Buyang Huanwu Decoction reduces mitochondrial autophagy in rheumatoid arthritis synovial fibroblasts in hypoxic culture by inhibiting the BNIP3-PI3K/Akt pathway

doi:10.12122/j.issn.1673-4254.2025.01.05

Abstract

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

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.

Figures/Tables 10

References 30

1	Buchanan WW, Kean CA, Kean WF, et al. Rheumatoid arthritis[J]. Inflammopharmacology, 2024, 32(1): 3-11.
2	Hu YD, Liu J, Jiang H, et al. Regulation of autophagy by circular RNAs in rheumatoid arthritis: potential targets of action[J]. Int J Rheum Dis, 2023, 26(5): 831-40.
3	Cai SH, Sun Y, Wang Y, et al. Exploring the effect of LncRNA DANCR to regulate the Keap1-Nrf2/ARE pathway on oxidative stress in rheumatoid arthritis[J]. Immun Inflamm Dis, 2024, 12(1): e1163.
4	丁仙红, 朱红国. 类风湿关节炎患者血清炎症因子和氧化应激指标治疗前后水平的变化[J]. 中国现代医生, 2020, 58(22): 135-8.
5	Liu FF, Wang Y, Huang D, et al. LncRNA HOTAIR regulates the PI3K/AKT pathway via the miR-126-3p/PIK3R2 axis to participate in synovial angiogenesis in rheumatoid arthritis[J]. Immun Inflamm Dis, 2023, 11(10): e1064.
6	Dervisevic A, Fajkic A, Jahic E, et al. Systemic immune-inflammation index in evaluation of inflammation in rheumatoid arthritis patients[J]. Medeni Med J, 2024, 39(3): 183-91.
7	Tang Z, Meng SY, Yang XX, et al. Neutrophil-mimetic, ROS responsive, and oxygen generating nanovesicles for targeted interventions of refractory rheumatoid arthritis[J]. Small, 2024, 20(20): e2307379.
8	Balogh E, Veale DJ, McGarry T, et al. Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis[J]. Arthritis Res Ther, 2018, 20(1): 95.
9	Mukhopadhyay K, De S, Kundu S, et al. Evaluation of levels of oxidative stress as a potential biomarker in patients with rheumatoid arthritis[J]. J Family Med Prim Care, 2021, 10(5): 1981-6.
10	张铌雪, 苏晓慧, 田雅格, 等. 自噬在类风湿关节炎中的作用及中药干预研究进展[J]. 中国实验方剂学杂志, 2021,27(6):169-75.
11	廉坤, 李鑫, 宁博, 等.线粒体自噬研究的文献计量学可视化分析[J]. 中西医结合心脑血管病杂志, 2024, 22(3): 425-32.
12	刘煜, 岳婷, 杨东宇, 等. 自噬在类风湿关节炎发病机制中的研究进展[J]. 中国全科医学, 2023, 26(29): 3710-4.
13	Cid-Castro C, Hernández-Espinosa DR, Morán J. ROS as regulators of mitochondrial dynamics in neurons[J]. Cell Mol Neurobiol, 2018, 38(5): 995-1007.
14	Kato M, Ospelt C, Gay RE, et al. Dual role of autophagy in stress-induced cell death in rheumatoid arthritis synovial fibroblasts[J]. Arthritis Rheumatol, 2014, 66(1): 40-8.
15	Ramzan R, Dolga AM, Michels S, et al. Cytochrome c oxidase inhibition by ATP decreases mitochondrial ROS production[J]. Cells, 2022, 11(6): 992.
16	Deng R, Wang Y, Bu YH, et al. Geniposide augments apoptosis in fibroblast-like synoviocytes by restoring hypoxia-enhanced JNK-BNIP3-mediated autophagy[J]. Inflamm Res, 2023, 72(8): 1745-60.
17	Ney PA. Mitochondrial autophagy: origins, significance, and role of BNIP3 and NIX[J]. Biochim Biophys Acta, 2015, 1853(10 Pt B): 2775-83.
18	Fan DD, Tan PY, Jin L, et al. Bioinformatic identification and validation of autophagy-related genes in rheumatoid arthritis[J]. Clin Rheumatol, 2023, 42(3): 741-50.
19	Deng R, Wang Y, Bu YH, et al. BNIP3 mediates the different adaptive responses of fibroblast-like synovial cells to hypoxia in patients with osteoarthritis and rheumatoid arthritis[J]. Mol Med, 2022, 28(1): 64.
20	Li H, Wu QY, Teng XH, et al. The pathogenesis and regulatory role of HIF-1 in rheumatoid arthritis[J]. Cent Eur J Immunol, 2023, 48(4): 338-45.
21	Li S, Chen JW, Xie X, et al. Autophagy inhibitor regulates apoptosis and proliferation of synovial fibroblasts through the inhibition of PI3K/AKT pathway in collagen-induced arthritis rat model[J]. Am J Transl Res, 2017, 9(5): 2065-76.
22	程伟刚, 李浩林, 杨娟娟, 等.中医药调控PI3K/Akt信号通路治疗类风湿关节炎的研究进展[J].中药新药与临床药理, 2024, 35(5):756-64.
23	Chen YM, Chang CY, Chen HH, et al. Association between autophagy and inflammation in patients with rheumatoid arthritis receiving biologic therapy[J]. Arthritis Res Ther, 2018, 20(1): 268.
24	陈宏道, 丁一帆, 谢师旅, 等. 中医药治疗类风湿关节炎临床研究进展[J]. 江西中医药, 2023, 54(4): 73-6.
25	曾苹, 侯雷, 宁乔怡, 等. 马武开教授从气虚血瘀论治类风湿关节炎临床经验[J]. 风湿病与关节炎, 2018, 7(4): 51-3.
26	蒋总, 姚晓玲, 唐芳, 等. 系统评价补阳还五汤对强直性脊柱炎临床疗效的Meta分析[J]. 贵州中医药大学学报, 2020, 42(2): 68-73.
27	郑卓铭, 李坚. 补阳还五汤治疗骨科相关疾病的研究进展[J]. 中医临床研究, 2023, 15(24): 96-102.
28	谢有鑫,王晓霞, 李森贤.补阳还五汤加减治疗类风湿性关节炎临床研究[J].新中医, 2022, 54(23): 39-42.
29	展俊平, 孟庆良, 孟婉婷,等. 补阳还五汤配合西药治疗类风湿关节炎气滞血瘀证临床观察[J]. 山西中医, 2018, 34(8): 29-30.
30	展俊平, 孟庆良, 孟婉婷, 等. 补阳还五汤对类风湿关节炎小鼠MPO, NE mRNA表达及TNF-α, IL-1β, IL-6, IL-17的影响[J]. 中国实验方剂学杂志, 2018, 24(24): 158-63.

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^#