缺氧微环境下补阳还五汤通过抑制BNIP3-PI3K/Akt通路抑制类风湿关节炎滑膜成纤维细胞的线粒体自噬

doi:10.12122/j.issn.1673-4254.2025.01.05

摘要/Abstract

摘要：

目的在低氧微环境下，以BNIP3-PI3K/Akt信号通路为核心，研究补阳还五汤参与FLS-RA线粒体自噬的调控机制。方法将成纤维样滑膜细胞（FLS）体外培养，分为正常对照组（FLS-RA正常培养）、模型对照组（IL-1β诱导）、补阳还五汤含药血清低、中、高干预组（IL-1β诱导+浓度分别为5%、10%、20%含药血清），除正常对照组外，其余组细胞按照10% O₂浓度低氧处理。AnnexinV-APC/7-AAD双染测定细胞凋亡和T-AOC试剂盒检测细胞总抗氧化能力。探针分子测定细胞ROS，ATP试剂盒测定细胞ATP水平、线粒体膜电位（Δψm）、细胞Ca²⁺平衡稳态的变化。Western blotting法检测BNIP3、PI3K、AKT蛋白表达水平，RT-q PCR法检测BNIP3、PI3K、AKT及自噬相关因子LC3、Beclin-1、P62 mRNA 的表达。结果补阳还五汤组能够降低细胞的凋亡百分比（P<0.05），且呈浓度依赖性。补阳还五汤组总抗氧化力降低，T-AOC浓度（U/mL）降低，ROS产生增加。观察到自噬小体的形成，ATP 酶水平释放量增加（P<0.05）；线粒体膜电位、Ca²⁺水平均呈下降趋势。补阳还五汤组BNIP3蛋白表达升高，PI3K、AKT表达降低；与模型组相比，补阳还五汤组BNIP3、P62、mRNA表达升高（P<0.05）；PI3K、AKT、LC3、Beclin-1、mRNA表达降低，Beclin-1的表达差异无统计学意义（P>0.05）；LC3的表达仅在中药小剂量组差异无统计学意义（P>0.05）。结论在低氧环境下，补阳还五汤可能通过抑制BNIP3介导的PI3K/AKT通路，从而抑制了自噬因子的表达。

关键词: 低氧, 补阳还五汤, 类风湿关节炎, 滑膜成纤维细胞, BNIP3-PI3K/Akt, 线粒体自噬

Abstract:

Objective To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. Methods Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. Results Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. Conclusion BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.

Key words: hypoxia, Buyang Huanwu Decoction, rheumatoid arthritis, synovial fibroblast, BNIP3-PI3K/Akt, mitochondrial autophagy

展俊平, 黄硕, 孟庆良, 范围, 谷慧敏, 崔家康, 王慧莲. 缺氧微环境下补阳还五汤通过抑制BNIP3-PI3K/Akt通路抑制类风湿关节炎滑膜成纤维细胞的线粒体自噬[J]. 南方医科大学学报, 2025, 45(1): 35-42.

Junping ZHAN, Shuo HUANG, Qingliang MENG, Wei FAN, Huimin GU, Jiakang CUI, Huilian WANG. Buyang Huanwu Decoction reduces mitochondrial autophagy in rheumatoid arthritis synovial fibroblasts in hypoxic culture by inhibiting the BNIP3-PI3K/Akt pathway[J]. Journal of Southern Medical University, 2025, 45(1): 35-42.

图/表 10

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Gene	Primer	Sequence （5'-3'）	PCR products
Homo GAPDH	Forward	TCAAGAAGGTGGTGAAGCAGG	115 bp
Homo GAPDH	Reverse	TCAAAGGTGGAGGAGTGGGT	115 bp
Homo Bnip3	Forward	CAGCAATAATGGGAACGGGG	160 bp
Homo Bnip3	Reverse	GGTATCTTGTGGTGTCTGCG	160 bp
Homo PI3K	Forward	CCTTATTATTACCCTCCCT	349 bp
Homo PI3K	Reverse	GTTCTTCATCACTCATCTGTC
Homo AKT	Forward	TATTGTGAAGGAGGGTTGGC
Homo AKT	Reverse	CAGTCTGGATGGCGGTTGT	302 bp
Homo p62	Forward	GGCTGATTGAGTCCCTCTCCCAGAT	140 bp
Homo p62	Reverse	CGGCGGGGGATGCTTTGAATACTGG	140 bp
Homo Beclin-1	Forward	GTCACTGGGGACCTTTTT	177 bp
Homo Beclin-1	Reverse	GTCATCCTCATTCATTTGC	177 bp
Homo LC3	Forward	TCCTGGACAAGACCAAGTTTTT	212 bp
Homo LC3	Reverse	CTGGGAGGCGTAGACCATATAGAG	212 bp

Gene	Primer	Sequence （5'-3'）	PCR products
Homo GAPDH	Forward	TCAAGAAGGTGGTGAAGCAGG	115 bp
Homo GAPDH	Reverse	TCAAAGGTGGAGGAGTGGGT	115 bp
Homo Bnip3	Forward	CAGCAATAATGGGAACGGGG	160 bp
Homo Bnip3	Reverse	GGTATCTTGTGGTGTCTGCG	160 bp
Homo PI3K	Forward	CCTTATTATTACCCTCCCT	349 bp
Homo PI3K	Reverse	GTTCTTCATCACTCATCTGTC
Homo AKT	Forward	TATTGTGAAGGAGGGTTGGC
Homo AKT	Reverse	CAGTCTGGATGGCGGTTGT	302 bp
Homo p62	Forward	GGCTGATTGAGTCCCTCTCCCAGAT	140 bp
Homo p62	Reverse	CGGCGGGGGATGCTTTGAATACTGG	140 bp
Homo Beclin-1	Forward	GTCACTGGGGACCTTTTT	177 bp
Homo Beclin-1	Reverse	GTCATCCTCATTCATTTGC	177 bp
Homo LC3	Forward	TCCTGGACAAGACCAAGTTTTT	212 bp
Homo LC3	Reverse	CTGGGAGGCGTAGACCATATAGAG	212 bp

Group	Early apoptosis	Late apoptosis	Total apoptosis
Blank	3.24±0.51	0.52±0.20	3.76±0.71
Model	19.94±0.96*	0.94±0.07	20.88±0.95*
Buyang huanwu
5%	13.97±1.02^#	1.60±0.23^#	15.57±1.24^#
10%	9.89±0.64^#	1.19±0.06^#	11.08±0.69^#
20%	5.95±0.61^#	0.53±0.05^#	6.49±0.59^#

Group	Early apoptosis	Late apoptosis	Total apoptosis
Blank	3.24±0.51	0.52±0.20	3.76±0.71
Model	19.94±0.96*	0.94±0.07	20.88±0.95*
Buyang huanwu
5%	13.97±1.02^#	1.60±0.23^#	15.57±1.24^#
10%	9.89±0.64^#	1.19±0.06^#	11.08±0.69^#
20%	5.95±0.61^#	0.53±0.05^#	6.49±0.59^#

Group	ATP(μmol/gprot)	Potential change (%)	Ca²⁺ equilibrium homeostasis
Blank	163.82±1.83	5.28±0.28	4219.4±99.6
Model	50.91±6.54*	28.64±1.24*	17100.0±825.2*
Buyang huanwu
5%	84.40±8.49^#	21.46±0.96^#	16972.3±309.5
10%	113.312±4.91^#	16.92±0.85^#	10244.6±184.1^#
20%	129.75±6.22^#	9.16±0.13^#	7973.0±477.1^#