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Objective To investigate the action mechanism of Xibining decoction on mitigation of pain of knee osteoarthritis (KOA) through regulation of redox homeostasis of synoviocytes by carnitine palmitoyl transferase 1 (CPT1) enzyme. Methods Fibroblast-like synovial cells (FLS) were extracted from rats' knee joint and the optimal concentration of the freeze-dried powder of Xibining decoction on FLS was selected by CCK-8. Subsequently, the cells were divided into control group, KOA group and Xibining group. The inflammatory environment of KOA was induced by 5 μg/ml lipopolysaccharide (LPS) in the last two groups, and in Xibining group, 100 μg/ml Xibining were added and cultured for 24 hours. The mRNA and protein expressions of CPT1 and carnitine/organic cation transporters 1 (OCTN1) were detected by Real-time PCR and Western blotting. The activities of CPT1 and superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected by assay kits. Reactive oxygen species(ROS) level was detected by a 2',7'-dichlorofluorescein diacetate assay. The dorsal root ganglion (DRG) neurons were extracted from rats and identified by βⅢ-tubulin and glial fibrillary acidic protein (GFAP) immunofluorescence. The neurons were divided into control group, KOA group and Xibining group, the FLS supernatants of the three groups were added to the DRG for 24 hours. The mRNA and protein expression of transient receptor potential A1 ion channel (TRPA1) were detected by Real-time PCR and Western blotting. Ca2+ influx in DRG neurons after TRPA1 opening was observed by Real-time fluorescent calcium imaging. ELISA assay was used to detect the content of calcitonin gene related peptide (CGRP) and substance P (SP) in DRG supernatant. Results The ideal freeze-dried powder of Xibining decoction concentration was identified as 100 μg/ml. Real-time PCR and Western blotting results showed that the expression levels of mRNA and protein in CPT1 and OCTN1 of Xibining group were significantly higher than those in KOA group (P<0.05). Compared with control group, the average fluorescence intensity and MDA contents increased significantly in KOA group [(5.52±0.78) AU vs. (26.46±2.07) AU; (2.77±0.03) nmol/ml vs. (3.13±0.02) nmol/ml] (P<0.05); the activities of CPT1 and SOD were significantly downregulated [(11.50±0.21) nmol/min vs. (4.98±0.02) nmol/min; (17.6±0.07) U/ml vs. (11.38±0.05) U/ml] (P<0.05). Compared with KOA group, the fluorescence intensity and MDA contents significantly decreased in Xibining group [(14.07±1.41) AU and (2.87±0.01) nmol/ml] (P<0.05), the activities of CPT1 and SOD [(7.94±0.21) nmol/min and (13.81±0.07) U/ml] were significantly upregulated (P<0.05). Compared with KOA group, after treatment with FLS supernatant in Xibining group, the mRNA and protein expression of TRPA1, and Ca2+ influx after TRPA1 opening were inhibited in DRG (P<0.05). In addition, the expression of CGRP and SP were also reduced in Xibining group [(19.93±1.2) ng/L vs. (30.19±1.58) ng/L, P<0.05; (84.23±1.26) ng/L vs. (123.16±2.95) ng/L, P<0.05]. Conclusion Xibining can regulate the local redox homeostasis of FLS through CPT1 enzyme, which can further influence the Ca2+ influx on the TRPA1 ion channel of DRG and reduce the secretion of pain factor to alleviate KOA pain.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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