Effects of VX765 on osteoarthritis and chondrocyte inflammation in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 HUANG Wanran , TU Junxue , QIAO Aiqing , HE Chujun

Department of Pharmacy, Yuying Children’s Hospital, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou Zhejiang, 325000, P. R. China;

Corresponding author：

HUANG Wanran, Email: xc324289783@163.com

Keywords：

VX765; nuclear factor erythroid 2-related factor 2 pathway; osteoarthritis; chondrocytes; rat

DOI：

10.7507/1002-1892.202308053

Video：

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Objective To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. Methods Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors—transforming growth factor β₁ (TGF-β₁), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. Results The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L (P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β₁, IL-6, and TNF-α in cells significantly increased when compared with the control group (P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group (P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 (P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression.Conclusion VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.

Citation： HUANG Wanran, TU Junxue, QIAO Aiqing, HE Chujun. Effects of VX765 on osteoarthritis and chondrocyte inflammation in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2024, 38(1): 74-81. doi: 10.7507/1002-1892.202308053 Copy

1.	Jagua-Gualdrón A, Peña-Latorre JA, Fernadez-Bernal RE. Apitherapy for osteoarthritis: Perspectives from basic research. Complement Med Res, 2020, 27(3): 184-192.
2.	Berenbaum F, Walker C. Osteoarthritis and inflammation: a serious disease with overlapping phenotypic patterns. Postgrad Med, 2020, 132(4): 377-384.
3.	艾尔西丁·阿不来提, 徐昌, 马强, 等. 膝关节骨关节炎患者全膝关节置换后影响关节功能恢复的因素. 中国骨与关节杂志, 2020, 9(2): 104-107.
4.	Zhang Q, Bai X, Wang R, et al. 4-octyl Itaconate inhibits lipopolysaccharide (LPS)-induced osteoarthritis via activating Nrf2 signalling pathway. J Cell Mol Med, 2022, 26(5): 1515-1529.
5.	Zhang Z, Wang S, Liu X, et al. Secoisolariciresinol diglucoside ameliorates osteoarthritis via nuclear factor-erythroid 2-related factor-2/nuclear factor kappa B pathway: In vitro and in vivo experiments. Biomed Pharmacother, 2023 Aug: 164: 114964.
6.	Cheng R, Feng Y, Zhang R, et al. The extent of pyroptosis varies in different stages of apical periodontitis. Biochim Biophys Acta Mol Basis Dis, 2018, 1864(1): 226-237.
7.	Liu W, Chen Y, Meng J, et al. Ablation of caspase-1 protects against TBI-induced pyroptosis in vitro and in vivo. J Neuroinflammation, 2018, 15(1): 48.
8.	马玉珍, 彭朋, 秦永生, 等. NLRP3炎性小体抑制剂VX-765对急性心肌梗死大鼠心功能及线粒体能量代谢的影响. 武警后勤学院学报(医学版), 2017, 26(9): 742-745.
9.	郑仕清, 洪旭东, 陈甜胜, 等. 半胱氨酸天冬氨酸蛋白酶1抑制剂VX765对冷束缚诱导小鼠急性胃溃疡的作用. 中华烧伤杂志, 2017, 33(11): 688-693.
