含笑内酯通过抑制NF-κB/NLRP3轴改善单侧输尿管梗阻模型小鼠的肾脏病变

doi:10.11958/20230150

天津医药 ›› 2023, Vol. 51 ›› Issue (10): 1059-1064.doi: 10.11958/20230150

含笑内酯通过抑制NF-κB/NLRP3轴改善单侧输尿管梗阻模型小鼠的肾脏病变

雷向宏¹(), 严文君¹, 熊梅梅¹, 龙海波², 陈斯佳³^,^△()

1.赣南医学院第一附属医院肾内科（邮编341000）
2.南方医科大学珠江医院肾内科
3.长沙市第一医院肾病风湿科

收稿日期:2023-02-13 修回日期:2023-04-21 出版日期:2023-10-15 发布日期:2023-10-18
通讯作者: ^∆E-mail：huicsj@163.com
作者简介:雷向宏（1977），男，副主任医师，主要从事慢性肾脏病方面研究。E-mail：531817899@qq.com
基金资助:
国家自然科学基金-广东联合基金项目(U1801288);江西省卫生健康委科技计划项目(202210049)

Micheliolide ameliorates renal lesion of unilateral ureteral obstruction mice by inhibiting NF-κB/NLRP3 axis

LEI Xianghong¹(), YAN Wenjun¹, XIONG Meimei¹, LONG Haibo², CHEN Sijia³^,^△()

1. Department of Nephrology, the First Affiliated Hospital of Gannan Medical University, Ganzhou 341000, China
2. Department of Nephrology, Zhujiang Hospital, Southern Medical University
3. Department of Nephropathy and Rheumatology, the First Hospital of Changsha

Received:2023-02-13 Revised:2023-04-21 Published:2023-10-15 Online:2023-10-18
Contact: ^∆E-mail：huicsj@163.com

雷向宏, 严文君, 熊梅梅, 龙海波, 陈斯佳. 含笑内酯通过抑制NF-κB/NLRP3轴改善单侧输尿管梗阻模型小鼠的肾脏病变[J]. 天津医药, 2023, 51(10): 1059-1064.

LEI Xianghong, YAN Wenjun, XIONG Meimei, LONG Haibo, CHEN Sijia. Micheliolide ameliorates renal lesion of unilateral ureteral obstruction mice by inhibiting NF-κB/NLRP3 axis[J]. Tianjin Medical Journal, 2023, 51(10): 1059-1064.

摘要/Abstract

摘要：

目的探究含笑内酯（MCL）对单侧输尿管梗阻（UUO）模型小鼠肾脏病变的干预作用及机制。方法将雄性C57BL/6J小鼠随机分为假手术（Sham）组、UUO组、UUO+MCL组，UUO+MCL组建立UUO模型前1 d以25 mg/kg MCL灌胃。术后8 d采集肾组织标本进行HE、Masson、TUNEL染色；蛋白免疫印迹（Western blot）和免疫组化检测核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）、胱天蛋白酶1（caspase-1）、白细胞介素（IL）-1β、肿瘤坏死因子（TNF）-α、核因子（NF）-κB p65蛋白表达情况。结果 HE染色结果显示，Sham组小鼠肾组织无明显病变；UUO组肾小管进行性扩张，间质水肿伴有少量的炎性细胞浸润；UUO+MCL组肾脏病变较UUO组明显改善。Masson染色结果显示，与Sham组相比，UUO组胶原容积分数明显增加，而UUO+MCL组胶原容积分数较UUO组明显减少。TUNEL染色结果显示，与Sham组相比，UUO组细胞凋亡率升高，UUO+MCL组较UUO组细胞凋亡率明显下降。Western blot和免疫组化结果显示，与Sham组相比，UUO组肾组织中NLRP3、caspase-1、IL-1β、TNF-α、p-NF-κB p65的表达水平均升高；与UUO组相比，UUO+MCL组上述因子均显著下降。结论 UUO小鼠肾组织NLRP3炎症小体及相关炎性因子表达增加，MCL通过抑制NF-κB通路及NLRP3炎症小体的活化，减轻肾小管间质的炎症反应，从而改善肾纤维化。

关键词: 输尿管梗阻, 纤维化, NLR家族, 热蛋白结构域包含蛋白3, NF-κB, 含笑内酯

Abstract:

