Mechanisms Of HMGB1 In Inflammation Of Tendinopathy Induced By Mechanical Overloading

Objective Tendinopathy is a very common disease which is often induced by repeated mechanical overloading.Tendinopathy affects patients' quality of life seriously.Unfortunately,there is still no specific effective treatment or definite mechanism for tendinopathy.With the development of modern molecules and immunology,inflammation is paid more and more attention during the etiology and pathogenesis of tendinopathy.The theory of damage-associated molecular pattern(DAMP)is adopted to explain various of aseptic inflammation in which alarmins play the key role.Extracellular high mobility group box-1(HMGB1)is one of the most common alarmins which extensively exists in kinds of tissues.To understand pathogenesis of tendinopathy and to detect new treatment targets,researches were carried out on the inflammation mechanism of tendinopathy induced by HMGB1.Methods Part one:Tendon cells and tendon stem cells were primarily cultured through short-time enzyme digestion plus tissue block method.High-low density planting and spot-digestion methods were used to purify tendon cells and tendon stem cells.Cell counting kit-8(CCK-8)was adopted to measure proliferation while cell viability was confirmed by trypan blue.The two types of cells were determined and discriminated by immunocytochemistry,flow cytometry and qRT-PCR.Tendon cells from different origins were also compared.Part two:Tendon cells were experimented with cell stretching system Flexcell-FX5000 in vitro.HMGB1 was confirmed in tendon cells with immunocytochemistry and Western blot.Cell proliferation and viability of tendon cells were observed after stretched with four different elongation%(0%,4%,8%,12%).Intracellular and extracellular HMGB1 concentrations were determined by Western blot and enzyme-linked immunosorbent assay(ELISA)individually.HMGB1 concentrations change in culture medium were also measured with ELISA at 5 different time points(0h,6h,12h,24h)after stretching.HMGB1 concentration changes were also observed after tendon cells were stimulated with lipopolysaccharide(LPS)as pathogen associated molecule pattern(PAMP),which were compared with DAMP.Part three:The effects of endogenous or exogenous HMGB1 on inflammation of Achilles tendinopathy were studied in vivo with rat animal models.Overloading treadmill rat model was set up.Intra-nucleus and extra-nucleus HMGB1 concentrations were measured at different time points(0h,6h,12h,48h)after overloading.One-dose or continuous exogenous HMGB1 was injected around bilateral Achilles tendons of rats with or without intraperitoneal injection of glycyrrhizic acid(HMGB 1 inhibitor).Gene expression change of IL-1 ??IL-6?TGF-? and Tenascin?Col??Col ? were measured by qRT-PCR.Part four:Cellular and molecular mechanisms for the role of HMGB 1 in inflammation of tendinopathy were studied.The effect of HMGB1 with different concentration on proliferation of tendon cells was determined by CCK-8.The proteins of receptors(TLR4,RAGE)and signal pathway(MAPK,NF-?B)were measured with Western blot after tendon cells stimulated with different concentration of exogenous HMGB 1.Results Part one:Tendon cells primarily cultured in this study appeared flat and spindle shape,while tendon stem cells showed cobblestone appearance.Population doubling times of the two kinds of cells were 2.71d and 2.18d individually.Tendon cells from 1-month old rat proliferated more rapidly than cells from 18-months old rat.Trypan blue staining results showed over 90%of all the cells were viable.Cell clones can be seen by crystal violet staining.Clones of tendon stem cells were bigger and more compact than clones of tendon cells.Collagen I was strongly positive in tendon cells.Cell surface markers of tendon stem cells tested by flow cytometry showed CD44(+)/CD90(+),CD45(-)/CD106(-).Results of qRT-PCR showed high expression of Scleraxis gene in tendon stem cells and Tenomodulin gene in tendon cells.Part two:Proliferation of tendon cells grew faster after cells were stretched in vitro.Cells numbers of 4%,8%and 12%group were 1.3,1.5 and 1.1 times comparing with 0%control group.But cell viability decreased when stretch percent was larger than 4%.Results of immumohistochemical staining showed that HMGB1 existed in tendon cells,especially in cell nucleus.The amount of HMGB 1 in tendon cell nucleus was decreased after stretching.Western blot and ELISA also showed that intracellular HMGB1 contents decreased and extracellular HMGB1 contents increased as the stretching percent increased.HMGB1 contents in culture mediums reached peak value at 6h after 8%stretching.While the time of peak concentration was 24h when tendon cells were stimulated by LPS.Part three:Achilles tendon of continued mechanical overloading group presented typical tendinopathy after treadmill training for 8w.The pathological sections of Achilles showed increased tendon cell number,disordered fibers and proliferated vascular.Bonar score was higher than control group significantly.Extracellular HMGB1 contents of transient mechanical overloading group gradually increased with peak value appearing at 12h after training.The effects of HMGB1 were further studied by local injection of exogenous HMGB1.One-dose group just showed a little vascular proliferation.Continuous injection group showed tendinopathy-like appearance with the genes of IL-1 ?,IL-6,TGF-?,Tenascin,Col I and Col III higher expression than control group.But the inflammatory response was partly inhibited by glycyrrhizic acid in HMGB1+GA group.Part four:Tendon cells of low-concentration HMGB1 group proliferation slightly increased,while high concentration of HMGB1 inhibited proliferation significantly.Both TLR4 and RAGE were found on tendon cells with Western blot.Expression of TLR4 increased with higher concentration of HMGB1.While high-concentration HMGB1 decreased expression of RAGE.IKK ? and NF-?B P65 in NF-?B pathway was promoted by HMGB1,especially with low and medium concentration.There was little change with JNK and P38 in MAPK pathway.ERK1/2 pathway of MAPK was elevated in low and medium concentration groups.Conclusions Part one:Tendon cells and tendon stem cells can be primarily cultured through short-time enzyme digestion plus tissue block method.High-low density planting and spot-digestion methods can purify tendon cells and tendon stem cells efficiently.Tendon cells and tendon stem cells had different appearance,proliferation rate and cell surface markers.Tendon cells from different origins varied in function.Part two:Long-time mechanical overloading did harm to the proliferation and viability of tendon cells.HMGB1 did exist in tendon cells with especial high expression in nucleus.Intracellular HMGB1 was released out of tendon cells after being stretched with mechanical overloading(elongation%>4%).LPS can also promote the releasement of HMGB1,but the time of peak value was later than the mechanical overloading group.Part three:Transient mechanical overloading resulted in tendon inflammation,while long-time mechanical overloading led to tendinopathy.Long-time high concentration of extracellular HMGB1 can induce tendinopathic appearance.Gene expression of IL-1??IL-6?TGF-??Tenascin and Col III in Achilles tendons increased significantly after continuously injected with exogenous HMGB1,which were partly inhibited by glycyrrhizic acid.Part four:High-dose HMGB1 might have cytotoxicity which inhibited proliferation of tendon cells.Both TLR4 and RAGE were found on the surface of tendon cells that were promoted expression by low-dose HMGB1.While high-dose HMGB1 decreased RAGE.NF-?B pathway was activated by HMGB1.HMGB1/TLR4/NF-?B may be the main signal pathway which account for the inflammation of tendinopathy?...