Study Of The Therapeutic Effects And Underlying Mechanisms Of Epigallocatechin-3-gallate In The Treatment Of Rotator Cuff Tendinopathy

Part 1Levels and Clinical Significance of the Expression of Pain-and Inflammatory-related Genes in Subacromial Bursa in TendinopathyBackgroundShoulder pain is one of the most common complaints in orthopaedic clinics.There are many diseases can cause shoulder pain,such as trauma,rotator cuff syndrome,acromial impingement syndrome,rotator cuff tendinopathy,in which rotator cuff tendinopathy attracts wide attention in recent years.Tendinopathy is a chronic tendon disorder,usually caused by tendon overuse injury.The supraspinatus,biceps brachii,forearm extensor and flexor,patella and Achilles tendons are the most commonly involved tendons when tendinopathy occurs.According to epidemiology studies,30-50%of the injuries in sports are related to tendinopathy.While causing painful symptoms,rotator cuff tendinopathy can also lead to loss of productivity and increasement of medical expectation.However,there are only limited methods in the treatment of rotator cuff tendinopathy,thus it is necessary to explore novel therapeutic approaches for the disease.The exact mechanisms involved in the development of rotator cuff tendinopathy pain are not fully understood yet.Some researches demonstrate that in normal tendon tissue,the nerve fiber and neuro-mediators are mainly in para-and peri-tendon.During the tendon repair after injury,the nerve fibers will invasively grow into tendon and produce a large number of neuro-mediators,leading to tendon tissue degradation and pain.These researches suggest tendon itself could be the source of pain in rotator cuff tendinopathy.In clinical practice,however,the pain symptom sometimes doesn’t match the severity of tears in rotator cuff tendinopathy patients with tears,suggesting tendon is not the only source of pain in rotator cuff tendinopathy.We still need to explore the other potential tissue sources.The subacromial bursa is a synovial cavity anatomically adjacent to the rotator cuff.Recent studies revealed that there were significant histological and radiological changes in the subacromial bursa of patients with rotator cuff tears.This suggests the subacromial bursa might be related to the development of pain in rotator cuff diseases,while the related study of this topic is still blank.Therefore,we addressed our question on what kind role does the subacromial bursa play in rotator cuff tendinopathy and its clinical significance.ObjectiveTo investigate the histological features and the levels of expression of pain-and inflammatory-related genes of subacromial bursa in rotator cuff tendinopathy patients,giving direction and theoretical support to the studies on exploring therapeutic methods for rotator cuff tendinopathy in part 2.Methods1.Twelve pairs of diseased rotator cuff tendon and subacromial bursa were collected from patients with pain symptoms during rotator cuff repair.Twelve control bursa samples were collected from asymptomatic patients during shoulder stabilization procedures.The samples were used for histological study,gene expression study,primary cells isolation and continues in vitro studies.2.H&E staining and immunohistochemistry staining were used to analyze the histological features and immune cells infiltration in the bursa tissue in painful patients.3.Quantitative real-time polymerase chain reaction,western blot and immunohistochemistry staining were used to investigate the levels of expression of pain-and inflammatory-related genes of subacromial bursa in rotator cuff tendinopathy patients.Results1.There was significant alteration in subacromial bursa in rotator cuff tendinopathy patients.H&E staining showed obvious neoangiogenesis and fibroblasts proliferation in tissue,while small blood vessels invaded the superficial layer of the tissue with high endothelial cells on the wall.IHC staining showed that there were many monocytes/macrophages,mast cell infiltration,while B lymphocytes,T lymphocytes or plasma cells were barely observed.2.Elevated levels of pain-and proinflammatory-related genes in subacromial bursa in rotator cuff tendinopathy patients compared to the control group.The expression of TAC1,MCP1,TNF-α,IL-1β,IL-6,MWP1,MMP13,S100A4,S100A10 and SI00A11 were higher in subacromial bursa from patients with pain symptom(p<0.05).3.Compared to the tendon tissue of rotator cuff,subacromial bursa expressed more TAC1,MCP1,PANX1,P2X7,TNF-α,IL-6,MMPl,S100A4,S100A10,S100A11(p<0.05).IHC staining and western blot confirms the expression of substance P(encoded by TAC1 gene),IL-1β and IL-6 in protein level.Conclusions1.In painful rotator cuff tendinopathy patients,the subacromial bursa was obviously affected and was in sever inflammatory status.2.The expression of pain-related and proinflammatory mediators in diseased bursa were significantly higher compared to rotator cuff tendon or normal tendon tissue,suggesting subacromial bursa might serve as an important source of pain and inflammation in