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OBJECTIVE To investigate the improvement effect and potential mechanism of “Layers adjusting external application” paste on synovial fibrosis （SF） in rats with knee osteoarthritis （KOA）. METHODS Male SD rats were randomly divided into sham operation group， KOA group and Layers adjusting external application group， with 8 rats in each group. KOA model was induced by the anterior cruciate ligament disruption method in KOA group and Layers adjusting external application group. Fourteen days after modeling， the Layers adjusting external application group was given “Layers adjusting external application” paste [Sanse powder （8 g for every 100 cm2）， Compound sanhuang ointment （5 g for every 100 cm2）] on the knee joint， 8 h every day， for 28 d in total. After the last administration， the degree of synovitis and fibrosis in rats was observed， and Krenn scoring was performed in each group. The expressions of collagen Ⅰ， high mobility group protein B1 （HMGB1） and phosphorylated nuclear factor-κB p65 （p-NF-κB p65） were detected in the synovial membrane； the contents of interleukin-1β （IL- 1β）， IL-6 and tumor necrosis factor-α （TNF-α） in serum as well as the expressions of fibrosis-related and HMGB1/Toll-like receptor 4 （TLR4）/NF-κB signaling pathway-related proteins and mRNA were detected in synovial tissue. RESULTS Compared with the sham operation group， the synovial lining cells in the KOA group showed significant proliferation and disordered arrangement， the inflammatory cell infiltration and collagen fiber deposition were obvious； the positive expressing cells of collagen Ⅰ， HMGB1 and p-NF-κB p65 were increased significantly； the contents of IL-1β， IL-6 and TNF-α in serum， the expressions of fibrosis-related protein （transforming growth factor-β， collagen Ⅰ， tissue inhibitor of metalloproteinase 1， α-smooth muscle actin） and their mRNA as well as theexpressions of HMGB1， TLR4 protein and their mRNA， the expressions of p-NF-κB p65 protein and NF-κB p65 mRNA were all increased significantly in synovial tissues of rats （P＜0.01）. Compared with the KOA group， the pathological changes in the synovial tissue of rats in Layers adjusting external application group were significantly improved， and the above quantitative indicators were significantly reversed （P＜0.05 or P＜0.01）. CONCLUSIONS “Layers adjusting external application” paste could significantly improve SF in KOA rats， the mechanism of which may be associated with the inhibition of the activation of HMGB1/ TLR4/NF-κB signaling pathway.
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OBJECTIVE To investigate the improvement effect mechanism of Xibining prescription （XBN） on knee osteoarthritis （KOA） model rats based on AMP-activated protein kinase（AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. METHODS Totally 36 rats were randomly divided into blank group， model group， XBN group （12.56 g/kg）， XBN+metformin （AMPK agonist） group （12.56 g/kg XBN+100 mg/kg metformin）， with 9 rats in each group. Except for blank group， KOA model was induced by anterior cruciate ligament transection in other groups. After modeling， each group was given relevant medicine/normal saline， XBN and normal saline intragastrically， once a day， and metformin intraperitoneally， every other day， for 4 consecutive weeks. The pathomorphological changes of cartilage tissue in rats were observed and Mankin scoring was conducted. The expression level of Aggrecan in rat cartilage， mRNA and protein expressions of platelet reactive protein disintegrin and metalloproteinase with thrombospondin motifs 4 （ADAMTS-4）， ADAMTS-5， matrix metalloproteinase 3 （MMP-3） and MMP- 13， and the phosphorylation level of AMPK and mTOR proteins were detected. RESULTS Compared with blank group， the structure of cartilage tissue in the model group was disordered， the matrix of cartilage layer was lightly stained，the tide line was distorted or interrupted， and Mankin score was significantly increased （P＜0.05）. The protein expression of Aggrecan in cartilage tissue and the phosphorylation level of AMPK protein were all decreased significantly （P＜0.05）； mRNA and protein expressions of ADAMTS-4， ADAMTS-5， MMP-3 and MMP-13 and the phosphorylation levels of mTOR protein were significantly increased in cartilage tissues （P＜0.05）. Compared with model group， the pathological morphology of cartilage was improved significantly in each administration group， and above score or indexes were reversed significantly （P＜0.05）. Compared with XBN group， the degree of cartilage lesions in rats was further alleviated in XBN+ metformin group， and the levels of above score or indicators were further improved （P＜0.05）. CONCLUSIONS XBN can ameliorate cartilage injury in KOA model rats， promote cartilage synthesis and reduce cartilage degradation， the mechanism of which may be associated with activating AMPK/mTOR signaling pathway.
