Effects Of Different Cyclic Strengths On Expression Of Phospholipase A2/Cyclooxygenase/Prostaglandin E2in Human Tenocytes In Vitro

Tendinopathy is a common disease in orthopaedics and sports medicine, with cyclicalstretch overloading being its direct reason. Pathology has confirmed that overused andrepeated injury of strengthening led to the tendon tissue inflammation, degeneration, andeven the final micro-injury, fracture. Numerous studies demonstrate that expression level ofprostaglandin E2(PGE2) produced by tenocytes in the case of cyclical stretch loading invitro significantly increases which plays an important role in the inflammatory reaction ofTendinopathy. PGE2is mainly regulated and controlled by PLA2/COX/PGE2modularnetwork. At present, in the development of Tendinopathy, it is still not clear whether tendoncells regulate and control PLA2/COX/PGE2modules under different stretch strengths.A preliminary study of our study group showed tenocytes may, through thetransformation from extracellular mechanical signal into intracellular biochemical signal,start PLA2/COX/PGE2molecular network system, promote the production of PGE2, andcause tendinopathy. On the basis of preliminary studies, this research explores influnces ofdifferent stretch strengths on proliferation and apoptosis of tenocytes and expression ofCytosolic phospholipase A2, Secreted Phospholipase A2, cyclooxygenase1,cyclooxygenase2and Prostaglandin E2, makes clear the regulation and control of differentstretch strengths on tenocytes PLA2/COX/PGE2modular network system and provides anew idea for Tendinopathy prevention and treatment.This study preliminarily analyzes the regulation and control of different stretchstregths on tenocytes PLA2/COX/PGE2modular network from the following three aspects:1. To establish the model of tenocytes stretching in vitro and evaluate the tenocyteproliferation and apoptosis between different stretching groups.This part of the experiment is to establish an effective model that simulates the normalstate of tenocytes in vitro stretching and evaluate the proliferation and apoptosis betweendifferent stretching groups on human tenocytes in vitro. 1.1Methods1.1.1The human tenocytes were isolated by the means of enzymic digestion,morphology of the cells were assayed with inverted microscope,collagen typeⅠ,Ⅲ andVimentin of the tenocytes were charactered by immunofluorescence stain and confocallaser scanning microscope. A vitro cyclic stretching model system was applied in thisstudy.1.1.2GroupingAdherent growth of tenocytes grown in microgrooved silicone dishes, under0.5Hz,4hconditions along one-way loop stretching, placed in a cell incubator. Microgrooved siliconedishes of the same strength group were cultured in pairs. The human tenocytes wereuniaxially stretched4hours at0.5Hz with different intensity of stretching (4%,8%and12%). Non stretched tenocytes were applied to control group. The tenocytes and nutrientsolution were harvested after four hours.1.1.3To evaluate proliferation and apoptosis of human tenocytes between differentstretching group.CCK-8was applied to evaluate the tenocyte proliferation, and flow cytometry wasapplied to evaluate cell cycle.1.2Results1.2.1Culture and identification of human tenocytesUnder an inverted microscope the general shape of the tenocytes were long spindle,showing the arrangement of the tendon-like cluster, Immunofluorescence staining showedthat vimentin was positive, type Ⅰcollagen was positive, type III collagen was negative.1.2.2Human tenocyte morphology before and after stretchingTenocytes was grown in microgrooved silicone dishes. After4hours some cells beganto adhere, after12hours95%of tenocytes were adherent along the bottom of the dishes,arranged to irregular. After stretching, the tenocytes aligned along the stretching bottom ofthe dishes.1.2.3CCK-8evaluated tenocyte proliferationOD value of4%，8%，12%stretching group were decreased significantly (P <0.05).1.2.4Flow cytometry evaluated tenocytes cycle and apoptosisThere was no significant difference about G1/S phase between test and control groups in the apoptotic rate. Compared with increased stretching group, the apoptotic ratewas increased significantly; especially8%and12%stretching group.1.3Summary1.3.1Enzyme digestion method successfully isolated and cultured human primarytenocytes. Tenocytes were identified by the immune phenotype so that it can providesufficient tenocytes.1.3.2The vitro cyclic stretching model system was effective in this study in order tosimulate in vivo human conditions.1.3.3Tenocytes proliferation and apoptosis were changed with