Study On The Intervention Effect And Mechanism Of N3 Polyunsaturated Fatty Acids And Wheat Oligopeptides On Sarcopenia Based On Proteomics

Sarcopenia is a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability,poor quality of life and death.Studies in Europe and the United States indicated that skeletal muscle mass decreases with age after 50 years old,and decreases by 6.6-23.3% at the age of 80.However,the data of Chinese population are relatively few,and the trend data of skeletal muscle attenuation of large Chinese population are rare.In recent years,the improvement effect of n-3 polyunsaturated fatty acid(PUFA)on sarcopenia in the elderly has received much concern,but the results remain controversial.More high-quality intervention studies are needed to evaluate the improvement effect and explore the possible mechanism.Protein supplementation has always been the focus of the study of muscle decay population,but the intervention effect showed inconsistent results.As a small molecule active peptide,wheat oligopeptide has rich biological activity and gastrointestinal protection,which can improve the morphology of gastrointestinal tract and promote protein absorption.At the same time,it is also a good source of protein supplement,which has important research value.However,the intervention of wheat oligopeptides on sarcopenia has not been reported.Therefore,it is necessary to further study the effect of wheat oligopeptides on sarcopenia and explore its possible mechanism.This study collected the data of physical examination survey population of our research group from September 2017 to February 2021.A total of 2069 volunteers were included to analyze and explore the change trend of skeletal muscle,fat,water and other body components with age in different gender groups in China.The elderly was supplemented with fish oil for six months to explore the effect of fish oil on muscle and body fat of the elderly.The effects of fish oil,wheat oligopeptide and their combined intervention on muscle atrophy and fat infiltration were studied,and the possible mechanism was explored by proteomics.The research content is divided into three parts.Part 1 Distribution characteristics of skeletal muscle mass and fat mass of adults in Jiangsu ProvinceObjective: To explore the changes of skeletal muscle mass and fat mass with age in adults with different genders in Jiangsu Province,and the relationship between BMI and central obesity and skeletal muscle mass and fat mass in different age groups.Methods: The data of 2115 volunteers were collected from September2017 to February 2021.The data of height,weight,waist circumference,hip circumference and body composition were collected.Results: the basic information of 2115 volunteers were collected,46 of them were excluded,and 2069 volunteers were included for analysis.(1)FFM,TSMM,ASM,segmental muscle mass and its index of adult male were significantly higher than those of female.The body fat mass,body fat index,body fat percentage and visceral fat area of women were significantly higher than those of men.(2)FFM,TSMM,ASM and segmental muscle mass of adults over 30-39 years old showed a downward trend with the increase of age,and TSSM showed a significant decline at 60 years old.ASM and SMM(lower extremity)of male began to decline significantly at the age of 50,while that of female began to decline significantly at the age of 60.(3)TSMMI,ASMI and SMMI(lower limbs)of adults over 30 years old showed a trend of first rising and then declining,reaching a peak at 40-49 years old.TSMMI began to decline significantly at 60 years old,and ASMI and SMMI(lower limbs)began to decline significantly at 50 years old.(4)The body fat mass,body fat rate,waist hip ratio and body fat index of adults over 30 years old showed an upward trend with the increase of age.The body fat mass,body fat rate and percentage of fat free body weight increased significantly at the age of 40,while the fat free body weight and fat free body mass index decreased significantly at the age of 60.The body fat index of men increased significantly at the age of 40,while that of women increased significantly at the age of 60.