Mechanism Of LOXL2 Regulating D-galactose-induced Skeletal Muscle Fibrosis Through TGF-BETA1/P38 MAPK Pathway

PART ONE THE EFFECTS OF AGING ON THE EXPRESSION LEVEL OF LOXL2 AND THE STRUCTURE AND FUNCTION OF SKELETAL MUSCLE IN MICEObjective Lysyl oxidase-like 2(LOXL2)is a copper-dependent monoamine oxidase which function is to catalyze the crosslinking of elastin and collagen in the extracellular matrix(ECM).Studies have confirmed that LOXL2 is involved in the occurrence and progression of epithelial-mesenchymal transition,ECM deposition,and fibrosis-related diseases.However,the role of LOXL2 in the development of skeletal muscle fibrosis remains unclear.This study investigated the effects of aging on the expression level of LOXL2,and the structure and function of skeletal muscle in mice,to provide theoretical support for further exploration of LOXL2 as a therapeutic target for sarcopenia.Methods The body weight and grip strength of C57BL/6J mice in young group(4 months old)and old group(20 months old)were measured;The body composition of mice in young group and old group was measured using dual energy X-ray absorptiometry(DEXA).Based on skeletal muscle strength and mass,mice with sarcopenia were selected.Three mice were randomly selected from each group,anesthetized according to body weight,and then perfused with universal tissue fixative.After fixation,the bilateral gastrocnemius muscles of the mice were dissected,dehydrated with sucrose solution of gradient concentrations,and then sectioned by paraffin-embedded and stained with H&E,Sirius red and LOXL2 immunohistochemical staining.The rest of mice were sacrificed after anesthesia,the bilateral gastrocnemius muscles were rapidly isolated on ice.About 50 mg of gastrocnemius muscle tissue was cut and added into RIPA lysate solution with 1% PMSF,fully lysed by a low-temperature ultrasonic homogenizer,and then centrifuged at high speed to obtain the tissue supernatant.After the protein concentration of the supernatant was determined,the protein expression levels of myogenin,Fbx O32,TGF-β1,α-SMA and LOXL2 were detected by Western blotting.Results 1.The result of body weight showed that compared with the young group,the body weight of mice in the old group decreased significantly.2.The result of grip strength showed that compared with the young group,the relative grip strength of mice in the old group was significantly decreased.3.The DEXA test results showed that compared with the young group,the lean mass and hindlimb lean mass of mice in the old group were significantly reduced.4.The result of H&E staining showed that compared with the young group,the cross-sectional area of muscle fibers in the old group was significantly reduced;the expression level of myogenin was significantly decreased;the expression level of Fbx O32 was significantly increased.5.The result of Sirius red staining of gastrocnemius muscle showed that compared with the young group,the collagen deposition area in the old group was significantly increased;the relative expression levels of TGF-β1 and α-SMA in the gastrocnemius muscle of the aged group mice were significantly increased.6.The result of LOXL2 immunohistochemical staining in gastrocnemius muscle showed that compared with the young group,the LOXL2-positive cells in the gastrocnemius muscle of the aged mice were significantly increased;the expression level of LOXL2 protein in the gastrocnemius muscle of the aged mice was significantly increased.Conclusion 1.Aging caused a decline in skeletal muscle function and mass in mice.2.Aging caused significant skeletal muscle fibrosis in mice.3.The expression level of LOXL2 in skeletal muscle was increased due to aging.PART TWO OVEREXPRESSING AND SILENCING OF LOXL2 MEDIATE FIBROBLAST CELLS REGULATING SKELETAL MUSCLE FIBROSISObjective To explore the effects of overexpressing and silencing of LOXL2 expression in skeletal muscle fibroblast cells on skeletal muscle fibrosis,which provided a theoretical basis for the subsequent exploration of the signaling pathway of LOXL2 involved in skeletal muscle fibrosis.Methods(1)Establishment of overexpressing LOXL2 fibroblast cell model: skeletal muscle fibroblast cells were cultured in complete medium containing 20% fetal bovine serum.When cells grew to 50-60% confluency,fibroblast cells were transfected with LOXL2 overexpression plasmid,cells were collected after 72 hours of incubation.