吡喹酮对日本血吸虫急性感染小鼠肾脏功能损伤的影响

Effect of praziquantel on impaired renal function in mice with acute infection of Schistosoma japonicum

目的研究日本血吸虫感染急性期小鼠肾脏炎症的发生、发展进程,评价吡喹酮对急性期感染小鼠肾脏功能损伤的治疗效果。方法取55只C57BL/6J雌性小鼠,随机分为健康对照组(20只)、健康对照给吡喹酮组(5只)、感染组(20只)、感染给吡喹酮组(5只)和吡喹酮杀虫组(5只)等5组,其中后3组每只小鼠均经腹部皮肤感染(14±2)条日本血吸虫尾蚴。感染后第6周开始给予吡喹酮治疗,健康对照给吡喹酮组和感染给吡喹酮组给药剂量为300 mg/kg,每12小时给一次药,连续给药2周;吡喹酮杀虫组给药剂量为250mg/kg,连续给药3天。健康对照组和感染组小鼠在感染后第1、3、6和8周各剖杀5只,健康对照给吡喹酮组、感染给吡喹酮组和吡喹酮杀虫组在感染后第8周剖杀,收集各组各时间点小鼠的尿样和血样,检测尿蛋白和血清肌酐水平,以评价肾功能变化。取小鼠肾脏,制备病理切片,经HE染色后,观察各组小鼠肾脏组织病理学改变;经F4/80免疫组化染色,观察各组小鼠肾脏中巨噬细胞浸润情况。分别用实时荧光定量PCR(qRT-PCR)和Western blotting检测各组小鼠肾脏炎症相关因子(TNF-α、TGF-β、IL-1β、IL-6)和巨噬细胞趋化因子(CCL2)的mRNA相对转录水平和蛋白表达水平。结果感染后第1、3、6和8周,感染组小鼠的尿蛋白含量分别为(1.67±0.52)、(2.17±0.41)、(3.00±0.89)和(4.00±0.63) g/L,均高于健康对照组小鼠(P<0.01);感染组小鼠的血清肌酐水平分别为(10.01±1.45)、(13.00±2.12)、(14.33±1.21)和(19.00±2.54)μmol/L,均高于健康对照组小鼠(P>0.05或P<0.01)。qRT-PCR检测结果显示,感染组小鼠的炎症因子TNF-αmRNA相对转录水平分别为2.47±0.51、3.14±1.43、1.83±0.31和7.48±5.54, TGF-βm RNA相对转录水平分别为0.94±0.24、2.80±1.07、2.72±0.88和3.03±0.36, IL-1βmRNA相对转录水平分别为1.49±0.28、4.32±1.85、2.08±0.69和2.81±0.73, IL-6 mRNA相对转录水平分别为1.96±0.31、1.48±0.35、2.38±1.17和2.85±1.32,均高于对应时间点的健康对照组小鼠(P<0.05或P<0.01)。Western blotting分析结果显示,感染组小鼠的TNF-α和IL-1β蛋白表达水平均较健康对照组升高。感染组小鼠的巨噬细胞趋化因子CCL2 mRNA相对转录水平为1.84±0.17、3.42±0.85、3.03±1.00和2.63±0.43,均高于健康对照组小鼠(P<0.01)。肾脏病理切片HE染色结果显示,感染组小鼠肾脏组织中大量炎性细胞浸润,肾小球增大,肾小球系膜细胞增生。免疫组化分析结果显示,感染组小鼠肾脏F4/80阳性区域面积为(1.99±0.49)%、(4.27±0.90)%、(6.71±1.38)%和(9.83±1.91)%,均高于健康对照组(P<0.01)。经吡喹酮2周疗程治疗后,感染给吡喹酮组小鼠的肾脏组织损伤减轻,肾小球系膜细胞增生缓解。与感染组比较,感染给吡喹酮组小鼠的尿蛋白含量[(2.67±0.52) g/L]减少,血清肌酐水平[(14.17±2.88)μmol/L]下降,F4/80阳性区域面积[(5.92±1.81)%]减少,TGF-β(0.99±0.23)和IL-6(1.24±0.53)m RNA相对转录水平下降(P<0.05或P<0.01), TNF-α和IL-1β的蛋白表达减少;而吡喹酮杀虫组与感染组比较差异无统计学意义(P>0.05)。与感染组(5.29±2.98、2.81±0.73、2.63±0.43)比较,感染给吡喹酮组(0.98±0.36、0.95±0.33、0.80±0.27)和吡喹酮杀虫组的TNF-α(1.56±0.66)、IL-1β(1.76±0.76)和CCL2mRNA(1.41±0.48)相对转录水平均下降(P<0.05或P<0.01)。结论日本血吸虫感染小鼠在感染初期和急性期发生肾脏炎症,肾脏组织中伴有巨噬细胞浸润,肾小球和肾功能损伤。吡喹酮2周疗程治疗可明显改善急性日本血吸虫感染小鼠的肾脏炎症和肾功能。

