Mechanistic Study on Tougu Xiaotong Capsules in Regulating PANoptosis to Delay Degeneration of Chondrocytes in Knee Osteoarthritis
10.13422/j.cnki.syfjx.20251903
- VernacularTitle:透骨消痛胶囊调控泛凋亡延缓膝骨关节炎软骨细胞退变的机制
- Author:
Jinxia YE
1
;
Yixin LIN
1
;
Xiaoqing LEI
1
;
Yanfeng HUANG
2
;
Changlong FU
2
;
Desen LI
3
;
Wenyi WANG
4
;
Lan WANG
5
Author Information
1. College of Integrative Medicine， Fujian University of Traditional Chinese Medicine （TCM）， Fuzhou 350122，China
2. Fujian Key Laboratory of Integrative Medicine on Geriatrics， Fuzhou 350122，China
3. College of Pharmacy， Fujian University of TCM， Fuzhou 350122，China
4. Innovation and Transformation Center， Fujian University of TCM， Fuzhou 350122，China
5. Institute of Chinese Materia Medica， China Academy of Chinese Medical Sciences， Beijing 100700，China
- Publication Type:Journal Article
- Keywords:
Tougu Xiaotong capsules;
osteoarthritis;
chondrocytes;
PANoptosis
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2026;32(8):149-161
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo investigate the effect of Tougu Xiaotong capsules （TGXTC） on the regulation of chondrocyte PANoptosis， delay of chondrocyte degeneration， and improvement of the symptoms in knee osteoarthritis （KOA）. MethodsIn vivo experiments： 50 male C57BL/6 mice were randomly assigned into five groups （n=10 per group）： sham operation group， model group， low-dose TGXTC group （7.2 g·kg-1）， high-dose TGXTC group （14.4 g·kg-1）， and diclofenac sodium group （0.05 g·kg-1）. Except for the sham group， KOA models were established in all other groups using the modified Hulth method. Following successful model induction， the TGXTC groups received daily oral gavage of 7.2 or 14.4 g·kg-1 for 6 weeks， while the diclofenac sodium group received 0.05 g·kg-1 solution daily over the same duration. Model evaluation was performed using Lequesne MG score； micro-computed tomography （micro-CT） was used to scan the knee， hematoxylin-eosin （HE） staining and safranin O-fast green staining were used to observe the morphology of cartilage， transmission electron microscopy （TEM） was used to determine ultrastructural changes of PANoptosis. Multiple immunofluorescence （IF） co-localization assays was performed to detect the co-localization of cleaved Caspase-3， receptor-interacting protein 3 （RlPK3）， and the N-terminal domain of gasdermin D （GSDMD-N） in cartilage tissue， while western blot was employed to detect the expression levels of cleaved Caspase-3， RIPK3， and GSDMD-N. In vitro experiments： The knee cartilages of 4-week-old SD rats were isolated， and a chondrocyte in vitro culture system was established through mechanical digestion with 0.2% type Ⅱ collagenase. Second-generation chondrocytes were divided into three groups： the control group， the model group （pretreated with 10 mg·L-1 lipopolysaccharide （LPS） for 24 h followed by treatment with 1 μmol·L-1 nigericin for 4 h）， and the TGXTC treatment group （pretreated with 10 mg·L-1 LPS for 24 h， followed by exposure to 1 μmol·L-1 nigericin for 4 h and subsequently treated with 100 mg·L-1 TGXTC for an additional 24 h）. The levels of reactive oxygen species （ROS）， apoptosis， necroptosis， and pyroptosis of chondrocytes were evaluated via fluorescence microscopy following staining with ROS detection， AO/EB and YO-PRO-1/PI staining kits. Transmission electron microscopy was utilized to investigate the ultrastructural changes associated with PANoptosis in cartilage tissue of KOA mice. Inflammatory cytokine levels （IL-1β and IL-18） were measured using ELISA. Western blot was conducted to assess protein expressions related to PANoptosis， including cleaved Caspase-3， cleaved Caspase-8， RIPK3， ZBP1， GSDMD-N， and NLRP3. ResultsCompared with the sham group， the Lequesne MG scores were significantly up-regulated（P<0.01） in the model group， and the pathological changes of cartilage were significantly， with joint spaces narrower， osteophyte formation increased， secere abrasion of cartilage surface. Ultrastructural analysis revealed pronounced chondrocyte apoptosis， necroptosis， and pyroptosis， along with markedly elevated expression of cleaved Caspase-3， RlPK3， and GSDMD-N in cartilage tissue （P<0.01）. In addition， The mean fluorescence intensities of ROS， orange-red fluorescence in AO/EB staining， green fluorescence and red fluorescence in YO-PRO-1/PI staining were increased of chondrocyte in the model group （P<0.01） . The levels of inflammatory factors IL-1β and IL-18 in the supernatant were increased （P<0.01）. The expression of PANoptosis related proteins （cleaved Caspase-3， cleaved Caspase-8， RIPK3， ZBP1， GSDMD-N， and NLRP3） were also significantly upregulated（P<0.05）. Compared to the model group， the TGXTC group demonstrated a significant improvement in various parameters of mice. These included a reduction in the Lequesne MG score， an increase in joint space， a decrease in osteophyte formation， diminished cartilage damage， reduced release of ROS， and alleviation of apoptotic， necroptotic， and pyroptotic processes in chondrocytes. Additionally， mitochondrial swelling and endoplasmic reticulum dilation were also mitigated. The levels of ROS as well as IL-1β and IL-18 were significantly decreased （P<0.05）. Furthermore， the expression levels of proteins associated with PANoptosis in cartilage tissue showed marked reductions （P<0.05）. Similar results were observed in chondrocytes： cleaved Caspase-3， cleaved Caspase-8， RIPK3， ZBP1， GSDMD-N， and NLRP3 exhibited significant decreases as well （P<0.05）. ConclusionTGXTC may mitigate chondrocytes degeneration and alleviate KOA symptoms by reducing oxidative stress and suppressing the activation of PANoptosis pathways.
