黄苓昔对狼疮鼠肾炎的治疗作用和对滤泡辅助T细胞的调控作用

Therapeutic effects of baicalin on lupus nephritis in a lupus-prone mouse model and its regulatory role in follicular helper T cell differentiation

目的研究黄苓昔对狼疮鼠肾炎的治疗作用和对滤泡辅助T(Tfh)细胞的调控作用方法8只12周龄雌性狼疮鼠按随机数字表法随机分为两组,每组4只.黄苓昔组腹腔注射200 mg/kg黄苓昔每日1次,共治疗4周,对照组注射生理氯化钠溶液治疗结束时.考马斯亮蓝法检测24h尿蛋白含量,处死小鼠.取出脾脏称重,分离单个核细胞,流式细胞仪检测Tfh细胞比例;取出肾脏行苏木精-伊红染色,评估肾脏损伤情况从上述对照组狼疮鼠脾脏中分离单个核细胞.免疫磁珠分选幼稚性CD4+ T细胞,分3组培养,空白对照组不做处理.诱导组加入10 μg/L IL-21 IL-6和3 μg/L抗CD3及CD28抗体培养5 d,干预组在诱导组基础上同时加入40μmol/L黄苓齐体外培养5d 用50 μg/L佛波酯,750μg/L离子霉素和20 mg/L布雷非德菌素A刺激在上述体系中培养的部分幼稚CD4+T细胞5 h:流式细胞仪检测CD4+CXCR5+PI)-1+绷胞和CDM4+ IL-21+胞比例.采用SPSS20.0进行统计分析.定量资料通过方差分析和Student检验进行组间比较:结果黄苓昔治疗能有效改善狼疮鼠肾脏损伤,黄苓井组尿蛋白含量(1416 土 171)μg/24 h显著低于对照组[(2 623 ± 278)μg/24 h、P =0.022],脾脏中Tfh细胞的比例也低于对照组(12.6%±2.3%比40.2%±1.1%,P= 0.005)。体外黄苓昔能抑制Tfh细胞分化,干预组CD4+CXCR5+PD-1+Tfli细胞比例(13.3%± 0.8%)低于诱导组(17.6%±0.9%. P = 0.04).CD4+IL-21细胞比例(1.0%± 0.4%)亦低于诱导组(2.7%士 0.2%, P < 0.01 )-结论黄苓秆治疗能有效改善狼疮肾炎的损伤.可能与抑制Tfh细胞分化有关.

Objective To evaluate the therapeutic effect of baicalin on lupus nephritis in a lupusprone mouse model, and to investigate its regulatory role in the differentiation of follicular helper T (Tfh) cells. Methods Eight 12-week-old female MRL/lpr lupus-prone mice were randomly and equally divided into two groups by a random number table i.e.. baicalin group and control group intraperitoneally injected with 200 mg/kg baicalin sodium and chloride physiological solution, respectively, once even day for 4 weeks. After the end of treatment. Coomassie brilliant blue staining was performed to detect the level of 24- hour urine protein. Then, the mice were sacrificed, and the spleens were resected and weighed. Mononuclear cells were isolated from these spleens, and flow cytometry was conducted to determine the proportion of Tfli cells. Additionally, the kidneys were resected and subjected to hematoxylin and eosin (HE) staining for the evaluation of kidney impairment. Moreover, some other mononuclear cells were isolated from the spleens of the lupus-prone mice in the control group, and magnetic activated cell sorting (MACS) was performed to isolate naive CD4* T cells, which were divided into 3 groups: blank control group receiving no treatment, in duction group treated with 10 |xg/L anti ?i nterleukin (IL)- 21 and anti ?I L - 6 antibodies and 3 |xg/L anti-CD3 and anti-CD28 antibodies for 5 days, and intervention group additionally treated with 40 p,mol/L baicalin for 5 days besides the above treat me nt. Then, 50 |ig/L phorbol ester, 750 |xg/L ionomycin and 20 mg/L brefeldin A were used to stimulate some cultured naive CD4* T cells in the above groups. Flow cytometry was conducted to determine the proportion of CD4*CXCR5'PD-1 * cells and CD4*IL-21 * cells. Statistical analysis was carried out with SPSS20.0 software by using one-way analysis of variance (ANOVA) and student t test for the comparison of quantitative data between groups. Results The baicalin treatment could effectively improve the kidney impairment in the lupus-prone mice. Compared with the control group, the baicalin group showed significantly decreased 24 - hour urine protein level ([1 416 ± 171 ] vs.[2 623 ± 278 J |ig/24 h, P 二 0.022), and significantly decreased proportion of Tfli cells in the spleen (12.6%± 2.3% vs. 40.2%± 1」%, P = 0.005 ). In vitro baicalin could further inhibit the differentiation of Tfh cells. Compared with the induction group, the intervention group showed significantly decreased proportion of CD4*CXCR5 PD-1" Tfh cells (13.3%± 0.8% vs. 17.6%± 0.9%, P = 0.04) and CD4TL-21 * cells (1.0%± 0.4% vs. 2.7%± 0.2%, P < 0.01). Conclusion Baicalin can effectively ameliorate lupus nephritis, which may be associated with the inhibition of Tfh cell differentiation.