Objective To investigate the occurrence and development of renal inflammation in mice with acute infection of Schistosoma japonicum and evaluate the therapeutic effect of praziquantel on the impairment of renal function in the infected mice. Methods Fifty-five female C57 BL/6 J mice were randomly divided into 5 groups including the health control group(20), health control with praziquantel-treatment group(5), infection group(20), infection with praziquantel-treatment group(5) and praziquantel treatment group(5). Mice in the latter 3 groups were infected with 14 ± 2 cercariae of S. japonicum. Praziquantel treatment was given from the 6 th week after infection. In the health control with praziquantel-treatment group and the infection with praziquantel-treatment group, praziquantel was given at a dosage of 300 mg/kg every 12 h for two consecutive weeks, and the praziquantel dosage in the praziquantel treatment group was 250 mg/kg for 3 consecutive days. Five mice in each of the health control group and the infection group were dissected at weeks 1, 3, 6 and 8 after infection. Five mice in each of the health control with praziquantel-treatment group, the infection with praziquantel-treatment group and the praziquantel treatment group were dissected at week 8 after infection. Urine and blood samples were collected to detect the levels of urinary protein and serum creatinine to evaluate renal function of the mice. The histopathological changes of kidney in each group were observed by HE staining. The infiltration of macrophages in the kidneys of mice was assessed by F4/80 immunohistochemical staining. Quantitative real-time PCR(qRT-PCR) and Western blotting were used to detect the relative transcription and protein expression level of mRNA of inflammatory cytokines(TNF-α, TGF-β, IL-1β and IL-6)and macrophage chemokine CCL2. Results At weeks 1, 3, 6 and 8 after infection, the urine protein contents in the infection group were(1.67 ± 0.52) g/L,(2.17 ± 0.41) g/L,(3.00 ± 0.89) g/L and(4.00 ± 0. 63) g/L, respectively,all higher than those in the health control group(P<0.01). The serum creatinine levels in the infection group were(10.01 ± 1.45)μmol/,(13.00 ± 2.12)μmol/,(14.33 ± 1.21) and(19.00 ± 2.54) μmol/L, which were higher than those in the health control group(P>0.05 or P<0.01). qRT-PCR results showed that in the infection group the relative transcription level of inflammatory factor TNF-α mRNA were 2.47 ± 0.51, 3.14 ± 1.43, 1.83 ± 0.31 and 7.48 ± 5.54,the relative transcription level of TGF-β mRNA were 0.94 ± 0.24, 2.80 ± 1.07, 2.72 ± 0.88 and 3.03 ± 0.36, the relative transcription level of IL-1β mRNA were 1.49 ± 0.28, 4.32 ± 1.85, 2.08 ± 0.69 and 2.81 ± 0.73, the relative transcription level of IL-6 mRNA were 1.96 ± 0.31, 1.48 ± 0.35, 2.38 ± 1.17 and 2.85 ± 1.32, respectively, which were all higher in the health control group(P<0.05 or P<0.01). Western blotting analysis showed that the expression of TNF-α and IL-1β in the infection group were higher than those in the health control group. The relative transcription level of macrophage chemokine CCL2 mRNA in the infection group were 1.84 ± 0.17, 3.42 ± 0.85,3.03 ± 1.00 and 2.63 ± 0.43, respectively, which were also higher than that in the health control group(P<0.01).HE staining of renal pathological sections showed massive inflammatory cell infiltration, glomerular enlargement, and mesangial cell proliferation. Immunohistochemical results showed that the F4/80-positive areas in the kidneys of the infection group were(1.99 ± 0.49)%,(4.27 ± 0.90)%,(6.71 ± 1.38)% and(9.83 ± 1.91)%, which were higher than those in the health control group(P<0.01). After treatment with praziquantel for two weeks, the renal tissue impairment and glomerular mesangial cell proliferation were alleviated. Compared with the infection group, the urinary protein content [(2.67 ± 0.52) g/L], serum creatinine level [(14.17 ± 2.88) μmol/L], F4/80-positive area [(5.92 ±1.81)% ], mRNA relative transcription of TGF-β and IL-6(0.99 ± 0.23, 1.24 ± 0.53), and the protein levels of TNF-α and IL-1β were decreased in the infection with praziquantel-treatment group(P<0.05 or P<0.01), but did not change significantly in the praziquantel treatment group(P>0.05). Compared with the infection group(5.29 ±2.98, 2.81 ± 0.73, 2.63 ± 0.43), the mRNA relative transcription of TNF-α, IL-1β and CCL2 were decreased(P<0.05 or P<0.01) in the infection with praziquantel-treatment group(0.98 ± 0.36, 0.95 ± 0.33, 0.80 ± 0.27) and the praziquantel treatment group(1.56 ± 0.66, 1.76 ± 0.76, 1.41 ± 0.48). Conclusion In mice infected with S. japonicum, renal inflammation may occur in the early and acute stages of infection, accompanied by infiltration of macrophages in renal tissues, and impairment in glomerular and renal functions. Treatment with praziquantel for two weeks can significantly ameliorate renal inflammation and renal function in mice infected with S. japonicum.