黄芩清热除痹胶囊通过PTEN/PI3K/AKT信号通路改善痛风性关节炎大鼠的炎症反应及尿酸、脂质代谢失衡

doi:10.12122/j.issn.1673-4254.2024.08.03

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (8): 1450-1458.doi: 10.12122/j.issn.1673-4254.2024.08.03

• • 上一篇

黄芩清热除痹胶囊通过PTEN/PI3K/AKT信号通路改善痛风性关节炎大鼠的炎症反应及尿酸、脂质代谢失衡

张先恒¹^,²(), 刘健¹^,³(), 韩琦¹^,², 陈一鸣¹^,², 丁香¹^,², 陈晓露²

^1.安徽中医药大学第一附属医院，安徽合肥 230011
^2.安徽中医药大学，安徽合肥 230012
^3.安徽省中医药科学院风湿病研究所，安徽合肥 230009

收稿日期:2024-04-28 出版日期:2024-08-20 发布日期:2024-09-06
通讯作者: 刘健 E-mail:1148465915@qq.com;liujianahzy@126.com
作者简介:张先恒，在读博士研究生，E-mail: 1148465915@qq.com
基金资助:
国家中医药管理局高水平中医药重点学科(ZYYZDXK-2023100);现代中医内科应用基础与开发研究安徽省重点实验室(2016080503B041);安徽省第12批“115”创新团队(皖人才办[2019]1号);安徽省名中医刘健工作室建设项目(中医药发展秘[2018] 11号)

Huangqin Qingrechubi Capsule alleviates inflammation and uric acid and lipid metabolism imbalance in rats with gouty arthritis by inhibiting the PTEN/PI3K/AKT signaling pathway

Xianheng ZHANG¹^,²(), Jian LIU¹^,³(), Qi HAN¹^,², Yiming CHEN¹^,², Xiang DING¹^,², Xiaolu CHEN²

^1.The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230011, China
^2.Anhui University of Chinese Medicine, Hefei 230012, China
^3.Institute of Rheumatology, Anhui Academy of Chinese Medicine, Hefei 230009, China

Received:2024-04-28 Online:2024-08-20 Published:2024-09-06
Contact: Jian LIU E-mail:1148465915@qq.com;liujianahzy@126.com

摘要/Abstract

摘要：

目的探究黄芩清热除痹胶囊（HQC）对痛风性关节炎（GA）大鼠炎症反应及尿酸、脂质代谢的影响，并探讨其可能机制。方法随机将60只SD大鼠分为6组：正常组，模型组，HQC低、中、高剂量组，秋水仙碱组（n=10）；以单钠尿酸盐注射右踝关节腔进行GA大鼠造模，HQC低、中、高剂量组及秋水仙碱组予相应剂量药物灌胃，模型组予等量生理盐水，连续7 d。造模后4、8、24、48、72 h检测各组大鼠足趾肿胀度；HE染色法进行滑膜组织学分析；ELISA检测血清中白细胞介素（IL）-10、IL-18、肿瘤坏死因子（TNF）-α、转化生长因子（TGF）-β1、脂联素（APN）、瘦素（LP）、抵抗素（RS）、内脂素（VF）的表达，检测滑膜中APN、LP、RS、VF的表达；生化法检测血清中高密度脂蛋白胆固醇（HDL-C）、甘油三酯（TG）、总胆固醇（TC）、血尿酸（BUA）的水平；RT-qPCR检测滑膜中磷酸酶与紧张素同源物（PTEN）、磷脂酰肌醇-3-激酶（PI3K）、蛋白激酶B（AKT） mRNA的表达；Western blotting检测PTEN、PI3K、p-PI3K、AKT、p-AKT 蛋白表达。结果与正常组比较，模型组足趾肿胀度升高（P<0.05），且在48 h达到高峰；HE染色显示大量炎性细胞浸润和滑膜组织增生；TNF-α、TGF-β1、IL-18、TC、TG、LP、RS、VF、BUA、p-PI3K、p-AKT的表达增加（P<0.05），IL-10、APN、HDL-C、PTEN的表达降低（P<0.05）。与模型组比较，HQC和秋水仙碱可降低TNF-α、TGF-β1、IL-18、TC、TG、LP、RS、VF、BUA、p-PI3K、p-AKT的表达（P<0.05），升高IL-10、APN、HDL-C、PTEN的表达（P<0.05），减轻滑膜组织病理损伤，改善足趾肿胀度。结论 HQC可改善GA大鼠炎症反应及尿酸、脂质代谢失衡，可能与抑制PTEN/PI3K/AKT信号通路有关。

关键词: 痛风性关节炎, 黄芩清热除痹胶囊, 炎症, 尿酸代谢, 脂质代谢, PTEN/PI3K/AKT信号通路

Abstract:

Objective To investigate the effects of Huangqin Qingrechubi Capsule (HQC) on inflammation and uric acid and lipid metabolism in rats with gouty arthritis (GA) and its mechanism. Methods SD rat models of GA established by injecting monosodium urate into the right ankle joint were treated with saline, colchicine and HQC at low, medium and high doses (n=10) by gavage for 7 days. Toe swelling of the rats was detected at 4, 8, 24, 48 and 72 h after modeling, and synovial histological changes were observed with HE staining. Serum levels of interleukin-10 (IL-10), IL-18, tumor necrosis factor‑α (TNF‑α), transforming growth factor-β1 (TGF-β1), adiponectin, leptin, resistin and visfatin were measured by ELISA, and the levels of high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), total cholesterol (TC), and uric acid (BUA) were detected. RT-qPCR and Western blotting were used to detect the mRNA expressions of phosphatase and tensin homolog (PTEN), phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) and the protein expressions of PTEN, PI3K, p-PI3K, AKT and p-AKT. Results The rat models of GA showed obvious toe swelling, which reached the peak level at 48 h. HE staining revealed massive inflammatory cell infiltration and synovial tissue hyperplasia. The rat models showed significantly increased expressions of TNF-α, TGF-β1, IL-18, TC, TG, leptin, resistin and visfatin, BUA, p-PI3K, and p-AKT and lowered levels of IL-10, APN, HDL-C, and PTEN. Treatment with HQC and colchicine obviously improved these changes and alleviated synovial pathologies and toe swelling in the rat models. Conclusion HQC can improve inflammation and correct the imbalance of uric acid and lipid metabolism in GA rats possibly by inhibiting the PTEN/PI3K/AKT signaling pathway.