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OBJECTIVE To optimize the extraction technology for the raw drugs of Sanse powder gel paste. METHODS SD rats were divided into blank group， model group， traditional technology group， water extraction group and ethanol extraction group， with 5 rats in each group. Anterior cruciate ligament transection was used to construct knee osteoarthritis model， and the pharmacodynamic effects of different extraction methods on arthritic rats were investigated. Analgesic experiments were conducted using cold and hot pain thresholds and pain mediators calcitonin gene-related peptide （CGRP）， cyclooxygenase-2 （COX-2）， substance P （SP）， and prostaglandin E2 （PGE2） contents as indicators. HE staining was performed on the synovial membrane of rats to observe the degree of synovial cell proliferation， inflammatory infiltration and vascular invasion， and anti-inflammatory experiments were conducted using protein and mRNA expressions of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α） and IL-6 as indicators. The analgesic and anti-inflammatory effects were compared among those groups. In the orthogonal test， ethanol dosage， extraction time and extraction times were used as evaluation factors， and the contents of casticin， strychnine and toxiferine were taken as evaluation indicators； comprehensive score was calculated. The validation experiments were carried out after optimizing the extraction technology of the raw drugs of Sanse powder gel paste. RESULTS Compared with the model group， the cold and heat pain thresholds of drug administration groups （except for the traditional technology group） were all increased significantly （P＜0.05）， while the contents of pain （No.Y2021rc02） mediators CGRP， COX-2， SP and PGE2 were all decreased significantly （P＜0.05）. HE staining showed that inflammatory cell infiltration， fibrosis and collagen deposition were 炎。E-mail：liuzixiu3221@126.com decreased in the administration groups； a small amount of capillary proliferation could be found； the protein and mRNA expressions of inflammatory factors such as IL-1β， IL-6 and TNF-α were decreased significantly in synovial tissue of rats in administration groups （P＜0.05）. Compared with the traditional technology group， most indicators of the ethanol extraction group were significantly reduced （P＜0.05）， and only heat pain threshold and mRNA expression of IL-6 in rats were decreased significantly in the water extraction group （P＜0.05）. The optimal extraction technology of the raw drugs of Sanse powder gel paste included suitable dose of Sanse powder， 8-fold 55% ethanol， heating reflux extraction for 90 minutes， extracting twice. The results of 3 times of verification experiments showed that the average contents of casticin， strychnine and toxiferine were 0.007%， 0.092%， and 0.214%， respectively； RSD were all less than 5%. CONCLUSIONS The optimized extraction technology for the raw drugs of Sanse powder gel paste is stable and feasible， which can improve the efficacy of the preparation.
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OBJECTIVE：To discuss the mechanism of synovitis of knee osteoarthritis （KOA）relieved by “Sanse powder ” volatile oil based on the activation of Nod-like receptor family 3（NLRP3）inflammasome. METHODS ：“Sanse powder ”volatile oil was extracted by distillation- condensation process and the components of it were analyzed by GC-MS. Fibroblast-like synovial cells（FLS）were extracted from the knee joint of male SD rats. The effects of 10，25，50，100，250，500，1 000 μg/mL“Sanse powder”volatile oil on the viability of FLS were examined by CCK-8 assay. KOA inflammatory cell model was induced by lipopolysaccharide for 12 h. The effects of 10，250 μg/mL“Sanse powder ”volatile oil on the protein and mRNA expression of NLRP3，caspase-1 and apoptosis-associated speck-like protein （ASC）were detected. The levels of IL- 1β and IL-18 in supernatants of FLS were determined . RESULTS ：Totally 0.51-0.61 g volatile oil with yellow clear and unique aromatic odor were extracted from“Sanse powder ”，and the extraction rate was 0.33％-0.41％（n＝9）. A total of 41 chemical components were isolated ，and 30 of them were identified and their peak area accounted for 90.073 6％ of the total peak area. The components with high relative content were gingerol flavonoids （17.573 9％），δ-junionene（15.434 5％），gingerol（11.509 5％），etc. When the concentration of volatile oil was 10-250 μg/mL，there was no significant effect on survival rate of FLS （P＞0.05）. Compared with blank group ，the relative protein and mRNA expression of NLRP 3，caspase-1 and ASC and the levels of IL- 1β and IL-18 in supernatant were significantly increased in model group （P＜0.05）. Compared with model group ，relative expression or levels of above indexes were all decreased significantly in FLS and supernatant of “Sanse powder ”volatile oil 10 and 250 μg/mL groups（P＜0.05）. CONCLUSIONS：“Sanse powder ”volatile oil can inhibit NLRP 3 inflammasome activation in FLS and reduce the downstream inflammatory cascade response ，thus exerting its efficacy in ameliorating synovial inflammation of KOA.