different intensity ofstretching (0%,4%,8%and12%) under0.5Hz,4h condition. With the increase of intensityof stretching, the cell apoptosis rate was increasing; the cell proliferation was decreasing.However, the tenocyte G1/S phase did not change.2. To evaluate the expression of PLA2/COX between different stretching groupson human tenocytes in vitroThis study was based on the first part of the stretching model of the human tenocytesin vitro. It aimed to evaluate human tenocytes from gene and protein levels of cPLA2, ofsPLA2, to COX1, of COX2trends between different stretching groups.2.1Methods2.1.1Western blot was applied to evaluate the cPLA2, COX1and COX2proteinexpression differences.The rabbit anti-human of cPLA2, COX1and COX2monoclonal antibodies andHRP-labeled secondary antibody was used. The image density was determined bymeasuring the Western blot the expression of gray value. The relative gray value was usedto evaluate the protein expression differences.2.1.2SPLA2was measured according to ELISA manufacturer instructions. Theamount of sPLA2was analyzed by optical density (OD) with enzymic linkedimmunocemetry.2.1.3RT-PCR were performed to investigate the difference of genetic transcription ofcPLA2、COX1and COX2, Gelation Image to scan, Quantity one software to measuretargeted Gershgorim band and grey level, and relative grey level to analyze difference trendof gene expression. 2.2Results2.2.1RT-PCR was applied to evaluate cPLA2, COX1and COX2gene transcriptionunder different stretching strengthsThe level of cPLA2、COX1and COX2mRNA significantly increased between controlgroup and4%or8%or12%stretch group (P<0.05).2.2.2Western blot was applied to evaluate cPLA2, COX1and COX2proteinexpression under different stretch strengthsThe gray scale of cPLA2and COX1protein increased significant in8%and12%stretch group (P﹤0.01), but there was no difference between4%stretch and control groupin cPLA2and COX1protein (P＝0.135and P＝0.387). The gray scale of COX2increasedin different cyclic strengths (P﹤0.01).2.2.3ELISA was applied to evaluate the secretion of sPLA2secretionThere was no different expression of sPLA2between the4%stretch and the controlgroup (P＝0.260) in ELISA results. However, sPLA2released in8%and12%stretch groupmore than in control group (P﹤0.01).2.3SummaryThe level of PLA2、COX1and COX2increased between control group and4%or8%or12%stretch group under0.5Hz,4h condition. With different cyclic stretching groups, theexpression of phospholipase A2/cyclooxygenase/Prostaglandin E2pathway play animportant role in human tenocytes stretching in vitro.3. To evaluate the role of research the AACOCF3with tenocytes proliferationand, apoptosis and PGE2expressionBased on the results of the second part of PLA2/COX/PGE2molecular network, thispart researched influence of cPLA2-specific inhibitor AACOCF3on PGE2and thetenocytes proliferation and apoptosis.3.1Methods3.1.1Western blot was applied to evaluate different concentrations of AACOCF3inthe12%stretching group cPLA2protein expressionAdditon of0.1μM、1μM and10μM AACOCF3to12%stretching group was stretched4hours at0.5Hz. Tenocytes with non AACOCF3were applied to control group. The suitableconcentration of AACOCF3was decided by Western blot and the protein band of cPLA2 was observed.3.1.2CCK-8was applied to evaluate tenocytes proliferationExperimental methods: see the first part.3.1.3Flow cytometry was applied to evaluate tenocytes cycle and apoptosisExperimental methods: see the first part.3.1.4ELISA evaluated PGE2expressionExperimental methods: see the second part of the ELISA for sPLA2, method.3.2Results3.2.10.1μM AACOCF3can not inhibit cPLA2protein expression effectively;however1μM,10μM AACOCF3strongly inhibited cPLA2protein expression, especially inthe10μM concentration. So we chose10μM AACOCF3as the inhibition of cPLA2in nexttest.3.2.2Between group and control group, the OD values significantly increased.10μMpromoted cell proliferation.3.2.3Under12%stretching group, tenocytes cycle apoptotic peak significantlydecreased, compared with the control group (P <0.05). There was no significant differenceabout G1/S phase percentage (P=0.427).3.2.4Compared with control group, the experimental group of PGE2secretionincreased significantly (P <0.05).3.3Summary3.3.1The protein expression of cPLA2increased between the12%stretching groupwith10μM AACOCF3, especially with10μM AACOCF3.3.3.2The cell proliferation increased; however the cell apoptosis rate decreased, andthe tenocyte cycle did not change.3.3.310μM AACOCF3stimulated the tenocytes PGE2secretion in the12%stretchinggroup, related with cell proliferation and apoptosis.