| Knee Osteoarthritis(KOA)is a common disabling disease in middle-aged and elderly population,which seriously affects the quality of life of the affected population.The pathogenesis of OA has not been fully clarified,and it is currently believed that mechanical factors,inflammation,aging and metabolic factors are involved in the occurrence and progression of OA.In recent years,the research on the metabolic disorder of OA cartilage has become a hot topic.Under OA microenvironment and inflammatory,chondrocytes undergo metabolic switch and pathological changes of cartilage remodeling,which are characterized by the enhancement of glycolysis pathway,mitochondrial dysfunction and cartilage senescence.Lactate has always been considered as a metabolic by-product of glycolysis,but in recent decades,more and more studies have shown that lactate plays an important role in regulating physiological and pathological processes of the body.Lactate can enter and exit cells through its specific transporter and bind its specific Hydroxy Carboxylic Acid Receptor 1(HCAR1)on the cell membrane.And its signal transduction involves energy metabolism,immune response and inflammatory response.At the same time,lactate can serve as respiratory fuel for mitochondria.promote aerobic oxidation,increase Reactive oxygen species(ROS)production,and cause cellular oxidative stress.At present,lactate is not only the by-product of glycolysis,but also a signal molecule involved in cell energy metabolism,immune regulation and tumor growth.Although lactate has been widely studied in tumor,immunity and other fields,the role of lactate in many other fields is still unknown and needs further exploration.Studies have shown that the levels of lactate in arthritic joints are significantly higher than normal physiological concentrations.In Rheumatoid Arthritis(RA),lactate regulates immune function by affecting immune cell metabolism,potentially affecting the occurrence of RA.OA itself presents a chronic low-grade inflammation,but there is a lack of studies on the effect of lactate on OA.Therefore,our study mainly explored the role of lactate in the pathological process of OA and its regulatory mechanism,further improved the pathogenesis of OA,looked for potential targets,and provided new ideas for the prevention and treatment of OA.In our study,we first found that OA chondrocytes had enhanced glycolysis capacity and increased expression of lactate transporter and lactate receptor through bioinformatics analysis.Therefore,we targeted the lactate molecule for exploration,and found that the serum lactate level of OA patients increased,and the lactate level in the synovial fluid of OA patients was positively correlated with the serum lactate level.The chondrocytes of KOA patients were isolated and cultured in vitro,and we found that lactate can damage the function of chondrocytes.We further explored the mechanism of lactate injury to chondrocytes,and found that lactate can make chondrocytes in a state of oxidative stress.By scavenging intracellular reactive oxygen species(ROS),the damage of lactate to chondrocytes can be improved.And NADPH oxidase 4(NOX4),a key molecule leading to oxidative stress,was locked.Next,we confirmed that lactate can activate the downstream PI3K/Akt signaling pathway by inducing and binding lactate receptor HCAR1,inducing the increase of NOX4 expression,thus promoting the increase of ROS level in chondrocytes and causing chondrocyte injury.In vivo experiments of SD rats have confirmed that long-term high concentration of lactate in the articular cavity can cause cartilage damage and aggravate the progression of OA.Finally,we analyzed the association between lactate levels in synovial fluid and serum of KOA patients and clinical metabolic markers.We found that there is a correlation between lactate levels and metabolic markers,suggesting that there is a correlation between lactate levels in synovial fluid and metabolic syndrome(MetS).It was found that the level of lactate in the synovial fluid of OA patients with metabolic syndrome(MetS-OA)was significantly higher than that of OA patients with non-metabolic syndrome(nMetS-OA),suggesting that lactate may be an important medium for the association between OA and MetS.This study is divided into five chapters:(1)Lactate damaged the function of chondrocytes in KOA patients;(2)The effects of lactate on glucolipid metabolism and oxidative stress of chondrocytes;(3)Lactate induced NOX4 expression by binding HCAR1 receptor to activate PI3K/Akt signaling pathway;(4)The effect of lactate on knee cartilage in SD rats;(5)Clinical correlation analysis of lactate levels in serum and synovial fluid of KOA patients.Chapter 2:Lactate damaged the function of chondrocytes in KOA patientsObjective:To investigate the level of lactate in serum and synovial fluid of OA patients,and to further explore the effect of lactate on chondrocyte function.Methods:GSEA enrichment analysis and protein interaction network analysis