IgA肾病细胞模型中的炎症表达及丙戊酸钠的抗炎作用

doi:10.3969/j.issn.1674-8115.2022.06.009

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (6): 751-757.doi: 10.3969/j.issn.1674-8115.2022.06.009

• 论著 · 基础研究 • 上一篇

IgA肾病细胞模型中的炎症表达及丙戊酸钠的抗炎作用

戴芹¹(), 王伟铭²()

^1.复旦大学附属徐汇医院肾内科，上海 200031
^2.上海交通大学医学院附属瑞金医院肾内科，上海 200025

收稿日期:2022-03-01 接受日期:2022-06-03 出版日期:2022-06-28 发布日期:2022-08-19
通讯作者: 王伟铭 E-mail:dai11qin@sina.com;wweiming@medmail.com.cn
作者简介:戴芹（1978—），女，副主任医师，博士；电子信箱：dai11qin@sina.com。
基金资助:
上海市高级中西医结合人才培养项目[ZY(2018-2020)-RCPY-2020];上海市徐汇区卫生健康系统人才培养项目(xhxtrc2019-2021)

Inflammatory expression in IgA nephropathy cell model and anti-inflammatory effect of sodium valproate

DAI Qin¹(), WANG Weiming²()

^1.Department of Nephrology, Xuhui hospital, Fudan University, Shanghai 200031, China
^2.Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2022-03-01 Accepted:2022-06-03 Online:2022-06-28 Published:2022-08-19
Contact: WANG Weiming E-mail:dai11qin@sina.com;wweiming@medmail.com.cn
Supported by:
Shanghai Senior Integrated Traditional Chinese and Western Medicine Talents Training Project [ZY(2018-2020)-RCPY-2020];Personnel Training Project of Health System in Xuhui District of Shanghai(xhxtrc2019-2021)

摘要/Abstract

摘要：

目的·研究来源于IgA肾病患者的聚集性IgA1（P-aIgA1）对人肾系膜细胞（human renal mesangial cells，HMCs）分泌炎症因子及细胞增殖的影响，并观察组蛋白去乙酰化酶（histone deacetylation，HDAC）抑制剂丙戊酸钠（sodium valproate，VPA）体外抗炎和抗细胞增殖作用。方法·应用亲和层析法制备N-IgA1（来自健康对照者的IgA1）和P-IgA1（来自IgA肾病患者的IgA1），用热聚合法制备P-aIgA1和N-aIgA1（来自健康对照者的聚集性IgA1）。取HMCs，加入不同类型的IgA1及同体积的PBS（对照组），或者VPA干预。应用人炎症因子蛋白芯片检测各组HMCs培养上清液中炎症因子表达。用MTT法检测HMCs增殖情况。采用免疫印迹法或ELISA法检测相关蛋白的表达。结果·炎症因子蛋白芯片结果显示：与对照组比较，P-aIgA1组HMCs培养上清液中白介素-6可溶性受体（interleukin-6 soluble receptor，IL-6sR）、受激活调节的正常T细胞表达和分泌因子（regulated upon activation normal T cell expressed and secreted factor，RANTES）、金属蛋白酶组织抑制因子1（tissue inhibitor of metalloproteinase 1，TIMP1）、金属蛋白酶组织抑制因子2（tissue inhibitor of metalloproteinase 2，TIMP2）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）和肿瘤坏死因子Ⅱ型受体（tumor necrosis factor type Ⅱ receptor，TNFRⅡ）的表达水平均显著上调，分别为对照组的34倍、124倍、269倍、100倍、1.6倍和52倍；与对照组比较，N-aIgA1，P-IgA1，P-aIgA1都能促进TIMP2的表达，差异均有统计学意义（P=0.000，P=0.000，P=0.001）。用MTT法观察HMCs的增殖程度，结果显示：① 与对照组相比，N-IgA1组对HMCs的增殖无显著的影响，P-IgA1及P-aIgA1都能显著促进HMCs的增殖（P=0.045和P=0.003）；与N-IgA1相比，P-aIgA1组HMCs出现了显著的增殖，差异有统计学意义（P =0.036）。② 不同浓度的 P-aIgA1对HMCs增殖影响的结果显示：25 μg/mL的P-aIgA1就可以显著促进HMCs的增殖（P =0.038），呈剂量依赖性。③ 400 μg/mL的VPA能够显著抑制HMCs的增殖（P=0.028）。不同的IgA1对HMCs中HDAC1的影响结果显示：各组HDAC1的表达量分别是对照组的（8.64±0.59）倍（P-aIgA1）、（5.42±0.16）倍（P-IgA1）和（5.87±0.58）倍（N-IgA1）；与N-IgA1组相比，P-aIgA1组显著增加了HDAC1表达（P=0.021）。VPA对系膜细胞分泌TNF-α蛋白的影响结果显示：与正常对照组相比，P-aIgA1组TNF-α蛋白表达明显升高（P=0.001）；与P-aIgA1组比较，P-aIgA1+VPA组TNF-α蛋白表达水平显著下降（P=0.035）。结论·P-aIgA1能显著促进系膜细胞释放促炎症因子和系膜细胞增殖，且促细胞增殖作用呈浓度依赖。体外IgA肾病细胞模型中存在乙酰化修饰异常，其参与了系膜细胞增殖和炎症反应。HDAC抑制剂VPA可以部分逆转上述反应，提示其在IgA肾病疾病干预方面有一定应用前景，可为从表观遗传学角度防治IgA肾病提供新的思路。