(2)Establishment of D-galactose-induced fibroblast cell senescence model: fibroblast cells were cultured in complete medium containing 20% fetal bovine serum.Different concentrations(10,15,20,30,40 g/L)of D-galactose were added to the complete medium to induce senescence in fibroblast cells when the cells were grown to 50-60% confluency,after 72 hours of incubation,cells were collected.(3)Silencing LOXL2 in D-galactose induced fibroblast cells:fibroblast cells were cultured in complete medium containing 20% fetal bovine serum.When cells grew to 50-60% confluency,fibroblast cells were transfected with si RNA-LOXL2 to silence LOXL2 and D-galactose was added to the complete medium to induce senescence,cells were collected after 72 hours of incubation.The obtained cells were subjected to CCK-8cell proliferation assay;senescence-associated β-galactosidase(SA-β-gal)staining;Collagen-1 and TGF-β1 immunofluorescence staining;intracellular ROS fluorescence staining and flow cytometry;intracellular adenosine triphosphate(ATP)content detection;mitochondria Mito SOX staining;mitochondrial membrane potential JC-1 staining;senescence-related proteins,fibrosis-related proteins,LOXL2 m RNA and protein expression level detection.Results 1.Compared with the Ad-NC group,the protein expression level of LOXL2 in the Ad-LOXL2 group was significantly increased;the fluorescence intensity of Collagen-I and TGF-β1 was significantly increased;the expression levels of fibrosis-related proteins were significantly increased;the ratio of p-p38 to p38 was significantly increased.2.Compared with the control group,40 g/L D-galactose significantly inhibited the growth of fibroblast cells;the SA-β-gal positive cells in the D-galactose group increased significantly;the expression level of senescence-related proteins p53 and P16INK4 a increased significantly;LOXL2 expression level was significantly increased;the expression level of fibrosis-related proteins were significantly increased;intracellular ROS production was significantly increased;intracellular ATP content was significantly reduced;mitochondria ROS production was significantly increased;mitochondrial membrane potential was significantly reduced.3.Compared with cells in si RNA-NC group,the expression level of LOXL2 was significantly decreased;SA-β-gal positive cells were significantly decreased;senescence-related proteins were significantly decreased;the fluorescence intensity of Collagen-I and TGF-β1 was significantly decreased;fibrosis-related proteins were significantly decreased;the fluorescence intensity of ROS was significantly decreased;mitochondrial membrane potential was significantly increased;the ratio of p-p38 to p38 was significantly decreased.Conclusion 1.Overexpressing of LOXL2 promoted collagen deposition and p38 phosphorylation in fibroblast cells.2.D-galactose induced senescence,extracellular collagen deposition,increased expression level of LOXL2,increased ROS production and impaired mitochondrial function in skeletal muscle fibroblast cells.3.Silencing LOXL2 inhibited D-galactose induced senescence,extracellular collagen deposition,ROS generation,mitochondrial dysfunction and p38 phosphorylation in fibroblast cells.PART THREE LOXL2 REGULATES SKELETAL MUSCLE FIBROSIS BY MEDIATING FIBROBLAST CELLS THROUGH THE TGF-BETA1/P38 MAPK PATHWAYObjective To investigate the effects of p38 MAPK inhibitor on(1)fibroblast cells overexpressing LOXL2 and(2)D-galactose-induced fibroblast cells treated with selective LOXL2 inhibitor,in order to clarify the signaling pathway of LOXL2 regulating skeletal muscle fibrosis,providing an experimental