(5)FFM,TSMM and TSMMI increased with the increase of BMI and waist circumference,but TSMM/wt and TSMM/BMI decreased with the increase of BMI.However,ASM/wt and ASM/BMI decreased with the increase of BMI and waist circumference.Conclusion: The skeletal muscle mass of adults over 30-39 years old showed a downward trend with age,and male’s skeletal muscle mass was higher than female’s.Fat mass showed an upward trend,and female’s fat mass was higher than male’s.The absolute value of skeletal muscle mass showed an upward trend with the increase of BMI level,but ASM/wt and ASM/BMI showed a downward trend with the increase of BMI level.ASM and ASMI had no relationship with waist circumference level.Part 2 Effect of fish oil intervention on skeletal muscle quality and blood lipid in the elderlyObjective: To investigate the effects of fish oil intervention on skeletal muscle and fat mass and blood lipid in the elderly.Methods: from June 2018 to August 2018,200 elderly volunteers aged 60 years or above were recruited and randomly divided into intervention group(fish oil)and control group(corn oil),with 100 people in each group.During the study period,double blind method was adopted.The fish oil group was supplemented with fish oil capsules(twice a day,two capsules each time,containing 650 mg n-3 PUFA)daily,and the control group was given corn oil capsules as placebo(twice a day,two capsules each time).The intervention lasted for 6 months.Physical examination,body composition test and blood biochemical test were performed at baseline,3 months and the end of the intervention.Physical examination indexes included height,weight,waist circumference,hip circumference and thigh circumference;Body composition tests include skeletal muscle,fat,body moisture,etc.Blood biochemical indexes included total protein,albumin,total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),urea nitrogen,creatinine,uric acid,etc.Results: A total of 187 people completed the 6-month intervention,including 97 in fish oil group and 90 in control group.Baseline data showed that There was no significant difference in age,gender,weight,BMI,waist circumference,hip circumference,WHR and body fat rate between the two groups(P>0.05).Compared with baseline,TSMM,ASM,TSMMI,ASMI,thigh circumference,grip strength and HDL-C in fish oil group increased significantly(P<0.05),and the TUG,body fat rate and TG level decreased significantly(P<0.05);Compared with control group,TSMM,ASM,TSMMI,ASMI,thigh circumference,grip strength and HDL-C in fish oil group increased significantly(P<0.05),and the TUG and TG level decreased significantly(P<0.05);Conclusion:fish oil intervention can significantly improve muscle attenuation and blood lipid level,significantly increase skeletal muscle mass,thigh circumference and grip strength,improve muscle function,significantly reduce serum TG level and increase HDL-C level.Part 3 Effects of fish oil and wheat oligopeptides and their combined intervention on the aged rats with sarcopenia and its mechanismObjective: To investigate the effect of fish oil and wheat oligopeptides and their combined intervention on skeletal muscle of aged rats with sarcopenia and its mechanism.Methods: one hundred 25-month-old SD rats were randomly divided into model control group,fish oil(FO)low-dose group,FO medium dose group,FO high-dose group,wheat oligopeptide(WP)low-dose group,WP medium dose group,WP high-dose group,WFM 1 group,WFM 2 group and WFM 3 group,with 10 rats in each group;Ten 6-month-old SD rats were used as young control group.The low,medium and high dose fish oil groups were given fish oil by gavage at the doses of 200mg/kg BW,400mg/kg BW and 800mg/kg BW respectively,the low,medium and high dose wheat oligopeptide groups were given WP by gavage at the doses of 100mg/kg BW,200mg/kg BW and400mg/kg BW respectively,and the WFM 1,WFM 2 and WFM 3 groups were given(800mg/kg FO+100mg/kg WP),(400mg/kg FO+200mg/kg WP)and(200mg/kg FO+400mg/kg WP).The rats were given intragastric administration for 10 weeks.At the end of the experiment,the grasping force of the rats was measured by the grasping force meter,and the muscle and fat infiltration of the rats were scanned and analyzed by the nuclear magnetic resonance imaging instrument.The rats fasted for 12 hours,blood sample was collected from the femoral artery after anesthesia,the skeletal muscle and white fat tissue were separated,and the serum TC,TG,HDL-C and LDL-C were measured.The quadriceps femoris and white adipose tissue were stained with he for pathological analysis.The Fbox-1,GDF-8,Murf-1,Myo D,Myo G,GH,IGF-1,IL-6,TGF-β,TNF-α,ROS,SOD and MDA of quadriceps femoris were detected by using kit.The differentially expressed proteins in FO medium dose group,WP medium dose group,WFM 2 group and model group were detected by proteomics,and some differentially expressed proteins between groups were detected by Western blot.Results: Compared with young rats,the grasping power and skeletal muscle mass of 27-month-old rats with sarcopenia decreased significantly;NMR