rotator cuff tendinopathy.Part 2The Therapeutic Effects and Underlying Mechanisms of Epigallocatechin-3-gallate in The Treatment of Rotator Cuff Tendinopathy:An in vitro StudyBackgroundTendon is a dense connective tissue conducting contractive force between skeletal muscle and bone.Tenocytes,the mature fibroblasts in tendon,are the predominated cell type in the tissue,accounting for 90-95%of the total number of the cells.Extracellular matrix(ECM)is significant for remaining the normal structure and function of tendon.The anabolic activity of ECM is mainly the process of synthesizing structure proteins,including type I,II and III collagen and various kinds of proteoglycans.The catabolic activity mainly depends on several proteolytic enzymes and inhibitors,such as MMPs,ADAM,ADAMTS,TIMPs.In normal tendons,anabolic and catabolic activities are in dynamic balance,called homeostasis,and the tendon will be diseased when homeostasis is broken.It has been reported that the expression of several proinflammatory cytokines such as IL-1β,IL-6 and TNF-αare up-regulated in tendon in tendinopathy.These cytokines can be synthesized both by the immune cells and tenocytes in the tendon,regulating the expression of downstream genes and finally leading to tendinopathy.Besides,the expression of pain-related genes can also be up-regulated by inflammatory reaction,which might be responsible for the chronic tendinopathic pain.Therefore,suppressing the inflammatory reaction at the early stage of tendinopathy is significant for its therapy.The methods in treatment of tendinopathy are limited.For now,systemic or local administration non-steroid anti-inflammatory drugs(NSAIDS)is the most common treatment in clinical practice.However,because of the sever side effects in digestive and cardiovascular system,long-term usage of NSAIDS in tendinopathy patients is not suggested.Epigallocatechin-3-gallate(EGCG),a polyphenol extracted from green tea,has been reported to have an anti-oxidative,anti-inflammatory and neuroprotective effect in many studies,exhibiting satisfied therapeutic effect and safety in the treatment of many diseases.But there is no study on the application of EGCG in treatment of tendinopathy.In the first part,we revealed that the subacromial bursa was in sever inflammatory status with an up-regulated expression of several pain-and inflammatory-related genes,suggesting the subacromial bursa might serve as an important source of pain and inflammation in rotator cuff tendinopathy.In this part,we will explore the therapeutic effects of EGCG on tendinopathy by targeting on human primary bursa cells and tenocytes.Objective1.To investigate the effect of EGCG on inflamed human primary bursa cells.2.To investigate if EGCG can also exhibit therapeutic effect on human primary tenocytes while targeting on bursa cells and explore the underlying mechanisms.Methods1.Immunofluorescence staining was used to investigate the expression and spatial distribution of substance P,IL-1β,IL-6 in tissue,especially in bursa cells which was highlighted by scleraxis,in subacromial bursa specimen from rotator cuff tendinopathy patients.2.Isolating the primary bursa cells and tenocytes from clinical specimens,providing materials for the in vitro study of EGCG.3.Human primary bursa cells were treated by 10ng/ml rhIL-1 P+25μM EGCG for 24h,and qRT-PCR was used to measure the expression of pain-and inflammatory-related genes in EGCG group as well as vehicle control(lOng/ml rhIL-1β+PBS)and naive control group.4.Conditioned medium from EGCG-treated(lOng/ml rhIL-1β+25μM EGCG,24h)and vehicle control(10ng/ml rhIL-1β+PBS,24h)bursa cells was collected respectively,marked as EGCG CM and Ctrl CM.Then rhIL-1β-pretreated(10ng/ml rhIL-1β,24h)human primary tenocytes were cultured with EGCG CM or Ctrl CM for 24h.qRT-PCR was used to measure the expression of pain-and inflammatory-related genes in tenocytes.Results1.In subacromial bursa from rotator cuff tendinopathy patients,the expression of substance P,IL-1β and IL-6 was mainly focusing in the areas around blood vessels and the superficial layer of tissue,while some of the cells with long spindle nucleus were identified as bursa cells by the expression of scleraxis.2.Compared to the vehicle control,EGCG significantly suppressed the levels of expression of TAC1,MCP1,PAN1,P2X7,TNF-α,IL-1β,IL-6,MMP1,MMP13,VEGF and NGF(p<0.05),while the expression of PANX1,TNF-α,IL-1β,IL-6,MMP1,MMP13 is still higher than the naive control((p<0.05).3.Conditioned medium from EGCG-treated bursa cells significantly suppressed the expression of pain-and inflammatory-related genes in rh IL-1β-pretreated human primary tenocytes,especially MMP1,MMP13,VEGF and NGF.Conclusions1.Bursa cells are significant cell source of pain and inflammatory mediators in subacromial bursa in rotator cuff tendinopathy.2.EGCG suppresses the inflammatory response in primary bursa cells.3.EGCG can also suppressed the inflammatory response in tenocytes via a paracrine mechanism when curing tendinopathy by targeting on bursa cells.