basis for subsequent animal studies.Methods(1)Mouse skeletal muscle fibroblast cells were cultured in complete medium containing 20% fetal bovine serum.When cells were grown to 50% confluency,fibroblasts were transfected with LOXL2 overexpression plasmid,and a selective p38 MAPK inhibitor(SB 203580)was administered,cells were collected after 72 hours of incubation;(2)Mouse skeletal muscle fibroblast cells were cultured in complete medium containing 20% fetal bovine serum.When cells were grown to 50%confluency,30 g/L D-galactose was added to the complete medium and different concentrations(0.25,0.5,1 μM)of selective LOXL2 inhibitor were added,and the cells were collected after 72 hours of culture.(3)Mouse skeletal muscle fibroblast cells were cultured in complete medium containing 20% fetal bovine serum.When cells were grown to 50%confluency,30 g/L D-galactose,selective LOXL2 inhibitor and p38 MAPK inhibitor(SB 203580)were added to the complete medium,and the cells were collected after 72 hours of culture.The obtained cells were stained for SA-β-gal staining;immunofluorescence staining for Collagen-I and TGF-β1;intracellular ROS flow cytometry detection;intracellular ATP content;mitochondria Mito SOX staining;mitochondrial membrane potential staining;detection of senescence-related proteins,fibrosis-related proteins and LOXL2,p38,p-p38 protein expression levels.Results 1.Compared with the Ad-LOXL2 group,the expression level of phosphorylated p38 in the SB 203580 group was significantly decreased;the fluorescence intensity of Collagen-I was significantly decreased;the expression levels of Vimentin and α-SMA were significantly decreased;the production of ROS was decreased;mitochondrial membrane potential was increased;2.Compared with the D-galactose group,as the concentration of selective LOXL2 inhibitor increased,SA-β-gal positive cells gradually decreased;0.5 and 1 μM LOXL2 inhibitors significantly decreased the expression levels of senescence-related proteins;the fluorescence intensity of Collagen-1 gradually decreased;1 μM LOXL2 inhibitor significantly decreased the expression levels of fibrosis-related proteins;intracellular ROS production was significantly decreased;intracellular ATP content was significantly increased;mitochondrial ROS production was significantly decreased;mitochondrial membrane potential was significantly increased;3.Compared with the LOXL2 inhibitor group,SB 203580 further inhibited the phosphorylation level of p38,and further inhibited the expression levels of Collagen-III,Collagen-I and α-SMA.Conclusion 1.SB 203580 ameliorates collagen deposition and mitochondrial function impairments in fibroblast cells overexpressing LOXL2;2.LOXL2 inhibitor alleviates D-galactose induced fibroblast cells senescence,extracellular collagen deposition and mitochondrial function impairments;3.Combined application of SB 203580 and LOXL2 inhibitor further alleviated D-galactose-induced collagen deposition in fibroblast cells;4.LOXL2 participates in the regulation of skeletal muscle fibrosis by mediating collagen deposition in fibroblast cells through the TGF-β1/p38 MAPK pathway.PART FOUR SELECTIVE LOXL2 INHIBITOR AMELIORATED D-GALACTOSE INDUCED IMPAIRMENT OF SKELETAL MUSCLE STRUCTURE AND FUNCTION IN C57BL/6J MICEObjective To explore the effects of selective LOXL2 inhibitors on skeletal muscle structure and function in D-galactose induced subacute aging mouse,and to provide animal research basis for the treatment of sarcopenia with LOXL2 inhibitor.Methods Fifty 8-week-old male C57BL/6J mice were randomly divided into groups after one-week adaptive feeding.According to whether D-galactose(200 mg/kg)was injected or not,the mice were divided into control group(12 mice)and D-galactose group(38 mice).After continuous injection of D-galactose for 8 weeks,the mice in D-galactose group were randomly divided into D-galactose group(12 mice)and low-dose inhibitor group(5 mg/kg,13 mice)and high-dose inhibitor group(10 mg/kg,13mice)according to whether selective