| Osteoarthritis(OA)is a joint degenerative disease with articular cartilage degeneration,subchondral osteosclerosis,synovial hyperplasia and osteophyte formation.The knee joint is the most common site of the disease,which is characterized by the decreased of the activity adaptability of the knee joint.Age,mechanical stress and inflammation are the main factors inducing joint degeneration in the pathogenesis of OA.Chronic inflammation plays an important role in the development of OA.Although the occurrence of inflammatory reaction is not necessarily the cause of articular cartilage degeneration,the damage caused by its cascade amplification reaction mechanism can accelerate cartilage degeneration and affect the function of chondrocytes.This can cause phenotypic change of chondrocytes and degradation of extracellular matrix(ECM),which in turn leads to structural changes and loss function of articular cartilage.At present,the treatment of OA is still limited to symptomatic treatment.In modern clinical drug treatment,non-steroidal anti-inflammatory and nutritional cartilage drugs are mainly selected to relieve symptoms,but long-term use of these drugs will lead to serious side effects.In view of the characteristics of the course of OA,long-term treatment may be needed to slow down its progress,so there is an urgent need to develop a safe method for long-term clinical use of OA.Studies have shown that traditional Chinese medicine and dietary plants,fruits or their products involved in some foods have played a positive role in relieving arthritis.There are many kinds of natural products including terpenoids,polyphenols,flavonoids,quinones,phenolic acids,polysaccharides,alkaloids,saponins and other natural product compounds.Among them,flavonoids are more studied,which are widely found in fruits,vegetables,nuts and grains.Sinensetin(SIN)is a kind of multi-methoxy flavonoid compound,which has the effects of anti-cancer,anti-inflammatory,anti-oxidation,anti-angiogenesis.It mainly exists in clerodendranthus spicatus and citrus fruits.Clerodendranthus spicatus is widely grown in Southeast Asia and tropical countries.Its leaves are also known as "Java tea",and are used to make common herbal tea.This plant is used in local traditional medicine to treat rheumatism,arthritis,edema,gout and urinary inflammation.Similarly,citrus fruits are often used as traditional medicine in the treatment of traditional Chinese medicine in China,such as Aurantii Fructus Immaturus,Citri Grandis Exocarpim,Fructus Aurantii,Pericarpium Citri Reticulatae and Citri Reticulatae Pericarpium Viride,etc.They are mostly used to treat indigestion,bronchitis,asthma,and some inflammatory diseases,among which Pericarpium Citri Reticulatae is also often used to make tea in daily life,and studies have shown that Pericarpium Citri Reticulatae plays a positive role in the treatment of O A with traditional Chinese medicine decoction.Many studies have shown that sinensetin not only shows good activity to cells,but also has very little toxicity to normal cells and high selectivity.Based on the above research background and the results of drug screening in the previous database,we propose to apply sinensetin to treat OA chondrocytes and OA rat models.Then we observe its effects and study its mechanism hoping to provide a new method and theoretical basis for the treatment of OA in the future.Part I:Effect of sinensetin on human OA chondrocytesObjective:To screen the research drug and explore its effect on human OA chondrocytes in vitro.Methods:(1)We first obtained the differential genes of patients with OA compared with normal people through the GEO database analysis,then obtained the potential drugs with interaction through the cMAP database.And then we conducted the limited screening according to the screening results of TCMSP database platform and MCE website,and finally got the research object.(2)Human OA chondrocytes were extracted and identified.Chondrocytes were treated with different concentrations of sinensetin(0,0.01,0.1,1,5,10,50,100 μg/ml)incubate chondrocytes for 24,48 and 72 hours.CCK-8 was used to detect the effect of sinensetin on cell viability.(3)The effect of sinensetin and IL-1β on the proliferation of human OA chondrocytes was detected by EdU method.(4)The cell cycle distribution of chondrocytes treated with sinensetin and IL-1β was detected by flow cytometry.(5)EnzymeLinked Immunosorbent Assay(ELISA)was used to detect the contents of inflammatory factors TNF-α and IL-6 in the culture supernatant of chondrocyte under different conditions.(6)qRT-PCR and Western blot were used to detect the expression changes of inflammatory factors and extracellular matrix synthesis and catabolism-related proteins in chondrocytes treated with different concentrations of sinensetin and IL-1β.(7)Detection of the expression of type Ⅱ Collagen in chondrocytes by immunofluorescence.Results:(1)A total of 15 small molecular compounds were screened by cMAP database,TCMSP database and MCE website.Among the top 5 compounds,sinensetin was finally selected as the eligible research object.