Key words: gouty arthritis, Huangqin Qingrechubi Capsule, inflammation, uric acid metabolism, lipid metabolism, PTEN/PI3K/AKT signaling pathway

张先恒, 刘健, 韩琦, 陈一鸣, 丁香, 陈晓露. 黄芩清热除痹胶囊通过PTEN/PI3K/AKT信号通路改善痛风性关节炎大鼠的炎症反应及尿酸、脂质代谢失衡[J]. 南方医科大学学报, 2024, 44(8): 1450-1458.

Xianheng ZHANG, Jian LIU, Qi HAN, Yiming CHEN, Xiang DING, Xiaolu CHEN. Huangqin Qingrechubi Capsule alleviates inflammation and uric acid and lipid metabolism imbalance in rats with gouty arthritis by inhibiting the PTEN/PI3K/AKT signaling pathway[J]. Journal of Southern Medical University, 2024, 44(8): 1450-1458.

图/表 8

表1 引物设计表

Tab.1 Primer design table

Gene	Amplicon size	Primer sequence (5'→3')
PTEN	140	F:TATCTTGTGCTCACCCTGAC R:CTGCTAGCCTCTGGATTTGA
PI3K	123	F:CGAAACAAAGCCGAGAACCT R:GACGCAATGTTTGACTTCGC
AKT	143	F:CAGGTTCACCCAGTGACAAC R:CTCCTTCACCAGGATCACCT
β-actin	150	F:CCCATCTATGAGGGTTACGC R:TTTAATGTCACGCACGATTTC

表2 抗体信息

Tab.2 Antibody information

Antibody	Dilution rate	Species
PI3K	1:1000	Rabbit
p-PI3K	1:5000	Mouse
AKT	1:1000	Rabbit
p-AKT	1:1000	Rabbit
PTEN	1:1000	Rabbit
GAPDH	1:2000	Mouse

图1 注射MSU 48 h后各组大鼠右踝关节图像

Fig.1 Right ankle joints of the rats in each group after MSU injection 48 h. A: Normal control group; B: Model group; C: HQC-L group; D: HQC-M group; E: HQC-H group; F: Colchicine group.

表3 HQC对GA大鼠足趾肿胀度的影响

Tab.3 Effect of HQC on toe swelling in GA rats (Mean±SD, mL, n=10)

Groups	4 h	8 h	24 h	48 h	72 h
Normal	1.856±0.025	1.796±0.023	1.702±0.027	1.650±0.025	1.645±0.029
Model	2.015±0.073*	2.156±0.045*	2.335±0.058*	2.547±0.059*	2.456±0.068*
HQC-L	1.945±0.059^△#	2.096±0.045^△#	2.170±0.059^△#	1.954±0.057^△#	1.741±0.065^△#
HQC-M	1.908±0.033^△#	1.995±0.036^△#	1.896±0.026^△#	1.805±0.040^△#	1.693±0.040^△#
HQC-H	1.896±0.038^△#	1.920±0.035^△	1.855±0.030^△	1.727±0.040^△	1.642±0.034^△
Colchicine	1.853±0.036^△	1.914±0.040^△	1.857±0.047^△	1.714±0.063^△	1.638±0.030^△

图2 各组大鼠滑膜组织HE染色图像

Fig.2 HE staining of synovial tissue of the rats in each group. Arrows indicate infiltration of inflammatory cells. A: Normal control group; B: Model group; C: HQC-L group; D: HQC-M group; E: HQC-H group; F: Colchicine group.

图3 各组大鼠TNF-α、TGF-β1、IL-18、IL-10表达比较

Fig.3 Comparison of TNF-α, TGF-β1, IL-18 and IL-10 expressions among the groups. *P<0.05 vs A; △P<0.05 vs B; #P<0.05 vs F. A: Normal control group; B: Model group; C: HQC-L group; D: HQC-M group; E: HQC-H group; F: Colchicine group.

图4 各组大鼠TC、TG、HDL-C、APN、LP、RS、VF、BUA水平比较

Fig.4 Comparison of TC, TG, HDL-C, APN, LP, RS, VF and BUA levels among the groups. *P<0.05 vs A; △P<0.05 vs B; #P<0.05 vs F. A: Normal control group; B: Model group; C: HQC-L group; D: HQC-M group; E: HQC-H group; F: Colchicine group.

图5 各组大鼠PTEN、PI3K、AKT mRNA表达比较

Fig.5 Comparison of mRNA expressions of PTEN, PI3K and AKT among the groups. *P<0.05 vs A; △P<0.05 vs B; #P<0.05 vs F. A: Normal control group; B: Model group; C: HQC-L group; D: HQC-M group; E: HQC-H group; F: Colchicine group.

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黄芩清热除痹胶囊通过PTEN/PI3K/AKT信号通路改善痛风性关节炎大鼠的炎症反应及尿酸、脂质代谢失衡

Huangqin Qingrechubi Capsule alleviates inflammation and uric acid and lipid metabolism imbalance in rats with gouty arthritis by inhibiting the PTEN/PI3K/AKT signaling pathway

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