Antagonistic Effect Of Naringin On Skin Injury Induced By Sodium Arsenite In Mice And Its Mechanism

Objective:Long-term low arsenic exposure is a serious threat to human health,which can cause many organ diseases,among which arsenic-induced skin damage is the most typical and common.Oxidative stress and oxidative damage induced by arsenic exposure may be one of the important mechanisms of arsenic-induced skin toxicity,and NF-κB pathway and apoptosis are also closely related to arsenic exposure.Naringin is a natural flavonoid glycoside,which has many pharmacological activities,such as antioxidation,anti-inflammatory,anti-apoptosis and anticancer.In order to further explore the antagonistic effect and mechanism of naringin on skin injury induced by sodium arsenite in mice,this study took Kunming mice exposed to arsenic as the intervention object,and took naringin as the intervention measure to observe the effects of naringin on organ coefficient,skin histopathological changes,and oxidative stress related indexes in serum and skin of mice exposed to arsenic.To investigate the expression of naringin on Nrf2 pathway and its downstream antioxidant enzyme gene,NF-κB pathway and apoptosis-related proteins,and to analyze the antagonistic effect of naringin on oxidative stress,immune response and apoptosis of mouse skin tissue caused by arsenic exposure and its possible mechanism.Methods:1.Experimental animals and grouping:60 female Kunming mice were randomly divided into 6 groups according to their body weight,with 10 mice in each group.The specific groups were as follows:Control group（Con）;Naringin group（Nar）;Simple arsenic exposure group（As）;Low dose Nar+arsenic exposure group（Nar 20 mg/kg/day）;Middle dose Nar+arsenic exposure group（Nar 40 mg/kg/day）;High dose Nar+arsenic exposure group（Nar 80mg/kg/day）.Mice in the control group and naringin group drank distilled water,while mice in other groups drank 50mg/L aqueous solution of sodium arsenite（Na As O₂）.Mice were exposed to arsenic for 12 weeks（84 days）,and then treated with drugs for 6 weeks（42 days）.Weigh and record the weight of mice every week,and observe the drinking situation of mice.After 42 days,naringin was given to mice in naringin intervention group at the dose of 20,40 and 80 mg/kg/day respectively.Mice in naringin group were given oral gavage at a dose of 80 mg/kg/day in the last 42 days,while mice in arsenic group were given the same volume of normal saline in the last 42 days.Urine was collected at the beginning,middle and end of Naringin intervention,and skin and blood samples were collected at the end（12 weeks）to be tested.2.Specific detection indexes and methods:（1）Morphological observation of mouse skin tissue:HE staining was used to observe the morphological changes of skin tissue;（2）Determination of oxidative damage index:SOD,MDA and CAT kit detection;（3）Determination of ROS in mouse skin tissue:fluorescent enzyme-labeled instrument;（4）The expression of Nrf2pathway and its downstream gene protein in mouse skin:Western Blot analysis;（5）Expression of NF-κB pathway protein in mouse skin:Western Blot analysis;（6）Expression of apoptosis-related proteins in mouse skin:Western Blot analysis;（7）Determination of keratin expression level in mouse skin tissue:Western Blot analysis;（8）Determination of various forms of arsenic in mouse urine:hydride generation-ultralow temperature deposition-atomic absorption spectrophotometry.3.In this study,SPSS software（version 19.0）was used for data analysis,and one-way ANOVA was used for comparison among groups.SNK or Dunnett’s T3 test was used to compare the two groups,and there was statistical significance when P<0.05.Results:1.Effect of Nar intervention on body weight and organ coefficient of mice induced by sodium arsenite.Three weeks before exposure,the weight of mice in all groups showed an increasing trend,and there was no obvious change between different groups.After exposure for 12weeks,the weight of mice did not change significantly.Compared with the control group,the organ coefficient of mouse spleen was significantly decreased in Nar group,arsenic group and Nar intervention group with 20 and 40 mg/kg（P<0.05）.There was no significant difference in organ coefficients of liver,kidney and lung in each group.2.Effect of Nar intervention on histopathologic