10.	常青, 王伟, 杨增华, 等. 黄芩苷对IL-1β诱导大鼠软骨细胞凋亡和炎症反应的抑制作用及相关机制研究. 中国免疫学杂志, 2020, 36(21): 2603-2607, 2618.
11.	Wannamaker W, Davies R, Namchuk M, et al. (S)-1-((S)-2-[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino-3, 3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R, 3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765), an orally available selective interleukin(IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J Pharmacol Exp Ther, 2007, 321(2): 509-516.
12.	Chen S, Huang Y, Chen W, et al. Protective effects of the Tougu Xiaotong capsule on morphology and osteoprotegerin/nuclear factor-κB ligand expression in rabbits with knee osteoarthritis. Mol Med Rep, 2016, 13(1): 419-425.
13.	Matsui H, Shimizu M, Tsuji H. Cartilage and subchondral bone interaction in osteoarthrosis of human knee joint: a histological and histomorphometric study. Microsc Res Tech, 1997, 37(4): 333-342.
14.	Shen J, Abu-Amer Y, O’Keefe RJ, et al. Inflammation and epigenetic regulation in osteoarthritis. Connect Tissue Res, 2017, 58(1): 49-63.
15.	Mendez ME, Sebastian A, Murugesh DK, et al. LPS-induced inflammation prior to injury exacerbates the development of post-traumatic osteoarthritis in mice. J Bone Miner Res, 2020, 35(11): 2229-2241.
16.	Wang T, He C. Pro-inflammatory cytokines: The link between obesity and osteoarthritis. Cytokine Growth Factor Rev, 2018, 44: 38-50.
17.	Gomez-Lopez N, Romero R, Xu Y, et al. A role for the inflammasome in spontaneous labor at term with acute histologic chorioamnionitis. Reprod Sci, 2017, 24(6): 934-953.
18.	Crowley DC, Lau FC, Sharma P, et al. Safety and efficacy of undenatured type Ⅱ collagen in the treatment of osteoarthritis of the knee: a clinical trial. Int J Med Sci, 2009, 6(6): 312-321.
19.	Zhang Y, Zheng Y. Effects and mechanisms of potent caspase-1 inhibitor VX765 treatment on collagen-induced arthritis in mice. Clin Exp Rheumatol, 2016, 34(1): 111-118.
20.	Wang L, He C. Nrf2-mediated anti-inflammatory polarization of macrophages as therapeutic targets for osteoarthritis. Front Immunol, 2022, 13: 967193.
21.	Zhang P, Jin Y, Xia W, et al. Phillygenin inhibits inflammation in chondrocytes via the Nrf2/NF-κB axis and ameliorates osteoarthritis in mice. J Orthop Translat, 2023, 41: 1-11.
22.	Chen Z, Zhong H, Wei J, et al. Inhibition of Nrf2/HO-1 signaling leads to increased activation of the NLRP3 inflammasome in osteoarthritis. Arthritis Res Ther, 2019, 21(1): 300.
23.	Zhou S, Shi J, Wen H, et al. A chondroprotective effect of moracin on IL-1β-induced primary rat chondrocytes and an osteoarthritis rat model through Nrf2/HO-1 and NF-κB axes. Food Funct, 2020, 11(9): 7935-7945.
24.	Cai D, Yin S, Yang J, et al. Histone deacetylase inhibition activates Nrf2 and protects against osteoarthritis. Arthritis Res Ther, 2015, 17: 269.

