| Objective: Inflamm-aging refers to a chronic inflammatory state in which pro-inflammatory cytokine levels increase during aging.It gradually increases with the increase of aging.It was considered to be an important factor that determines the progress of aging in the body and seriously related to many diseases,is a hotspot in medical research today.At present,the mechanism of inflamm-aging has not been fully elucidated.Oxidative-stress-inflammation-aging theory is one of the main mechanisms related to the occurrence mechanism of inflamm-aging.Fructus Schisandra chinensis is a traditional Chinese medicine.Its polysaccharide composition(Fructus Schisandrae Chinensis Polysacchard,FSCP)has various pharmacological effects such as liver protection,antioxidant,anti-inflammatory and anti-aging.However,there are few studies on the mechanism of anti-nflamm-aging effect of FSCP.This study aims to optimize the extraction process of FSCP,construction of cell inflamm-aging models and mice aging models,detect inflammatory cytokines,reactive oxygen species,mitochondrial membrane potential,antioxidant enzymes,aging genes and aging-related phenotypes in vivo and in vitro,explore the effect of FSCP on inflamm-aging from the cellular and overall levels.Through the detection of p65,I-κBα protein,explore whether FSCP can participate in the regulation of NF-κB signaling pathway,and clarify the molecular mechanism of FSCP against inflamm-aging,in order to provide a theory for the study of FSCP anti-inflamm-aging in accordance with.Methods:(1)Polysaccharide components of FSCP by hot water extraction.Purification of FSCP by repeated freeze-thaw,sevag and dialysis.Based on single-factor experiments,the four-factor three-level orthogonal analysis method was used to optimize the extraction process of FSCP.The effects of different univariate variables and interactions on extraction temperature,extraction time,materialliquidratio and number of times on the yield of polysaccharides from Fructus Schisandra chinensis were studied.(2)Establish an in vitro antioxidant model 1,1-diphenyl-2-picrylhydrazyl(DPPH)free radical scavenging experiment,hydroxyl free radical scavenging experiment and FRAP total antioxidant capacity test to determine the anti-oxidant activity of FSCP.(3)Lipopolysaccharide(LPS)induces mouse macrophage RAW264.7 to construct a cell inflamm-aging models,using MTT methods,aging-related β-galactosidase(SA-β-gal)staining,DAPI staining,ELISA,Reactive oxygen species(ROS)detection,mitochondrial membrane potential detection to explore the inhibition of FSCP on LPS-induced inflamm-aging of RAW264.7 cells.(4)D-galactose(D-gal)induced mice to build aging animal models,using HE staining,serum superoxide dismutase(SOD)and glutathione peroxidase(GSH-px)activity determination,malondialdehyde(MDA)content detection,ELISA and DAPI staining to study the protective effect of FSCP on D-gal-induced inflamm-aging mice and the inhibition of inflamm-aging in D-gal-induced aging mice.(5)Western blotting method was used to detect the effects of FSCP on LPS-induced RAW264.7 cells,D-gal-induced mouse senescence-related genes p53 and p16 expression,and detection of p65 and I-κBα proteins to determine whether FSCP inhibits inflamm-aging through the NF-κB signaling pathway.Results:(1)According to the orthogonal analysis experiment,the best extraction of FSCP was determined as follows:material-liquid ratio 1:40,extraction times 3times,extraction time 3h,extraction temperature 90°C.According to this scheme,the highest yield of FSCP can reach 10.72%.(2)The purified polysaccharidetotal sugar content was 84.28%±2.03%,and glucose content was 30.26%±1.12%,protein content was 1.7%±0.86%.(3)In vitro anti-oxidation experiments showed that:DPPH clearance rate of FSCP in 25-1600 μg/mL(9.78%±2.18%-71.92%±1.01%),hydroxyl radical clearance rate(40.16%±1.01%-83.06%±0.21%)and total antioxidant capacity(0.14±0.01-0.28±0.01)mM showed a dose-dependent increase and was significantly better than VE(DPPH clearance rate:9.34%±1.37%-59.24%±1.31%;hydroxyl radical clearance rate:40.16%±1.01%-83.06%±0.21%;total antioxidant capacity:(0.04±0.02-0.16± 0.001)mM)(p<0.05),confirming that FSCP has strong antioxidant activity in vitro.