were performed by screening osteoarthritis datasets from GEO database.KOA patients who underwent total knee arthroplasty in the department of Bone and Joint Surgery of our hospital from November 2019 to August 2021 were selected as the study group,and healthy people who underwent routine annual physical examination in the health examination center of our hospital were selected as the healthy control group according to age and gender matching.Serum samples from both groups and synovial fluid from OA patients were collected to detect lactate level.The knee joint chondrocytes of KOA patients were isolated and cultured in vitro.The concentration of lactate stimulation was confirmed by CCK-8 and apoptosis.The expression of lactate transporter was detected by qPCR and immunocytochemistry.The expression of matrix degrading enzyme was detected by ELISA and qPCR,and the effect of lactate on the expression of type II collagen was detected by immunocytochemistry.The expressions of inflammatory factors and chemokines were detected by ELISA and qPCR.The expression of genes related to cell hypertrophy and senescence was detected by qPCR,and the senescence of chondrocytes was detected by β-galactosidase staining.Results:(1)Bioinformatics analysis showed that the chondrocytes of patients with KOA showed enhanced glycolysis and increased expression of lactate transporter and lactate receptor.(2)Serum lactate level in KOA patients was significantly higher than that in healthy subjects,and was positively correlated with the level of lactate in synovial fluid(r=0.5420,p=0.005).(3)Lactate induced increased expression of lactate transporters Mctl,Mct2,Mct3,Mct4,Slc5a8 and Slc5a12 in KOA chondrocytes,and significantly increased Mctl protein level.(4)Lactate induced increased levels of Mmp3,Mmp13 and Adamts4 genes in chondrocytes,significantly increased protein levels of Mmp13 and Adamts4,and decreased expression of type Ⅱ collagen.(5)The levels of IL-6,IL-1β,Tnf-α,Ccl-3 and Ccl-4 genes were increased,and the protein levels of IL-6,CCL-3 and CCL-4 were significantly increased in chondrocytes induced by lactate.(6)Lactate induced chondrocyte hypertrophy related genes Col10a1,Alp,Runx2,Vegfa,Opn were significantly increased,the expression of senescence gene p53 was significantly increased,and the proportion of β-galactosidase positive cells was significantly increased,and increased chondrocyte hypertrophy and senescence.Conclusion:Lactate induced the expression of lactate transporter in chondrocytes and promoted the entry of lactate into cells.Lactate can induce extracellular matrix degradation by increasing the expression of chondrocyte matrix degradation enzyme and reducing the synthesis of type Ⅱ collagen,meanwhile induced secretion of inflammation and chemokines,and induced cartilage to hypertrophy phenotype and senescence,resulting in chondrocyte damage.Chapter 3:The effects of lactate on glucolipid metabolism and oxidative stress of chondrocytesObjective:To investigate whether lactate promotes ROS production by influencing cell metabolism and induces chondrocyte injury through ROS mediated oxidative stress.Methods:The effect of lactate on energy metabolism of chondrocytes was detected by SeahorseXF analyzer.Flow cytometry 2-NBDG labeling method and HK kit were used to detect the effect of lactate on the glucose uptake of chondrocytes.QPCR,flow cytometry and Western blot were used to detect the effects of lactate on the expression levels of key enzymes in glucolipid metabolism of chondrocytes.NADH detection kit and acetyl-CoA detection kit were used to detect the levels of intermediate metabolites NADH and acetyl-CoA.Flow cytometry was used to detect intracellular and mitochondrial ROS levels,and NAC was used to scavenge intracellular ROS to observe the effect on cell function.Glucose metabolism inhibitor 2-DG or lipid metabolism inhibitor Etomoxir were used to observe the effects on intracellular ROS and cell functions.The expression of NOXs was detected by qPCR and immunocytochemistry,and the NADPH level was detected by NADPH detection kit to further explore the source of ROS.Results:(1)Lactate significantly increased the OCR value and decreased the ECAR value of chondrocytes.Adding 2-DG in addition to lactate could not change the OCR value and ECAR value,suggesting that lactate could enhance the aerobic oxidation capacity of chondrocytes and inhibit the glycolysis pathway to some extent.(2)Lactate can induce the expression levels of glycolysis pathway genes Hk2,Pfkfb3,Ldha,G6pd significantly increased,Pdkl expression was decreased,Hk2 and LDH protein levels were increased,suggesting that lactate could divert glucose metabolism pathway to pentose phosphate pathway.(3)Lactate can increase NADH level,decrease NAD+/NADH ratio,decrease Pdkl expression,and increase acetyl CoA level,suggesting that lactate can generate pyruvate through LDHA into mitochondria to generate acetyl CoA,and enhance aerobic oxidation of chondrocytes.