changes of mice skin induced by sodium arsenite.In the skin histopathological sections of mice exposed to poison for 12 weeks and 6weeks,the skin structure of mice in the control group was intact,the epidermis was not significantly thickened,and the cuticle was not significantly keratosis.In the arsenic group,the epidermis thickened and the keratosis of the stratum corneum were observed.No pathological changes were observed in 40 mg/kg Nar and 80 mg/kg Nar intervention groups.It can be seen in the pathological sections of mouse skin tissue that the skin tissue structure of the control group is complete,the epidermis has no obvious thickening,and the stratum corneum has no obvious keratinization;However,in the arsenic exposure group,epidermis thickening and severe keratinization of stratum corneum appeared.40 and 80mg/kg Nar intervention group did not show the above pathological changes in skin tissue.3.Effect of Nar intervention on serum and skin tissue oxidative stress induced by sodium arsenite in mice.Compared with the control group,MDA in serum of mice exposed to arsenic alone increased significantly（P<0.05）,while SOD in 20 mg/kg Nar intervention group decreased（P<0.05）,MDA decreased after 40 mg/kg Nar and 80 mg/kg Nar intervention（P<0.05）,and SOD also rebounded.There was no significant difference in serum CAT activity between groups.Compared with the control group,the ROS in skin tissue of mice exposed to arsenic alone and 40 mg/kg Nar intervention group increased significantly（P<0.05）,and the ROS level decreased after 80 mg/kg Nar intervention compared with the corresponding arsenic alone group（P<0.05）.4.Effect of Nar intervention on sodium arsenite induced oxidative stress in mouse skin.Compared with the control group,the expression levels of Nrf2 protein in arsenic-only group,20 mg/kg and 40 mg/kg Nar intervention group were all up-regulated（P<0.05）,while the expression levels of NQO1 protein in arsenic-only group and 80 mg/kg Nar intervention group were up-regulated（P<0.05）.Compared with the arsenic exposure group,the protein expression level of Nrf2 in 40 and 80 mg/kg Nar intervention group was further increased（P<0.05）,and the protein expression level of NQO1 in 80 mg/kg Nar intervention group was further increased（P<0.05）,but the protein expression of HO-1was not obvious among the groups.5.Effect of Nar intervention on NF-κB of mouse skin tissue transcription factor induced by sodium arsenite.Compared with the control group,the protein expression level of NF-κB in the skin of mice exposed to arsenic alone increased（P<0.05）.Compared with arsenic exposure group,the expression level of NF-κB protein in 40 and 80 mg/kg Nar intervention group decreased in a dose-dependent manner（P<0.05）.6.Effect of Nar intervention on expression level of sodium arsenite induced apoptosis protein in mouse skin tissue.Compared with the control group,the expression of Bcl-2 was down-regulated（P<0.05）,and the protein expression levels of Caspase-3 and Bax were up-regulated（P<0.05）.Compared with the arsenic alone group,the protein expression level of Bcl-2 in 40 and 80mg/kg Nar intervention groups was up-regulated（P<0.05）,and the protein expression level of Caspase-3 was down-regulated（P<0.05）.7.Effect of Nar intervention on the expression level of keratin in mouse skin tissue induced by sodium arsenite.Compared with the control group,the protein expression levels of KRT1 and KRT10in the arsenic-only group increased significantly（P<0.05）,and the protein expression levels of KRT1 in the 20、40 and 80 mg/kg Nar intervention groups decreased（P<0.05）,while those in the 40 and 80 mg/kg Nar intervention groups decreased（P<0.05）.8.Effects of Nar intervention on urinary arsenic level and arsenic metabolism in mice induced by sodium arsenite.The results showed that with the extension of naringin intervention time,the contents of i As and t As in urine of mice in the naringin intervention group were significantly increased（P<0.05）,but the changes of arsenic methylation were not significant.Conclusion:1.Arsenic exposure can cause pathological and oxidative damage to the skin tissue of mice.2.Naringin can antagonize the skin damage induced by arsenic exposure in mice by regulating Nrf2 pathway and apoptosis pathway.3.Naringin can promote arsenic excretion in mice exposed to arsenic.