(4)Compared with the normal growth cell group,after LPS stimulated macrophages,macrophages showed inflammatory proliferation and the inflammatory cytokines interleukin1-α(IL-1α),interleukin6(IL-6),tumor Necrosis factor α(TNF-α)expression was significantly increased,SA-β-gal positive cells increased,SAHF fluorescence intensity was increased(p<0.01);compared with LPS model group,FSCP can effectively relieve LPS stimulated macrophages inflammatory proliferation and inhibit the expression of pro-inflammatory cytokines,reduce the occurrence of SA-β-gal positive cells and SAHF(p<0.05).(5)Compared with the normal growth cell group,after LPS stimulated macrophages,the expression level of ROS increased,the fluorescence intensity increased,mitochondrial membrane potential decreased,and the fluorescence intensity decreased(p<0.001);compared with the LPS model group,FSCP can effectively reduce after LPS stimulated macrophages,the expression level of ROS was declined,the fluorescence intensity was reduced,the mitochondrial membrane potential was reduced,and the fluorescence intensity was increased(p<0.001).(6)Compared with mice in the control group,D-gal stimulated mice with obvious signs of aging,showing slow movements,sparse hair,weight loss,significantly increased liver index.Mice in different concentrations of FSCP group aging symptoms improved significantly,body weight increased steadily,and liver index tended to be normal.(7)Compared with mice in the control group,after D-gal induced,the activities of SOD and GSH-px decreased,the MDA content increased in the serum of the mice,increased expression of IL-1α,IL-6 and TNF-α in liver tissue,the liver tissue damage was serious,inflammation was obvious,and SAHF increased(p<0.01);The FSCP low and high dose groups can effectively increase the activity of SOD and GSH-Px,reduce the expression level of MDA in the serum of mice induced by D-gal in a dose-dependent manner,and inhibit IL-1α,IL-6 and TNF-α expression,relieve liver tissue damage caused by SAHF and inflammation(p<0.01).(8)Western blotting experiments showed that compared with the control cells and mice,after LPS and D-gal stimulation,p53,p16 protein and phosphorylated p65,p53,p16,I-κBα protein expression increased,and I-κBα protein expression decreased(p<0.05);different concentrations of FSCP can effectively reduce the levels of p53,p16 protein and phosphorylated p65,p53,p16,I-κBα protein,and increase the expression of I-κBα protein(p<0.05).Conclusion:(1)Clarified the optimal extraction process of FSCP;(2)FSCP has strong DPPH free radical scavenging ability,hydroxyl radical scavenging ability and total antioxidant activity;(3)FSCP can significantly reduce LPS-induced RAW264.7 Cell inflammation and aging-related phenotypes,including inhibition of inflammatory proliferation,expression of inflammatory cytokines,SA-β-gal content and SAHF;(4)FSCP can protect mitochondrial membranes by reducing ROS production Potentials can inhibit LPS-induced inflamm-aging of RAW264.7 cells;(5)FSCP can significantly inhibit D-gal-induced aging and inflammation phenotypes in mice,improve body weight,liver index,protect liver tissue damage,increase SOD and GSH-px activity,reduce MDA,proinflammatory cytokine expression,decrease neutral Generation of lobulated nucleus granulocytes and SAHF,inhibits inflamm-aging in D-gal-induced aging mice;(6)FSCP can effectively inhibit the expression of senescence-related genes p53 and p16;(7)FSCP can inhibit the expression of p65 and I-κBα phosphorylated proteins and participate in the regulation of NF-κB signaling pathway;(8)FSCP can inhibit inflamm-aging,the possible mechanism is:by inhibiting the activation of NF-κB signaling pathway induced by LPS and D-gal,inhibiting the expression of pro-inflammatory cytokines,aging-related phenotypes and oxidative stress response. |
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