results showed that the skeletal muscle of elderly rats with sarcopenia showed obvious atrophy and fat infiltration,and the maximum cross-section of quadriceps femoris decreased significantly.At the same time,the cross-sectional area of skeletal muscle fibers and the number of white adipocytes also decreased significantly.After the intervention of medium and high doses of FO,low,medium and high doses of WP and intervention of WFM 1,WFM 2 and WFM 3,the maximum cross-section of quadriceps femoris,the cross-sectional area of muscle fiber and the number of white adipocytes increased significantly,and the number,size and area of fat droplets in muscle tissue decreased significantly(P<0.05),skeletal muscle atrophy and fat infiltration in aged rats were significantly improved.Compared with young rats,the content of serum HDL-C in aged rats with sarcopenia decreased significantly,and the contents of LDL-C,TC and TG increased significantly(P<0.05).Compared with the model control group,the intervention of low,medium and high doses of FO could significantly increase the content of serum HDL-C and reduce the contents of LDL-C,TC and TG(P<0.05).Compared with the model control group,the intervention of medium and high doses of WP could significantly increase the level of serum HDL-C and reduce the level of TG(P<0.05).The combined intervention could significantly increase the content of serum HDL-C and reduce the contents of LDL-C,TC and TG(P<0.05).Compared with young rats,the levels of ROS,MDA,Fbox-1,GDF-8,Murf-1,IL-6,TGF-β,and TNF-α in skeletal muscle tissue of senile sarcopenia rats increased significantly,the levels of SOD,Myo D,Myo G,GH and IGF-1 decreased significantly(P<0.05).The intervention of low,medium and high doses of FO could significantly reduce the levels of ROS,MDA,GDF-8 and IL-6,and significantly increase the levels of SOD,Myo D,Myo G,GH and IGF-1(P<0.05).The intervention of medium and high doses of FO could also significantly reduce the levels of Fbox-1,Murf-1,TGF-β,and TNF-α.Compared with the model control group,the intervention of low,medium and high doses of WP could significantly reduce the levels of ROS,MDA,Fbox-1,GDF-8,Murf-1,IL-6,TGF-β,and TNF-α,and significantly increase SOD and Myo G levels(P<0.05).The intervention of medium and high doses of WP could also significantly increase the level of Myo D(P<0.05).The intervention of WFM 1,WFM 2 and WFM 3 could significantly reduce ROS,MDA,Fbox-1,GDF-8,Murf-1,IL-6,TGF-β,and TNF-α levels in skeletal muscle,and significantly increased SOD,Myo D,Myo G,GH and IGF-1 levels(P<0.05).The mass of skeletal muscle,maximum cross-sectional area of quadriceps femoris,cross-sectional area of muscle fiber,number of adipocytes,expression level of Pfn1 and phosphorylation level of Akt in WFM 2intervention group were significantly higher than those in FO medium dose group(P<0.05),while lipid droplet area,expression level of Fabp1 protein,ROS,MDA,Fbox1,GDF-8,IL-6 and TNF-α were lower than those in FO medium dose group.The weight of tibialis anterior muscle,gastrocnemius muscle,muscle fiber cross-sectional area,levels of Myo D,Myo G,GH,IGF-1,and expression levels of Pycr3,Pfn1 and Akt in WFM 2 group were significantly higher than those in WP medium dose group(P<0.05),while the levels of MDA,LDL-C,GDF-8 and Fabp1 were significantly lower than those in WP medium dose group(P<0.05).The proteomic results of FO medium dose group and sarcopenia model showed that 81 differentially expressed proteins were screened,54 were up-regulated and 27 were down-regulated.The results of GO functional annotation showed that differential proteins were mainly involved in muscle contraction regulation,actin fiber regulation,skeletal muscle development,oxidative stress response,endoplasmic reticulum mediated protein degradation pathway,cellular lipid catabolism,fatty acid metabolism and transport,cholesterol homeostasis,lipid homeostasis and deposition regulation,cholesterol storage regulation.KEGG results showed that 81 differentially expressed proteins were involved in 114 signal pathways,mainly including PI3K-Akt,c AMP,PPAR,MAPK,JAK-STAT signaling pathway,actin cytoskeleton regulation and endoplasmic reticulum protein Quality processing,regulation of lipolysis in adipocytes,amino acid biosynthesis,ubiquitin mediated proteolysis,glyceride metabolism,cholesterol metabolism,peroxidase.Ten differentially expressed proteins were selected to carry out WB verification.The WB results showed that compared with young rats,the expression levels of Itgav,Fbln5,Qsox1,Tnnt1,Igfbp1 and Myosin in quadriceps femoris of senile sarcopenia rats were