Objective To investigate the effect and mechanism of micheliolide on renal lesions of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into the sham group, the UUO (model) group and the UUO+micheliolide (administration) group. Mice in the UUO+ micheliolide group were given micheliolide by gavage at 25 mg/kg body weight after the establishment of the animal model. Renal tissue samples were collected 8 days after operation, and the renal tissue was stained with HE, Masson and TUNEL staining. The protein expressions of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α and (phosphorylation)- nuclear transcription factor (NF)-κB p65 were detected by immunohistochemistry and Western blot assay. Results HE staining showed that there were no pathological changes in renal tissue in the sham group. Mice in the UUO group had progressive renal tubule dilation and interstitial edema with a small amount of inflammatory cell infiltration. The renal lesions were significantly improved in the UUO+MCL group compared with those of the UUO group. Masson staining showed that compared with the sham group, the collagen volume fraction (CVF) was significantly increased in the UUO group, while CVF was significantly reduced in the UUO+MCL group than that in the UUO group. TUNEL staining showed that compared with the sham group, the apoptosis rate was increased in the UUO group, and the apoptosis rate was significantly decreased in the UUO+MCL group than that of the UUO group. Western blot assay and immunohistochemistry result showed that the expression levels of NLRP3, caspase-1, IL-1β, TNF-α and p-NF-κB p65 were increased in kidney tissue of the UUO group compared with those of the sham group. Compared with the UUO group, NLRP3, caspase-1, IL-1β, TNF-α and p-NF-κB p65 were significantly decreased in the UUO+MCL group. Conclusion The expression of NLRP3 inflammasome and related inflammatory factors in renal tissue of mice with unilateral ureteral obstruction are increased. Micheliolide can reduce the inflammatory response of renal tubule interstitium by inhibiting NF-κB pathway and NLRP3 inflammasome activation, thereby improving renal fibrosis.

Key words: ureteral obstruction, fibrosis, NLR family, pyrin domain-containing 3 protein, NF-kappa B, micheliolide

中图分类号:

R692.5

图/表 10

图1 动物实验设计示意图

Fig.1 Schematic diagram of animal experiment design

图2 各组肾组织HE染色结果（×400）

Fig.2 HE staining results of renal tissue in each group (×400)

图3 各组肾组织Masson染色结果（×400）

Fig.3 Masson staining results of kidney tissue in each group (×400)

图4 各组肾组织TUNEL染色结果（×400）

Fig.4 TUNEL staining results of kidney tissue in each group (×400)

图5 各组肾组织NLRP3、caspase-1、IL-1β、TNF-α免疫组化结果（×400）

Fig.5 Immunohistochemical results of of NLRP3, caspase-1, IL-1β and TNF-ɑ in renal tissue (×400)

表1 各组肾组织NLRP3、caspase-1、IL-1β和TNF-α蛋白表达水平比较 (n=6,$\bar{x}±s$)

Tab.1 Comparison of the expression levels of NLRP3, caspase-1, IL-1β and TNF-α proteins in renal tissue between the three groups

组别	NLRP3	caspase-1	IL-1β	TNF-α
Sham组	0.356±0.031	0.166±0.018	0.226±0.040	0.205±0.034
UUO组	1.284±0.060^a	0.917±0.069^a	0.902±0.078^a	1.007±0.077^a
UUO+MCL组	0.454±0.021^ab	0.281±0.046^ab	0.491±0.056^ab	0.201±0.038^ab
F	947.521^＊＊	410.538^＊＊	193.414^＊＊	454.035^＊＊

图6 Western blot检测各组肾组织NLRP3、caspase-1、IL-1β、TNF-α蛋白表达情况

Fig.6 The expression levels of NLRP3, caspase-1, IL-1β and TNF-α proteins in renal tissue of each group detected by Western blot assay

表2 各组肾组织NLRP3、caspase-1、IL-1β和TNF-α蛋白表达水平比较 (n=6,$\bar{x}±s$)

Tab.2 Comparison of expression levels of NLRP3, caspase-1, IL-1β and TNF-α proteins in renal tissue between the three groups

组别	NLRP3	caspase-1	IL-1β	TNF-α
Sham组	0.422±0.091	0.155±0.044	0.367±0.095	0.401±0.118
UUO组	0.840±0.138^a	0.722±0.123^a	0.792±0.107^a	1.012±0.152^a
UUO+MCL组	0.470±0.110^b	0.462±0.093^ab	0.573±0.066^ab	0.646±0.156^ab
F	23.891^＊＊	56.410^＊＊	32.841^＊＊	27.745^＊＊

图7 各组肾组织NF-κB p65免疫组化结果（×400）

Fig.7 Immunohistochemical results of NF-κB p65 in renal tissue(×400)

图8 Western blot检测检测各组肾组织p-NF-κB p65蛋白表达情况

Fig.8 The expression of p-NF-κB p65 protein in kidney tissue of each group detected by Western blot assay

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含笑内酯通过抑制NF-κB/NLRP3轴改善单侧输尿管梗阻模型小鼠的肾脏病变

Micheliolide ameliorates renal lesion of unilateral ureteral obstruction mice by inhibiting NF-κB/NLRP3 axis

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