Comparative Study On Chondrocyte Apoptosis Signal Transduction Pathways, Gene Expression Profiles And Selenium Intervention Between Osteoarthritis And Kashin-Beck Disease

Background:Kashin Beck(KBD)is an endemic disease, with a high prevalence in north China. Thedisease is characterized by cartilage necrosis and local deformation of bone and joint. It wasfirst reported in 1849 by a Russian Yurenski. Since then a lot of work has been done on thisdisease but still its etiology and pathogenesis is unclear. Different aspects of Kashin Beckdisease studied so far include epidemiological as well as experimental studies includingautopsy pathology genetic interactions in the environment and the cultivation of living cells.Studies have shown that the pathogenesis of Kashin Beck disease is related to environmentalfactors (low selenium, T 2 toxin, etc.), cartilage necrosis, as well as abnormal expression ofgene and proteins.Osteoarthritis:Osteoarthritis (OA) is characterized by articular cartilage degeneration,causing joint pain and dysfunction. It occurs in knee, hip and other weight bearing joints. Itusually occurs in late years of life, mainly at the age of 60 years or older, while KBD startsin 3 12 year old child. They significantly differ in terms of abnormal thickening of thecartilage in joint's development disorders. Recent studies on OA show that the disease isrelated to apoptosis, disorder in cytokine and signal transduction mechanism. Tissue sectionexperiments have proved that KBD cartilage damage is also related to abnormal expressionof apoptotic factors. Thus both KBD and OA shares some mechanism of the diseasedevelopment but it needs further clarifications about differences in development of both thediseases.At present there are many unanswered questions about the pathogenesis and etiology ofKBD. Some of the questions which need further research include:?The etiology of KBD.Is it a single factor or an interaction between genes and environment??What is thedifference between the pathological changes of the articular cartilage in KBD and OA??What is the genetic basis of KBD and OA??What is the mechanism of the KBD prevention and treatment by selenium .Keeping in view the aim of differentiating these twodiseases the etiology and pathogenesis of KBD and OA was further which provided ascientific basis for KBD prevention and treatment. This aim in this study we focused on thecomparative study of KBD and OA chondrocyte apoptosis signal transduction, genome widescan screening differential gene profiles and selenium intervening on chondrocyte apoptosis;Objectives:1. To compare the differences between cartilage cells of KBD and OA in terms ofchondrocyte growth and proliferation, morphological structure, apoptosis and signaltransduction pathway by culturing the cartilage cells of KBD and OAin vitro;2. To identify the characteristic gene expression of KBD, which is different from the OA byAgilent Human 1AcDNAmicroarray;3. To investigate the effect of selenium on cell growth characters and apoptosis among KBD,OAand normal cartilage groups by exposing them to different levels of selenium.Method:1. The patients were divided into three groups: KBD, OAand the normal group. The patientsof control and OA groups were selected from the non KBD endemic area, while patients ofKBD were selected from KBD endemic area, aged 49 65 years old. The diagnostic criteriafor KBD included assessment on fingers, joints and immunohistochemistry analysis. Patientswith Stage?and?were included in this study. OA patients were diagnosed according toWOMAC, patients with genetic bone disease and rheumatoid arthritis were excluded fromthe study. All cartilage were taken from the same location in the joint as far as possible toachieve all aspects to be matched.2. In aseptic conditions, KBD, OA and normal cartilage cells were cultured in vitro. Wecontrast KBD, OA and normal chondrocytes of different growth characteristics and cellapoptosis pathway by MTT, flow cytometry, electron microscopy, immunofluorescence,laser confocal and immunohistochemistry; Selenium intervention on three groups ofcartilage cells was done to find the impact of different concentrations of selenium on cellgrowth and apoptosis in KBD and OA.3. We compared difference in gene expression between KBD and OA articular cartilage bygenome wide scan, and checked them by real time PCR; Microarray were scanned byGene Axon 4000B and data was analyzed by Genepix 3.0 software package. Special KBDgenes which were different to OAwere selected.Results:I. Comparision of cell apoptosis and signal transduction pathways between KBD and OA 1) The average cell proliferation of KBD and OA cartilage group were significantly lowerthan the normal group, while OA growth rate was less than KBD cartilage cell. Theultrastructure in KBD cartilage cell were as follows: irregular cell shape, distorted nuclei,glycogen gathered within the cell membrane, glycogen release, Golgi apparatus andsignifican mitochondrial swelling; OAgroup: cell swelling, cartilage cells, increased electrondensity, reduced membrane villus in cytoplasm with a large number of free lipid droplets,Golgi apparatus and endoplasmic reticulum swelling.2) The apoptosis in KBD and OA cartilage was significantly higher than normal group, cellcycle G1 / M