0.05）;but the total arsenic content in lung tissue decreased significantly compared with the corresponding arsenic exposure group（P<0.05）.2.Effect of curcumin intervention on pathological changes of lung tissue in mice exposed to arsenic in drinking water.In the pathological sections of lung tissue of mice at 6 and 12 weeks after the intervention,the alveolar structure of mice in the control group was complete and clear,while in the lung tissue of mice only exposed to curcumin,a large number of inflammatory cells infiltrated,while in the lung tissue of mice exposed to curcumin,there was no inflammatory cells infiltrated,and the lung had complete tissue morphology.3.Effect of curcumin intervention on serum cytokine content in mice exposed to arsenic in drinking water.Compared with the control group,the level of cytokines in the serum of the mice in each single exposure group decreased significantly after 6 weeks of feeding（P<0.05）.After curcumin intervention,the level of cytokines in the serum of the mice in the corresponding single exposure group also decreased（P<0.05）.The levels of IL-13and IL-17 in the serum of mice fed for 12 weeks after curcumin intervention were significantly higher than those in the corresponding group（P<0.05）.4.Effect of curcumin on oxidative stress in lung tissue of mice exposed to arsenic in drinking waterCompared with the control group,there was no significant difference in the GSH content in the whole blood of the mice fed for 6 weeks,but in the mice fed for 12weeks,the GSH content in the whole blood of the single exposure group was significantly lower than that of the control group（P<0.05）;the GSH content in the whole blood of the curcumin intervention group and the corresponding single exposure group was higher than that of the whole blood（P<0.05）.There was no significant difference in ROS of lung tissue between the control group and the control group after 6 weeks of feeding,but the level of ROS decreased slightly after the intervention of curcumin（P<0.05）;compared with the control group,ROS increased in the 10 mg/L group and decreased in the curcumin group after12 weeks of feeding（P<0.05）.5.Effect of curcumin on the inflammatory response of lung tissue in arsenic exposed mice.In the 6 and 12 weeks fed mice,compared with the control group,the MPO activity increased in arsenic groups（P<0.05）.The MPO activity decreased significantly after curcumin intervention compared with the corresponding group（P<0.05）.Compared with the control group,the total protein concentration of BALF was significantly higher in the arsenic exposure group（P<0.05）,and the protein concentration in BALF after curcumin intervention was lower than that in the corresponding exposure group（P<0.05）.6.Effect of curcumin on NF-κB and MAPK pathway in lung tissue of arsenic exposed miceThe lung tissue of mice in the curcumin intervention group was significantly lower than that in the control group（P<0.05）.Compared with the control group,the expression level of NF-κB protein in the lung tissue of mice fed for 12 weeks increased significantly（P<0.05）;compared with the control group,the expression level of NF-κB protein in the curcumin intervention group decreased significantly（P<0.05）.Compared with the control group,the protein levels of MAPK pathway in the lung tissue of the mice in the simple exposure group increased significantly（P<0.05）.Compared with the control group,the protein levels of p-JNK,p-ERK and p-p38 in the curcumin intervention group were significantly reduced（P<0.05）;compared with the control group,the protein levels of p-ERK in the curcumin intervention group were significantly increased（P<0.05）.Compared with the control group,the protein levels of p-ERK and p-p38 in the lung tissue of mice in the 12 weeks feeding group increased significantly（P<0.05）;compared with the control group,the protein levels of p-ERK and p-p38 in the curcumin intervention group decreased significantly（P<0.05）.Compared with the control group,the protein level of p-p38 increased significantly（P<0.05）.7.Effect of curcumin on Nrf2 pathway protein in lung tissue of arsenic exposed mice.In the lung tissue of mice fed for 6 weeks,the protein expression content of Nrf2 in the lung tissue of mice in the single exposure group was significantly increased compared with that in the control group（P<0.05）;at the same time,the protein expression content of NQO1 in the lung tissue of mice in the 25 mg/L single exposure group was significantly increased（P<0.05）.The expression of Nrf2 and NQO1protein in curcumin treated group was significantly higher than that in control group（P<0.05）.Compared with the control group,the expression of Nrf2 and NQO1 in lung tissue of mice treated with curcumin alone increased significantly（P<0.05）.The levels of Nrf2,GCLC and HO-1 protein in the lung tissues of the mice were significantly higher than those of the control group（P<0.05）.The protein levels of Nrf2,GCLC and HO-1 in each curcumin intervention group were significantly higher than those in the corresponding single exposure group（P<0.05）.Compared with the control group,the expression of Nrf2 and its downstream proteins GCLC and HO-1 in the lungs of mice after curcumin intervention was significantly higher（P<0.05）.Exposure to arsenic in drinking water did not induce Nrf2 protein to enter the nucleus,but increased after curcumin intervention.Curcumin may play a role in promoting Nrf2 protein into nucleus.8.Effect of curcumin on autophagy pathway in lung of arsenic exposed mice.Compared with the control group,Akt and mTOR protein phosphorylation was significantly increased in the lung tissue of mice in the simple exposure group（P<0.05）,while TFEB protein expression in the simple exposure group was significantly decreased（P<0.05）.Compared with the control group,Akt and mTOR protein phosphorylation decreased significantly after curcumin administration.Conclusion:1.Curcumin intervention can reduce the accumulation of arsenic in lung tissue,reduce the inflammatory damage,oxidative damage and autophagy dysfunction of lung tissue,which has a certain antagonistic effect on the lung injury of mice exposed to arsenic in drinking water.2.Curcumin may inhibit lung injury of mice exposed to arsenic by inhibiting NF-κB and MAPK pathway,activating Nrf2 pathway and activating autophagy pathway. |
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