(4)Lactate can increase intracellular total ROS level,but does not affect mitochondrial ROS level,suggesting that lactate can promote the increase of cytoplasmic ROS production;(5)Scavenging intracellular ROS can alleviate cell senescence caused by lactate,weaken the inhibitory effect of lactate on type Ⅱ collagen synthesis,and reduce the protein secretion levels of MMP3,MMP13,IL-6,CCL3,and CCL45 suggesting that lactate can induce chondrocyte injury by promoting intracellular ROS generation;(6)Inhibition of glucolipid metabolism does not affect the generation of ROS in mitochondria.The addition of 2-DG can inhibit the promoting effect of lactate on ROS,but the addition of Etomoxir does not significantly inhibit ROS,suggesting that the ROS increased by lactate may come from glucose metabolism.(7)2-DG can reduce the MMP13 protein level increased by lactate,but Etomoxir can not reduce the MMP13 level.Meanwhile,the addition of 2-DG can alleviate the senescence of cells,suggesting that inhibition of glucose metabolism can reduce the damage of lactate on chondrocytes.(8)Lactate can increase NOX4 gene and protein expression in chondrocytes.NADPH level and NADPH/NADP+ ratio increased after lactate stimulation,suggesting that lactate provides a substrate for NOX4-catalyzed ROS production by promoting NADPH generation.At the same time,the addition of 2-DG on the basis of lactate stimulation showed that the level of NADPH decreased and NADPH/NADP+ratio decreased Nox4-specific inhibitor GLX351322 can significantly reduce the secretion of MMP3,MMP13,CCL3 and CCL4 induced by lactate.Conclusion:Lactate can promote glucose metabolism to divert to pentose phosphate pathway,and increase NOX4 expression and cytoplasmic ROS level,thus inducing chondrocyte injury.Chapter 4:Lactate induced NOX4 expression by binding HCAR1 receptor to activate PI3K/Akt signaling pathwayObjective:To investigate the mechanism of lactate regulating NOX4 expression through PI3K/Akt.Methods:GEO database was used to screen data sets,and David bioinformatics resource website was used for enrichment analysis to screen underlying potential signal pathways.The chondrocytes were stimulated by lactate for 24h with or without the addition of PI3K inhibitor LY294002 or Akt inhibitor MK2206.Western blot was used to detect the effects of lactate on the expression of PI3K,p-PI3K,Akt,p-Akt and NOX4.qPCR and Western blot were used to detect the effect of lactate on HCAR1 expression.SiRNA-HCAR1 was transfected with Lipo2000 to silence HCAR1,and Western blot was used to verify the silencing effect.After HCAR1 was knocked out with or without lactate stimulation,the expression of PI3K,p-PI3K,Akt,p-Akt and NOX4 was detected by Western blot.Results:(1)Enrichment analysis of differential genes in OA cartilage was carried out by David bioinformatics resource website,as well as analysis of gene data sets with MCT1 and MCT4 knockout.The results showed that the differential pathways in the three data sets all contained PI3K/Akt signaling pathway.(2)After 24h of lactate stimulation,p-PI3K and p-Akt were significantly increased,while p-PI3K/PI3K ratio and p-Akt/Akt ratio were significantly increased.The addition of LY294002 at the same time of lactate stimulation could reduce p-Akt level and p-Akt/Akt ratio,while p-PI3K and p-PI3K/PI3K tended to increase,but there was no statistical difference.The level of p-PI3K and p-Akt,the ratio of p-PI3K/PI3K and p-Akt/Akt did not change significantly after the addition of MK2206 with lactate stimulation.Lactate can increase NOX4 expression,while the addition of LY294002 or MK2206 can attenuate the increased level of NOX4 by lactate.(3)Lactate can induce the elevation of HCAR1 gene and protein in chondrocytes;(4)The levels of p-PI3K and p-Akt,p-PI3K/PI3K and p-Akt/Akt ratio decreased when HCAR1-siRNA and lactate were used together.NOX4 levels were reduced by the use of HCAR1-siRNA and lactate.Conclusions:Lactate activates the downstream PI3K/Akt signaling pathway by inducing and binding lactate receptor HCAR1,and induces increased NOX4 expression,thus promoting the ROS level in chondrocytes and causing chondrocyte injurychapter 5:The effect of lactate on knee cartilage in SD ratsObjective:To verify the effect of lactate on knee cartilage in SD rats.Methods:Clean SD rats aged about 8 weeks and weighing between 240g and 280g were selected for OA modeling by ACLT+MMD(anterior cruciate ligament detachment+medial meniscus destabilization)group and type Ⅱ collagenase injection into the joint cavity.Thirty rats were randomly divided into 6 groups with 5 rats in each group,including 1 month saline injection group,3 months saline injection group,1 month lactate injection group,3 months lactate injection group,OA group and OA+lactate injection group.Daily indexes(body weight and knee joint diameter)of rats were recorded.After each group’s intervention,the group was executed at the scheduled time.The knee specimens were taken for gross observation score,and pathological sections were done.And HE staining,saffron O-solid green staining and Masson staining were performed.Finally,Mankin’s score and OARSI score were performed to evaluate the cartilage condition.Results:(1)The diameter of knee joint treated with type Ⅱ collagenase injection and ACLT+MMD was significantly increased compared with the healthy side.Compared with the healthy side,the gross score,Mankin’s score and OARSI score of type Ⅱcollagase injection and ACLT+MMD group were significantly increased,confirming that both OA modeling methods were successful.ACLT+MMD was selected as OA modeling method because of the similarity between surgical modeling methods and OA process.