关键词: IgA肾病, 人系膜细胞, 组蛋白去乙酰化酶, 丙戊酸钠, 炎症因子

Abstract:

Objective·To study the effects of aggregated IgA1 (P-aIgA1) from patients with IgA nephropathy on the secretion of inflammatory factors and cell proliferation of human renal mesangial cells (HMCs), and observe the anti-inflammatory and anti-proliferation effects of histone deacetylation (HDAC) inhibitor sodium valproate (VPA) in vitro.

Methods·N-IgA1 (IgA1 from health control) and P-IgA1 (IgA1 from patients with IgA nephropathy) were prepared by affinity chromatography, and P-aIgA1 and N-aIgA1 were prepared by thermal polymerization. The expression of inflammatory factors in HMCs culture supernatant was detected by human inflammatory factor protein chip. The proliferation of HMCs was detected by MTT assay. The expression of related proteins was detected by Western blotting or ELISA.

Results·The results of inflammatory factor protein chip showed that the concentration of interleukin-6 soluble receptor (IL-6sR), regulated upon activation normal T cell expressed and secreted factor(RANTES), tissue inhibitor of metalloproteinase 1 (TIMP1), tissue inhibitor of metalloproteinase 2 (TIMP2), tumor necrosis factor-α(TNF-α) and tumor necrosis factor type Ⅱ receptor (TNFR Ⅱ) in the culture supernatant of HMCs in the P-aIgA1 group were significantly up-regulated compared with the control group, which were 34, 124, 269, 100, 1.6 and 52 times higher than those in the control group, respectively; N-aIgA1, P-IgA1 and P-aIgA1 could promote the expression of TIMP2, and there were significant differences (P=0.000, P=0.000, P=0.001). The proliferation of HMCs was observed by MTT method. The results showed that: ① Compared with the control group, N-IgA1 group had no significant effect on the proliferation of HMCs, and P-IgA1 and P-aIgA1 could significantly promote the proliferation of HMCs (P=0.045 and P=0.003); compared with the N-IgA1, HMCs in the P-aIgA1 group had significant proliferation, and the differences was statistically significant (P=0.036). ② The results of the effects of different concentrations of P-aIgA1 on the proliferation of HMCs showed that: 25 μg/mL P-aIgA1 could significantly promote the proliferation of HMCs (P=0.038), in a dose-dependent manner. ③ 400 μg/mL VPA could significantly inhibit the proliferation of HMCs (P=0.028). The effect of different IgA1 on HDAC1 in HMCs showed that the expression of HDAC1 in each group was (8.64±0.59) times (P-aIgA1), (5.42±0.16) times (P-IgA1) and (5.87±0.58) times (N-IgA1) higher than that in the control group, respectively; compared with N-IgA1, the expression of HDAC1 was significantly increased in the P-aIgA1 group (P=0.021). The effect of VPA on TNF-α secretion by mesangial cells showed that the protein expression of TNF-α in the P-aIgA1 group was significantly increased compared with the normal control group (P=0.001); compared with the P-aIgA1 group, the protein expression of TNF-α in the P-aIgA1+VPA group decreased significantly (P=0.035).