phase was increased; The expression of Fas, Bax, Caspase 3, Caspase 9 andP53 increased; Caspase 8, P53 expression in KBD cartilage cells is relatively lower than theOA, while the expression of caspase 9 is relatively higher than OA.3) The comparision of apoptosis genes in KBD and OA cartilage by gene chips, indicatedthat KBD and OA have similar apoptosis pathway, KBD was prone to mitochondriaapoptosis signal pathway.II. The comparative study of genome wide scan and biological modeling in KBD and OAcartilage.In the comparative study of KBD and OA cartilage, there were 233 same array of genesexpression, in which 195 genes up regulated and 38 genes down regulated in the four pairgroups. They were divided according to their biological function into categories such ascellular metabolism, ion channels and transport proteins, signal transduction, cytokines, cellreceptors and the cytoskeleton .It was confirmed before gene selction that the genes selectedCSGALNACT, PIM2, EFNA1, SMAD 9, STK11, AQP, T cell factor / LEF, PTN, APCDD1,CAV and others in KBD were of great significance. Based on the results, KBD is mainlyrelated to metabolism, ion channel proteins, apoptosis and other aspects of gene expressionabnormalities. We build oxidative stress, amino acid metabolism, lipid metabolism andrelated pathways model.III. The effect of various concentrations of selenium (Se) on growth and apoptosis in KBDand OA.1) Selenium in high concentrations i.e Se>0.05 g/ml produced toxic damage to normalcartilage cells while the low concentration of selenium also have an impact on the growthrate and the proliferation rate became negative.Studies on KBD cartilage have shown that acertain concentration of selenium can promote the KBD cartilage cells growth, but the highconcentrations of selenium have some of its toxic effects. The concentration of seleniumbetween 0.25 g/ml?0.10 g/ml significantly promote cell proliferation, but the low environment selenium can reduce the proliferation rate. Studies on OA have showedtolerance to high concentrations of selenium and toxic effects of selenium on cellproliferation, but still the cells will be able to maintain growth and proliferation; inSe<0.25 g/ml cell proliferation and growth rate were also high.2) Normal cartilage after exposure to higher concentration of selenium showed toxic effectson the growth with more damage to cell growth; in terms of KBD, 1.0 g/ml?0.50 g/ml ofselenium produces toxic effect on cell growth, and a concentration of 0.25 g/ml?0.10 g/mlpromoted cell growth; while environmental selenium with a concentration of <0.05 g/mlwas the safety range; for OA, 1.0 g/ml?0.50 g/ml of selenium produced toxic effects oncell growth, Se<0.25 g/ml promoted cell proliferation.3) The tolerance of KBD and OA to selinum was higher than the normal group, and amongthem KBD had higher tolerance than OA group and the tolerance of OA group is higher thanthe normal group. Microstructure indicated that cartilage cells were tolerant to Se<0.10 g/ml in KBD group and to Se<0.05 g/ml in OA group. While in the normal group thesub structure was by a concentration of Se>0.025 g/ml.4) Effect of selenium on apoptosis: In normal cartilage cells the the apoptosis rate was higherwith different concentrations of selenium as compared to apoptosis rates without selenium.In the KBD group, the higher concentrations of selenium produced higher apoptotic rate butthe average apoptotic rate declined with selenium. In the OA group, the higher concentrationof selenium resulted in high apoptosis rate but the certain concentration of selenium mayreduce the apoptosis rate.5) Immunohistochemistry of Fas, Bax, Caspase 3, Caspase 9, P53 and other apoptosisindicators also showed that a certain concentration of selenium added to the KBD and OAchondrocyte in culture can inhibit apoptosis protein expression. The tolerance to selenium ofKBD was higher than OA, and OA was higher than normal cartilage cells. Beyond a certainconcentration selenium can induce apoptosis.Conclusion:1. The rate of apoptosis in KBD and OA articular cartilage was significantly higher than thenormal group, and expression of Fas, Bax, Caspase 3, Caspase 9 and P53 increased. In thecomparison of KBD and OA, Caspase 8 and P53 expression in KBD were relatively lowerthan OA, while the caspase 9 expression is relatively higher than OA. Micro structuralchanges in KBD by electron microscopy confirmed that KBD apoptosis pathway is similarto OA, and may be more prone to mitochondrial apoptosis pathway.2. There is significant difference in 233 gene expressions in KBD and OA, out of these 38genes were down regulated and 195 genes were up regulated in KBD. CSGALNACT, PIM2, EFNA1, SMAD 9, STK11, AQP, T cell factor / LEF, PTN, APCDD1, CAV and other geneswere confirmed to be of greater significance in KBD, and that is mainly related tometabolism, ion channel proteins, apoptosis and other abnormal genes expression.3. A certain concentration of selenium has protective effect on the KBD and OAchondrocytes. Under the same dose of selenium, the tolerance of KBD is higer than OA andOA tolerance is higher than normal group.Higher concentrations of selenium have damagingand toxic effects on the normal cells and the three groups of chondrocytes have differentconcentrations of selenium tolerance.