(2)There were no significant differences in gross score,Mankin’s score and OARSI score between the lactate injection group and the saline injection group after 1 month.After 3 months of lactate injection,cartilage damage occurred,and the gross score,Mankin’s score and OARSI score were statistically different from those of the normal saline injection group.Compared with OA group,the degree of cartilage damage after 3 months of lacta,te treatment was less than OA group,and there were statistical differences in gross score and Mankin’s score,suggesting that long-term injection of lactate in articular cavity could affect the occurrence of OA.(3)Knee joint diameter of OA group and OA+lactate injection group was increased compared with control group(sham operation+normal saline injection),but there was no significant difference in weight Compared with the control group,OA group and OA+ lactate injection group had statistical differences in gross score,Mankin’s score and OARSI score.Compared with the OA group,the OA+lactate injection group had more severe cartilage damage,with statistical differences in Mankin’s score and OARSI score,but no statistical differences in gross score,suggesting that the injection of lactate in the joint cavity can aggravate the development of OA.Conclusion:Long-term injection of high concentration of lactate in articular cavity can cause cartilage injury,and lactate can aggravate the progression of OA.Chapter 6:Cinical correlation analysis of lactate levels in serum and synovial fluid of KOA patients.Objective:To investigate whether there is a difference in lactate levels in the synovial fluid and serum of KOA patients,and whether there is a correlation between lactate levels and MetS related factors and other clinical factors.Methods:Correlation analysis between the lactate levels of serum and synovial fluid in KOA patients and clinical factors(height,weight,BMI,waist circumference,blood pressure),metabolic indicators(fasting blood glucose,triglyceride,high-density lipoprotein,low-density lipoprotein,cholesterol)and metabolic syndrome scores METS-IR and Z-Score was performed.According to the 2005 NCEP-ATP Ⅲ criteria for the diagnosis of metabolic syndrome in Asian patients,patients with metabolic syndrome in KOA were enrolled into MetS-OA group,and patients with non-metabolic syndrome were enrolled into MetS-OA group.The levels of lactate in synovial fluid and serum of the two groups were compared.Synovial tissues and cartilage tissues of patients with MetS-OA and nMetS-OA were cultured in vitro to observe the acidification of culture medium.And the gene and protein expression levels of LDHA in primary fibroblast-like synoviocytes of the two groups were detected by qPCR and Western blot.Results:(1)Compared with nMetS-OA group,the lactate level of the synovial fluid in MetS-OA group was significantly higher,but there was no statistical difference in serum level of lactate between the two groups.(2)Compared with the nMetS-OA group,the medium for synovial tissue culture in MetS-OA group was more yellow,indicating that the medium was more acidic,suggesting that MetS-OA synovial tissue had a stronger ability to secrete acid.There was no significant difference between MetS-OA and nMetS-OA in cartilage.(3)Compared with nMetS-OA group,the LDHA gene level and protein level of fibroblast-like synoviocytes in MetS-OA group were significantly increased.suggesting that MetS-OA fibroblast-like synoviocytes had stronger glycolysis ability.(4)Correlation analysis showed that lactate level in OA patients was negatively correlated with BMI(r=-0.3766,p=0.0368).It was positively correlated with systolic blood pressure(r=0.1528,p=0.027).It was positively correlated with fasting blood glucose(r=0.4081,p=0.0311).It was positively correlated with triglyceride(r=0.3668,p=0.0389).It was negatively correlated with high-density lipoprotein(r=-0.3460,p=0.0450);It was positively correlated with metabolic syndrome Z-score(r=0.4430,p=0.0098).There was no significant correlation between lactate level in synovial fluid and waist circumference,cholesterol,low density lipoprotein,or METS-IR.Conclusions:The difference in the level of lactate in the synovial fluid of KOA patients suggests that lactate may be an important mediator in the association of OA and MetS. |