Conclusion·P-aIgA1 can significantly promote the release of pro-inflammatory factors and mesangial cell proliferation, and the effect of promoting cell proliferation is concentration-dependent.Abnormal acetylation modification exists in IgA nephropathy cell model in vitro, which is involved in mesangial cell proliferation and inflammatory response. The HDAC inhibitor sodium valproate can partially reverse the above reaction. The above results suggest that sodium valproate has a certain application prospect in IgAN disease intervention, and provide a new idea for the prevention and treatment of IgA nephropathy from the perspective of epigenetics.

Key words: IgA nephropathy, human renal mesangial cell, histone deacetylation, sodium valproate, inflammatory factor

中图分类号:

R593.9

戴芹, 王伟铭. IgA肾病细胞模型中的炎症表达及丙戊酸钠的抗炎作用[J]. 上海交通大学学报（医学版）, 2022, 42(6): 751-757.

DAI Qin, WANG Weiming. Inflammatory expression in IgA nephropathy cell model and anti-inflammatory effect of sodium valproate[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 751-757.

图/表 7

图1 蛋白芯片检测不同IgA1干预HMCs后培养上清液中炎症因子的表达

Fig 1 Expression of inflammatory factor in cultured HMCs supernatant induced by different IgA1s

图 2 人炎症因子蛋白芯片的统计分析Note：①P=0.000, ②P=0.001, compared with the control group.

Fig 2 Statistical chart of the array

图 3 不同的IgA1对系膜细胞增殖的影响Note：①P=0.003, ②P=0.045, compared with the control group; ③P=0.036, compared with the N-IgA1 group.

Fig 3 Effects of different IgA1s on the proliferation of mesangial cell

图4 不同浓度的P-aIgA1对系膜细胞增殖的影响Note：①P=0.038, ②P=0.000, compared with the control group.

Fig 4 Effects of different concentrations of P-aIgA1 on the proliferation of mesangial cell

图5 不同IgA1对系膜细胞HDAC1蛋白的影响Note：①P=0.000, ②P=0.001, compared with the control group; ③P=0.021, compared with the N-IgA1 group.

Fig 5 Effects of different IgA1s on HDAC1 protein in HMCs

图6 不同浓度的VPA对P-aIgA1作用的HMCs增殖的影响Note：①P=0.009, compared with the control group; ②P=0.028, ③P=0.002, compared with the P-aIgA1 group.

Fig 6 Effects of different concentrations of VPA on the proliferation of mesangial cell induced by P-aIgA1

图7 不同干预成分对HMCs培养上清液TNF-α蛋白表达水平的影响Note：①P=0.001, compared with the control group; ②P=0.035, compared with the P-aIgA1 group.

Fig 7 Effects of different stimulators on TNF-α protein in HMCs cultured supernatant

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IgA肾病细胞模型中的炎症表达及丙戊酸钠的抗炎作用

Inflammatory expression in IgA nephropathy cell model and anti-inflammatory effect of sodium valproate

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