LOXL2 inhibitor was injected with D-galactose or not.LOXL2 inhibitors were injected for 8 weeks,during which the body weight and grip strength were measured every other week.Twenty-four hours after the last injection,the body composition of mice was measured by DEXA under anesthetized.Based on relative grip strength and skeletal muscle mass,mice with sarcopenia were selected. Subsequently,3 mice were randomly selected from each group,anesthetized according to body weight,and perfused with universal tissue fixative.After fixation,the bilateral gastrocnemius muscles of mice were dissected,dehydrated in a gradient of sucrose solution,and then sectioned by paraffin-embedded and stained with H&E,Sirius red,LOXL2 and Collagen-I immunohistochemical staining.The rest of the mice were sacrificed after anesthesia,and the blood was collected and centrifuged to obtain serum for the detection of oxidative stress biomarkers.The bilateral gastrocnemius muscles of the mice were rapidly separated on ice.About 50 mg of gastrocnemius muscle tissue was cut with tissue scissors and added to the RIPA lysis solution containing 1% PMSF,fully lysed by a low-temperature ultrasonic homogenizer,and then centrifuged at high speed to obtain the tissue supernatant.After the protein concentration of the supernatant was determined,the protein expression levels of GLB1,p53,P16INK4 a,Collagen-III,Collagen-I,LOXL2,α-SMA,TGF-β,p38 and p-p38 were detected by Western blotting.In addition,3 samples of gastrocnemius muscle with a size of 1 × 1 mm were randomly taken from each group,fixed with 2.5% glutaraldehyde,stained with osmium tetroxide and uranyl acetate,cut into a thickness of about 50 nm,and subjected to transmission electron microscopy.Results Compared with the control group,D-galactose injection for16 weeks caused C57BL/6J mice 1.Body weight decreased;2.The protein expression levels of senescence-related proteins GLB1,p53 and P16INK4 a in skeletal muscle increased;3.The expression level of LOXL2 protein in skeletal muscle increased;4.The deposition of collagen in skeletal muscle increased;5.The expression level of fibrosis-related proteins Collagen-III,Collagen-I,α-SMA and TGF-β1 in skeletal muscle increased,the ratio of p-p38 to p38 increased;6.The cross-sectional area of?muscle fibers decreased;7.The relative grip strength decreased;8.The hindlimb lean mass decreased;9.The oxidative stress biomarkers of skeletal muscle were abnormal;10.The number of damaged mitochondria in skeletal muscle increased.Compared with the D-galactose group,low-dose LOXL2 inhibitor injection for 8 weeks caused C57BL/6J mice 1.The expression levels of senescence-related proteins GLB1,p53 and P16INK4 a in C57BL/6J mice decreased;2.The expression levels of LOXL2 in skeletal muscle decreased;3.The deposition of collagen in skeletal muscle decreased;4.The cross-sectional area of skeletal muscle fiber increased;5.The relative grip strength increased.Compared with the D-galactose group,high-dose LOXL2 inhibitor injection for 8 weeks caused C57BL/6J mice 1.Body weight increased;2.The expression levels of senescence-related proteins GLB1,p53 and P16INK4 a decreased;3.The protein expression level of LOXL2 in skeletal muscle decreased;4.The deposition of collagen in skeletal muscle decreased;5.The expression level of fibrosis-related proteins Collagen-III,Collagen-I,α-SMA and TGF-β1 in skeletal muscle decreased,the ratio of p-p38 to p38 decreased;6.The cross-sectional area of skeletal muscle fibers increased;7.The relative grip strength increased;8.The hindlimb lean mass increased;9.The oxidative stress biomarkers of skeletal muscle improved;10.The number of damaged mitochondria in skeletal muscle decreased.Conclusion 1.D-galactose induced senescence,weight loss,skeletal muscle atrophy,skeletal muscle fibrosis and mitochondrial damage in mice.2.High-dose LOXL2 effectively delayed senescence in mice,reduced skeletal muscle atrophy and skeletal muscle fibrosis,and attenuated oxidative stress and mitochondrial damage.