摘要目的 观察砷暴露大鼠肝脏中CD4+CD25+Foxp3+调节性T细胞(regulatory T cell,Treg)的表达变化,探讨砷致肝损害的免疫调节机制.方法 选取健康Wistar大鼠32只,按体质量采用随机数字表法分为对照组,低、中、高砷剂量组,每组8只.对照组给予去离子水灌胃,染砷各组按体质量用2.00 g/L的亚砷酸钠(NaAsO2)水溶液进行灌胃,依次为1.25、2.50、5.00 ml/kg,每周6 d,染砷时间为4个月.实验终期收集各组大鼠肝脏组织,采用电感耦合等离子体质谱仪(ICP-MS)检测肝砷含量;免疫组织化学法检测大鼠肝脏组织内Treg细胞的表达;酶联免疫吸附试验(ELISA)检测大鼠肝匀浆白细胞介素-10(IL-10)、转化生长因子-β1(TGF-β1)、IL-6、IL-17、IL-2的表达.结果 低、中、高砷剂量组肝砷含量[中位数(四分位数):638.30(527.91~802.58)、591.64(513.82~723.16)、792.55(695.93~1074.41)μg/g]较对照组[28.57(17.64~35.64)μg/g]增高(P均 < 0.05),且高砷剂量组高于中砷剂量组(P<0.05).中、高砷剂量组大鼠肝脏Treg阳性细胞面数密度[(2.25 ± 0.50)、(4.00 ± 2.16)个/cm2]高于对照组[(0.60 ± 0.54)个/cm2,P均 <0.05],且高砷剂量组高于低砷剂量组[(1.50 ± 0.58)个/cm2, P<0.05].低、中、高砷剂量组大鼠肝脏IL-10表达[(5.58 ± 1.70)、(6.78 ± 1.09)、(7.18 ± 0.53)μg/L]高于对照组[(2.32 ± 0.83)μg/L,P均 <0.05],且高砷剂量组高于低砷剂量组(P < 0.05);高砷剂量组TGF-β1表达[(9.06 ± 3.60)μg/L]高于对照组[(1.46 ± 0.65)μg/L,P < 0.05];高砷剂量组炎性细胞因子IL-6表达[(5.03 ± 1.39)μg/L]高于对照组[(2.33 ± 0.66)μg/L,P<0.05]和低砷剂量组[(2.46 ± 1.71)μg/L,P<0.05];而对照组,低、中、高砷剂量组肝脏内IL-17表达[(4.87 ± 1.64)、(7.50 ± 2.74)、(6.21 ± 1.47)、(7.23 ± 2.68)μg/L]组间比较,差异无统计学意义(F = 1.429,P > 0.05);高砷剂量组肝脏 IL-2表达[(9.93 ± 2.65)μg/L]低于对照组[(16.30 ± 3.98)μg/L,P<0.05].肝砷含量与肝脏IL-10、TGF-β1、IL-17、IL-6表达呈正相关(rs=0.696、0.463、0.632、0.502,P均 <0.05),与IL-2表达呈负相关(rs=-0.522,P<0.05).结论 随着砷暴露水平的增加,大鼠肝砷含量、CD4+CD25+Foxp3+Treg表达增加,相应细胞因子分泌异常;砷致大鼠肝脏免疫微环境紊乱,形成免疫耐受,抑制免疫清除,可能是砷致大鼠免疫性肝损害发生发展的重要原因.

abstractsObjective To observe the differential expression level of CD4+CD25+Foxp3+regulatory T cells (Treg) in liver of arsenic-exposed rats, explore the regulatory mechanisms on immunological of hepatic injury induced by arsenic, and provide a basis for prevention and treatment of the disease. Methods Thirty-two healthy Wistar rats were selected and randomly divided into control,low,medium and high arsenic dose groups by weight,8 rats per group. Rats in control group were given oral gavage of deionized water, while the other groups were given oral gavage doses of 2.00 g/L sodium arsenite(NaAsO2) according to their body weight for 6 days every week, the concentrations were 1.25, 2.50 and 5.00 ml/kg. After 4 months, liver tissue samples of rats were collected, the content of arsenic in liver was detected by inductively coupled plasma mass spectrometry (ICP-MS);the expression of Treg cells in liver was detected by immunohistochemistry; enzyme-linked immunosorbent assay (ELISA) was applied to detect the levels of interloukin-10 (IL-10),transforming growth factor beta 1 (TGF-β1), IL-6, IL-17 and IL-2. Results Compared with the control group [28.57 (17.64 - 35.64)μg/g], the content of arsenic in liver in low,medium and high arsenic exposed groups[M(P25-P75):638.30(527.91-802.58),591.64(513.82-723.16),792.55 (695.93 - 1 074.41) μg/g] increased, the differences were statistically significant(P < 0.05). Compared with low arsenic group, the content of arsenic in liver in high arsenic group increased, the difference was statistically significant (P < 0.05). Numerical density on area (NA) of positive Treg cells in medium,high arsenic exposed groups [(2.25 ± 0.50),(4.00 ± 2.16)A/cm2]was higher than that of the control group[(0.60 ± 0.54)A/cm2,P<0.05];NA of positive Treg cells in high arsenic exposed group was higher than that of the low arsenic exposed group[(1.50 ± 0.58) A/cm2, P < 0.05]. The expressions of the IL-10 in low, medium and high arsenic exposed groups [(5.58 ± 1.70), (6.78 ± 1.09),(7.18 ± 0.53)μg/L]were higher than that of the control group[(2.32 ± 0.83) μg/L,P<0.05];compared with low arsenic group, the expression of IL-10 in high arsenic group increased (P < 0.05); compared with control group [(1.46 ± 0.65) μg/L], the expression of TGF-β1 in high arsenic exposed group increased[(9.06 ± 3.60)μg/L, P<0.05];compared with control group [(2.33 ± 0.66)μg/L], the expression of IL-6 in high arsenic exposed group increased [(5.03 ± 1.39) μg/L, P < 0.05], compared with low arsenic exposed group [(2.46 ± 1.71) μg/L], the expressions of IL-6 in high arsenic exposed group increased, the difference was statistically significant (P < 0.05);the expression of IL-17 among control, low, medium and high arsenic exposed groups[(4.87 ± 1.64),(7.50 ± 2.74), (6.21 ± 1.47),(7.23 ± 2.68)μg/L]were not statistically significant (F = 1.429, P > 0.05); compared with control group [(16.30 ± 3.98) μg/L], the expression of IL-2 in high arsenic exposed group decreased[(9.93 ± 2.65) μg/L, P <0.05]. The content of arsenic in liver was positively correlated with the expression of IL-10, TGF-β1, IL-17, IL-6 (rs=0.696,0.463,0.632,0.502,P<0.05),and negatively correlated with the expression of IL-2(rs=-0.522,P<0.05). Conclusion With increasing of arsenic exposure level, the content of arsenic in liver and the expression of CD4+CD25+Foxp3+Treg have increased,the cytokines are secreted abnormally,liver immunological micro environment is disordered,immune tolerance is formed,and immune clearance is inhibited,which may play an important role in the occur and development of immunological liver damage induced by arsenic in rat.