significantly decreased,and the expression levels of Lyric,Erlin1,Gpd2 and Fabp1 were significantly increased(P<0.05)Compared with the model control group,fish oil intervention significantly increased the expression levels of Itgav,Fbln5,Qsox1,Tnnt1,Igfbp1 and Myosin,and significantly decreased the expression levels of Lyric,Erlin1,Gpd2 and Fabp1(P<0.05).The WB results were completely consistent with proteomics.The proteomic results of WP medium dose group and sarcopenia model group showed that 48 differential proteins were screened,32 were up-regulated and 16 were down-regulated.The results of GO functional annotation showed that the differential proteins were mainly involved in amino acid biosynthesis,intracellular protein transport and biosynthesis,actin filament and stress fiber assembly,fatty acid metabolic process,negative regulation of lipid biosynthesis and protein hydrolysis metabolism,positive regulation of lipid metabolism and protein biosynthesis,endoplasmic reticulum mediated protein degradation pathway,oxidoreductase activity,and inhibition of IL-6,IL-1β and IFN-γ production.KEGG results showed that 48 differentially expressed proteins were involved in 142 signaling pathways,mainly including protein processing of endoplasmic reticulum,regulation of lipolysis of adipocytes,amino acid biosynthesis,ubiquitin mediated proteolysis,actin cytoskeleton regulation,PI3K-Akt,AMPK,MAPK,JAK-STAT,c AMP,TNF-α,m TOR signal pathway,oxidative phosphorylation,glyceride and cholesterol metabolism,protein digestion and absorption,etc.Ten differentially expressed proteins were selected for WB verification.The WB results showed that compared with young rats,the expression levels of Rbm3,Akt,p-Akt,Qsox1,Tnnt1,Myosin,Pycr3 and Pfn1 of sarcopenia rats were significantly down-regulated,and the expression levels of Lyric and Erlin1 were significantly up-regulated.Compared with the model control group,WP intervention significantly increased the expression levels of Rbm3,Akt,p-Akt,Qsox1,Tnnt1,Myosin,Pycr3 and Pfn1,and significantly decreased the expression levels of Lyric and Erlin1(P < 0.05).The WB results were completely consistent with proteomics.The proteomic results of WFM 2 intervention group and senile sarcopenia model showed that 52 differentially expressed proteins were screened between the groups,34 were up-regulated and 18 were downregulated.The results of GO functional annotation showed that the differential proteins were mainly involved in the regulation of lipid transport and lipoprotein particle level,fatty acid metabolism,muscle tissue regeneration,protein targeted membrane regulation,protein polymerization regulation,protein biosynthesis,inhibition of protein hydrolysis,regulation of cholesterol transport,fatty acid and glyceride metabolism,negative regulation of lipid localization and deposition,amp catabolism,peptide transport,intracellular protein transmembrane transport,extracellular tissue structure,etc.KEGG results showed that 52 differentially expressed proteins were involved in 145 signal pathways,mainly including the regulation of lipolysis of adipocytes,actin cytoskeleton regulation,AMPK,PI3K-Akt,c AMP,JAK-STAT,TNF-α,MAPK,m TOR signal pathway,ubiquitin mediated proteolysis,and growth hormone synthesis formation and secretion.Eight differentially expressed proteins were selected to carried out WB verification.WB results showed that compared with young rats,the expression levels of Itgav,Pycr3,Qsox1,Pfn1,Igfbp1,Akt and p-Akt protein of sarcopenia rats were significantly down-regulated,and the expression level of Fabp1 protein was significantly up-regulated(P<0.05).Compared with the model control group,the WFM 2 intervention group significantly increased the protein expression levels of Itgav,Pycr3,Qsox1,Pfn1,Igfbp1,Akt and pAkt,and significantly decreased the protein expression level of fabp1(P<0.05).The WB results were consistent with proteomics.Conclusion: fish oil and wheat oligopeptide and their combined intervention can significantly improve skeletal muscle atrophy,inhibit protein decomposition,promote protein synthesis and muscle regeneration,and improve oxidative stress and inflammation of skeletal muscle in age-related senile rats.Compared with the intervention group of fish oil or wheat oligopeptide alone,WFM 2(400mg / kg fish oil + 200 mg / kg wheat oligopeptide)showed a better intervention effect.This study made a valuable exploration for the intervention and treatment of sarcopenia in the elderly,and provided important theoretical support for the intervention research of sarcopenia in the elderly in China in the future.