(2)The extracted primary cells were identified to be consistent with the characteristics of chondrocytes.The results of CCK-8 detection showed that the concentration of 0.1-1 μg/mL was the safe application range of sinensetin,which could effectively protect the proliferation ability of chondrocytes.(3)EdU test results showed that the number of cells in the proliferation period in the IL-1β treatment group was significantly reduced,while the number of proliferation positive cells in the sinensetin treatment group was significantly increased(P<0.05).(4)The results of flow cytometry analysis showed that compared with IL-1β treatment group,the proportion of S phase in cells of sinensetin treatment group increased,the proportion of G1 phase decreased,and the activity of cell division increased significantly compared with IL-1β treatment group,suggesting that sinensetin can effectively improve the division ability of chondrocytes in OA state.(5)The results of ELISA detection showed that sinensetin inhibited the production of TNF-α and IL-6 induced by IL1β in a dose-dependent manner,and the difference was statistically significant(P<0.05).(6)The results of qRT-PCR and Western blot showed that sinensetin treatment increased the expression of type Ⅱ Collagen(Collagen Ⅱ)and decreased the expression of MMP-13,MMP9 and inflammatory factors such as COX2,TNF-α and IL-6 compared with IL-1β stimulation group.(7)Immunofluorescence results also showed that sinensetin significantly improved the expression of type Ⅱ collagen in chondrocytes.Conclusion:Sinensetin can reduce the release of inflammatory factors and metalloproteinases from OA chondrocytes,inhibit the degradation of ECM,improve the survival rate of OA chondrocytes and protect chondrocytes.Part Ⅱ:Mechanism of sinensetin on human OA chondrocytesObjective:To explore the molecular mechanism of sinensetin on human OA chondrocytes in vitro.Methods:(1)The gene differential changes of OA chondrocytes treated with sinensetin were analyzed by transcriptome sequencing,and potential targets were screened by molecular docking.Then the target in human OA chondrocytes was knocked down by lentivirus transfection technology.Western blot and immunofluorescence were used to detect and verify the expression changes of proteins related to ECM synthesis and catabolism.(2)The target is verified by database analysis and the signal pathway of target regulation is obtained,and then verified and detected by Western blot and immunofluorescence.(3)DNA damage and morphological changes of chondrocytes were detected by TUNEL staining and transmission electron microscope scanning.(4)Assessment of chondrocyte damage by detecting the release of LDH.Results:(1)According to the results of transcriptome sequencing,the target gene SERPINA3 was screened by molecular docking.The effect of sinensetin on increasing the target gene SERPINA3 was further detected and verified by qRT-PCR,Western blot and immunofluorescence technology.(2)The use of lentivirus to knock down the SERPINA3 target aggravated the injury of chondrocytes,upregulated the expression of matrix metalloproteinases and inflammatory factors,while decreased the synthesis of extracellular matrix,which could be reversed by sinensetin treatment.(3)According to the prediction of database and-the analysis of sequencing results,SERPINA3 has a regulatory effect on NFκB/NLRP3 signal pathway.The results of Western blot and immunofluorescence showed that sinensetin could inhibit the activation of IL-1β and target knockdown on NF-κB/NLRP3 pathway,and effectively inhibit the phosphorylation of IκBα and p65 and the nuclear translocation of p65.(4)TUNEL staining showed that the number of positive cells in the sinensetin group was significantly less than that in the IL-1β group,and the nuclear size was relatively uniform.(5)The results of transmission electron microscope showed that the chondrocytes induced by IL-1β were swollen,vacuolated and protuberant on the surface of the cell membrane.However,sinensetin treatment group could significantly alleviate cell injury and improve cell integrity.(6)Compared with IL-1β treatment group,sinensetin treatment group could effectively reduce the release of LDH,and the release of LDH decreased gradually with the increase of drug concentration.Conclusion:Sinensetin can increase the expression of SERPINA3,which is the target of action in OA chondrocytes.By regulating NF-κB/NLRP3 signal pathway,it can inhibit the degradation of extracellular matrix and the release of inflammatory factors,maintain cell integrity,reduce the release of LDH,alleviate chondrocyte pyroptosis,and thus play an important role in cartilage protection.Part Ⅲ:Cartilage protective effect and mechanism of sinensetin on knee osteoarthritis in ratsObjective:To study the protective effect of sinensetin on cartilage in vivo and its possible mechanism by establishing a rat model of OA.Methods:(1)Wistar male rats aged 4 weeks and weighing about 200g were selected to establish OA model,and were randomly divided into sham operation group,OA model group and sinensetin treatment group.After 6 weeks of feeding treatment,hot plate test was performed.Record the time when the rats will produce corresponding action response after thermal stimulation.