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ObjectiveTo investigate the mechanism of quercetin in regulating chondrocyte extracellular matrix metabolism and inflammatory response in knee osteoarthritis （KOA） from the perspective of autophagy. MethodChondrocytes were extracted and cultured， and the primary cells were identified by immunofluorescence staining with collagen Ⅱ. The chondrocytes induced by lipopolysaccharide （LPS） were divided into a control group （without any treatment）， a model group （10 mg·L-1 LPS treatment for 48 h）， and low-， medium-， and high-dose quercetin group （10 mg·L-1 LPS treatment for 48 h combined with 50， 100， and 150 mmol·L-1 quercetin for 24 h）. The inhibitory effects of LPS （2.5， 5， 7.5， 10， 12.5 mg·L-1） on the proliferation of chondrocytes for different periods （24， 48， 72 h） were detected by cell counting kit-8 （CCK-8）. The effects of quercetin （50， 100， 150， 200 mmol·L-1） on the LPS-induced proliferation of chondrocytes for different periods （12， 24， and 48 h） were investigated. The expression of microtubule-associated protein 1 light chain 3Ⅱ （LC3Ⅱ） and ubiquitin-binding protein p62 was detected by Western blot. LPS-induced chondrocytes were treated with 3-methyladenine （3-MA）. The resultant cells were divided into a control group （without any treatment）， a model group （10 mg·L-1 LPS）， a quercetin group （model group + 100 mmol·L-1 quercetin）， a 3-MA group （model group + 100 μmol·L-1 3-MA）， and a 3-MA + quercetin group （model group + 100 μmol·L-1 3-MA + 100 mmol·L-1 quercetin， specifically， LPS for 48 h， 3-MA for 2 h， and then quercetin for 24 h）. The content of interleukin （IL）-1β and tumor necrosis factor （TNF）-α was determined by enzyme-linked immunosorbent assay （ELISA）. The protein expression of matrix metalloproteinase 13 （MMP-13） and tissue inhibitor of metalloproteinase 1 （TIMP1） was detected by Western blot. ResultCollagen Ⅱ immunofluorescence staining showed that the extracted cells were consistent with the characteristics of chondrocytes. As revealed by CCK-8， the optimum concentration of LPS was 10 mg·L-1 with an action time of 48 h， and the optimum concentration of quercetin was 100 mmol·L-1 with an action time of 24 h. Western blot results showed that compared with the control group， the model group showed decreased expression of LC3Ⅱ （P<0.01） and increased expression of p62 （P<0.01）. The expression of LC3Ⅱ in the quercetin groups was higher than that in the control group （P<0.01）， especially in the medium-dose quercetin group. The p62 expression in the quercetin groups was lower than that in the control group （P<0.01）， especially in the medium-dose quercetin group. Compared with the control group， the model group showed increased expression of MMP-13 （P<0.05） and decreased expression of TIMP1 （P<0.01）. Compared with the model group， the quercetin groups and the 3-MA + quercetin group showed decreased expression of MMP-13 （P<0.05， P<0.01）， especially the quercetin groups， and increased expression of TIMP1 （P<0.01）， especially the quercetin groups. Morphological changes in chondrocytes under the inverted microscope showed that quercetin could restore the morphology of damaged chondrocytes. CCK-8 showed that compared with the control group， the model group showed inhibited chondrocyte proliferation （P<0.01）， and compared with the model group， the quercetin groups and the 3-MA + quercetin group showed promoted chondrocyte proliferation （P<0.01）， especially the quercetin groups. ELISA results showed that IL-1β and TNF-α levels in the model group were higher than those in the control group （P<0.01）， and the levels of IL-1β and TNF-α in the quercetin groups and the 3-MA + quercetin group were lower than those in the model group （P<0.05， P<0.01）， and the decrease in the quercetin groups was the most significant. ConclusionQuercetin can promote LPS-induced chondrocyte proliferation， regulate chondrocyte extracellular matrix synthesis and metabolic balance， inhibit the inflammatory response， and restore chondrocyte function. The mechanism may be related to the activation of autophagy by quercetin.
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Objective To study the relation between alcohol-induced osteonecrosis of femoral head (AIONFH) related with high morbidity TCM constitution type with CYP2C8 gene polymorphisms.Methods Totally 152 Han nationality NONFH cases from Feburary 2014 to September 2015 from outpatient and the inpatient departments in Gansu Province Hospital of TCM were collected. 50 AIONFH cases were set as medical case group; meanwhile, 45 healthy volunteers were enrolled as control group. Database for medical materials of all patients and volunteers was established. TCM distribution for AIONFH patients was determined. Solution DNA extraction kit was used to extract DNA, and detect the concentration and purity of DNA. The target gene was amplified by PCR and the target gene was amplified by gel electrophoresis. The length of the fragment was confirmed to conduct target gene sequencing. With the results of sequencing and gel electrophoresis, the relation of AIONFH with CYP2C8 gene polymorphism in AIONFH patients with phlegm-dampness syndrome and the control group.ResultsThe CYP2C8 gene loci rs17110453 gene polymorphism was not statistically significant between the two groups (χ2=0.253,P>0.05). There was no significant difference in allele between the two groups (χ2=0.077,P>0.05). The risk of disease in CC genotype was 1.37 times higher than the AA genotype (95%CI: 0.339-5.540), without statistical significance (P>0.05). There was no significant difference in genotype and allele distribution between AIONFH patients with phlegm-dampness and non-phlegm-dampness and the control group (P>0.05).Conclusion CYP2C8 gene loci rs17110453 gene polymorphism A/C mutation has no obvious relation with AIONFH risk. There is no clear relationship between CYP2C8 gene loci rs17110453 gene polymorphism with AIONFH.
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Objective To study the correlation between nontraumatic osteonecrosis of femoral head (NONFH) and ApoA1 polymorphism of blood stasis type.Methods Totally 93 cases of NONFH were selected as the case group, and 83 healthy volunteers were randomly selected as the control group. With TCM constitution questionnaire survey, the case group was screened out 32 cases of blood stasis NONFH type and 61 cases of non blood stasis NONFH type. In the case group and control group, the subjects took blood samples 2 mL, extracted DNA for PCR amplification, PCR products for DNA sequencing. G994T PAF-AH and rs9658282 gene NOS1 site polymorphism were detected for tatistical analysis.Results -75G/A gene AA ApoA1 genotype (OR: 2.578; 95%CI: 1.174-5.663;P=0.018) and A allele (OR: 1.726; 95%CI: 1.121-2.658;P=0.013) may be one of the risk factors of NONFH.Conclusion -75G/A gene ApoA1 may be related to the pathogenesis of NONFH. There was no correlation between the ApoA1 gene polymorphism of -75G/A gene and the pathogenesis of blood stasis NONFH. There was no correlation between the ApoA1 gene polymorphism of +83C/T gene and the pathogenesis of blood stasis NONFH.
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Objective To discuss the relationship between TCM constitutional types and the levels of transforming growth factor beta 1 (TGF-β1) of patients with knee osteoarthritis (KOA). Methods A total of 161 patients with KOA as a case group filled out questionnaires about 9 TCM constitution types, and 50 cases of unrelated healthy volunteers were selected randomly as control group. The serum samples of two groups were collected. The levels of TGF-β1 were detected and compared by double antibody sandwich ELISA. Results Compared with the control group, the level of TGF-β1 in the case group decreased, with statistical significance (P0.05). Conclusion The incidence of KOA with qi deficiency type may be related to the decrease of TGF-β1 level. The decreasing level of TGF-β1 may be one of the mechanisms of molecular biology that qi deficiency was linked to KOA.