1. Jagua-Gualdrón A, Peña-Latorre JA, Fernadez-Bernal RE. Apitherapy for osteoarthritis: Perspectives from basic research. Complement Med Res, 2020, 27(3): 184-192.
2. Berenbaum F, Walker C. Osteoarthritis and inflammation: a serious disease with overlapping phenotypic patterns. Postgrad Med, 2020, 132(4): 377-384.
3. 艾尔西丁·阿不来提, 徐昌, 马强, 等. 膝关节骨关节炎患者全膝关节置换后影响关节功能恢复的因素. 中国骨与关节杂志, 2020, 9(2): 104-107.
4. Zhang Q, Bai X, Wang R, et al. 4-octyl Itaconate inhibits lipopolysaccharide (LPS)-induced osteoarthritis via activating Nrf2 signalling pathway. J Cell Mol Med, 2022, 26(5): 1515-1529.
5. Zhang Z, Wang S, Liu X, et al. Secoisolariciresinol diglucoside ameliorates osteoarthritis via nuclear factor-erythroid 2-related factor-2/nuclear factor kappa B pathway: In vitro and in vivo experiments. Biomed Pharmacother, 2023 Aug: 164: 114964.
6. Cheng R, Feng Y, Zhang R, et al. The extent of pyroptosis varies in different stages of apical periodontitis. Biochim Biophys Acta Mol Basis Dis, 2018, 1864(1): 226-237.
7. Liu W, Chen Y, Meng J, et al. Ablation of caspase-1 protects against TBI-induced pyroptosis in vitro and in vivo. J Neuroinflammation, 2018, 15(1): 48.
8. 马玉珍, 彭朋, 秦永生, 等. NLRP3炎性小体抑制剂VX-765对急性心肌梗死大鼠心功能及线粒体能量代谢的影响. 武警后勤学院学报(医学版), 2017, 26(9): 742-745.
9. 郑仕清, 洪旭东, 陈甜胜, 等. 半胱氨酸天冬氨酸蛋白酶1抑制剂VX765对冷束缚诱导小鼠急性胃溃疡的作用. 中华烧伤杂志, 2017, 33(11): 688-693.
10. 常青, 王伟, 杨增华, 等. 黄芩苷对IL-1β诱导大鼠软骨细胞凋亡和炎症反应的抑制作用及相关机制研究. 中国免疫学杂志, 2020, 36(21): 2603-2607, 2618.
11. Wannamaker W, Davies R, Namchuk M, et al. (S)-1-((S)-2-[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino-3, 3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R, 3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765), an orally available selective interleukin(IL)-converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J Pharmacol Exp Ther, 2007, 321(2): 509-516.
12. Chen S, Huang Y, Chen W, et al. Protective effects of the Tougu Xiaotong capsule on morphology and osteoprotegerin/nuclear factor-κB ligand expression in rabbits with knee osteoarthritis. Mol Med Rep, 2016, 13(1): 419-425.
13. Matsui H, Shimizu M, Tsuji H. Cartilage and subchondral bone interaction in osteoarthrosis of human knee joint: a histological and histomorphometric study. Microsc Res Tech, 1997, 37(4): 333-342.
14. Shen J, Abu-Amer Y, O’Keefe RJ, et al. Inflammation and epigenetic regulation in osteoarthritis. Connect Tissue Res, 2017, 58(1): 49-63.
15. Mendez ME, Sebastian A, Murugesh DK, et al. LPS-induced inflammation prior to injury exacerbates the development of post-traumatic osteoarthritis in mice. J Bone Miner Res, 2020, 35(11): 2229-2241.
16. Wang T, He C. Pro-inflammatory cytokines: The link between obesity and osteoarthritis. Cytokine Growth Factor Rev, 2018, 44: 38-50.
17. Gomez-Lopez N, Romero R, Xu Y, et al. A role for the inflammasome in spontaneous labor at term with acute histologic chorioamnionitis. Reprod Sci, 2017, 24(6): 934-953.
18. Crowley DC, Lau FC, Sharma P, et al. Safety and efficacy of undenatured type Ⅱ collagen in the treatment of osteoarthritis of the knee: a clinical trial. Int J Med Sci, 2009, 6(6): 312-321.
19. Zhang Y, Zheng Y. Effects and mechanisms of potent caspase-1 inhibitor VX765 treatment on collagen-induced arthritis in mice. Clin Exp Rheumatol, 2016, 34(1): 111-118.
20. Wang L, He C. Nrf2-mediated anti-inflammatory polarization of macrophages as therapeutic targets for osteoarthritis. Front Immunol, 2022, 13: 967193.
21. Zhang P, Jin Y, Xia W, et al. Phillygenin inhibits inflammation in chondrocytes via the Nrf2/NF-κB axis and ameliorates osteoarthritis in mice. J Orthop Translat, 2023, 41: 1-11.
22. Chen Z, Zhong H, Wei J, et al. Inhibition of Nrf2/HO-1 signaling leads to increased activation of the NLRP3 inflammasome in osteoarthritis. Arthritis Res Ther, 2019, 21(1): 300.
23. Zhou S, Shi J, Wen H, et al. A chondroprotective effect of moracin on IL-1β-induced primary rat chondrocytes and an osteoarthritis rat model through Nrf2/HO-1 and NF-κB axes. Food Funct, 2020, 11(9): 7935-7945.
24. Cai D, Yin S, Yang J, et al. Histone deacetylase inhibition activates Nrf2 and protects against osteoarthritis. Arthritis Res Ther, 2015, 17: 269.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of VX765 on osteoarthritis and chondrocyte inflammation in rats

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