(2)We obtain the knee joint specimens of rats in each group and evaluated the pathological changes of knee osteoarthritis cartilage according to the International Osteoarthritis Research Society International(OARSI)grading system.(3)The knee joint specimens of rats were decalcified and embedded to prepare pathological sections.The pathological changes of rat knee cartilage were evaluated by HE staining,Alcian blue staining and safranin O fast green staining.(4)Evaluation of soft tissue lesions of knee joint in rats by Masson staining and Sirius red staining.(5)Immunohistochemical staining and Western blot were used to detect the expression of MMP-13,Collagen Ⅱ,SERPINA3,COX2,P65 and its phosphorylation levels as well as the expression of pyroptosis related proteins NLRP3,ASC and GSDMD in knee cartilage.(6)The changes of IL-1β and IL-18 secretion in plasma of rats in each group were detected by ELISA method.(7)The damage of chondrocytes was judged by TUNEL staining of rat knee joint specimens.(8)The abnormality of liver and kidney tissue was observed by HE staining,and the toxicity of the drug was evaluated.(9)Collect clinical samples to obtain cartilage,and further evaluate the effect of sinensetin by Western blot,qRTPCR,saffron O staining and quantitative detection of biochemical components after in vitro culture.Results:(1)The rat OA models were established successfully,and there was no death or joint infection occurred in all rats.The results of hot plate test showed that sinensetin could effectively alleviate the pain response of OA rats.The OARSI score of rats in sham operation group was close to normal.The surface of cartilage is smooth and flat.The rats in the OA model group had the highest OARSI score,the most serious cartilage damage,disorder of tissue structure,rough surface and more osteophytes formation.The score of sinensetin treatment group was significantly lower than that of O A model group,and the osteophyte and cartilage roughness were significantly improved.The difference was statistically significant(P<0.05).(2)The results of HE staining,Alcian blue staining and safranin O fast green staining of rat knee joint sections can clearly show that the cartilage layer of the knee joint in the sham operation group was thick and the surface was smooth,and the staining showed that the number of chondrocytes was large and evenly distributed.OA model group showed that the thickness of articular cartilage decreased,the surface was rough,the surface was irregular,and the number of chondrocytes decreased.Compared with the OA model group,the destruction of cartilage in the sinensetin treatment group was significantly alleviate,and the flatness of articular cartilage surface and the arrangement of chondrocytes in the articular cartilage were improved.(3)According to the results of HE staining,Masson staining and Sirius red staining,compared with the OA model group,the synovium in the sinensetin treatment group had a clear level of synovial tissue,no obvious hyperplasia,no obvious scar formation in the soft tissue around the joint,and the adhesion was significantly improved.(4)The results of immunohistochemistry staining and Western blot showed that among the three groups,the expression of SERPINA3 and Collagen Ⅱ was the lowest,the expression of MMP-13 and COX2 was the highest,and the phosphorylation level of P65,GSDMD and NLRP3 was also the highest in the OA model group.Compared with the OA model group,the sinensetin treatment group significantly improved the above indicators.(5)Through the analysis of ELISA test results,it can be found that the production of IL-1β and IL-18 in the OA model group was significantly higher than that in sham operation group,while the expression level of IL-1β and IL-18 in the sinensetin treatment group was significantly lower than that in OA model group.The difference was statistically significant(P<0.05).(6)TUNEL staining showed that the positive rate of TUNEL in knee joint of rats treated with sinensetin was significantly lower than that of OA model group.(7)The pathological results of HE staining showed that the structure of hepatic lobule in sinensetin treatment group was clear and normal,the morphology of glomerulus was normal,and there were no obvious signs of degeneration and necrosis in normal renal tubules.(8)The results of clinical samples showed that the expression of SERPINA3 in cartilage of patients with OA was significantly lower than that of healthy,people.The results of cartilage culture in vitro showed that sinensetin promoted the synthesis of GAG and the proliferation of chondrocytes.The results of qRT-PCR showed that sinensetin increased the expression of COL2A1 and SERPINA3,and inhibited the expression of p65,NLRP3,GSDMD and ASC.Conclusion:Sinensetin can relieve the pain of OA rats,reduce the expression of inflammatory factors and ECM decomposing factors,delay the-destruction of articular cartilage,regulate the NF-κB/NLRP3 signal pathway by regulating the expression of SERPINA3 in articular cartilage,and inhibit the scorching death of chondrocytes in the knee joint of rats.At the same time,sinensetin can promote the matrix synthesis of human cartilage and the proliferation of chondrocytes,and inhibit gene expression related to pyroptosis.It plays a positive role in protecting OA articular cartilage. |
PDF Full Text Request