A Study On The Effect Of Polygonum Capitatum On The Regulation Of NF-?B Acetylation By SIRT1 In Helicobacter Pylori Associated Gastritis

Objective: To establish a cell model of Helicobacter pylori-associated gastritis(HAG)to observe the effect of Polygonum capitatum on HAG cell deacetylases SIRT1 and NF-?B signaling pathway-related proteins,to inhibit SIRT1 activity to explore whether Polygonum capitatum can affect HAG by acetylating of NF-?B through SIRT1.Methods:(1)Recovered and identified H.pylori standard strain(ATCC700392)and human immortalized gastric epithelial cells(GES-1)were co-cultured at a ratio of 100:1 for 12 h.(2)MTT colorimetric assay was used to determine the inhibition of HAG cell proliferation by different concentrations of Polygonum capitatum(0,20,40,80,160,320,640 ?g/mL).(3)HAG cells were randomly divided into model group,drug group,inhibitor group,inhibitor+drug groupand,GES-1 cells without any treatment were set as normal control group;(4)Inverted microscope,scanning electron microscope and transmission electron microscopic were used to observe the morphological changes of HAG cells after Polygonum capitatum intervention;(5)Detection of SIRT1 activity in the experimental group;(6)The levels of SIRT1,NF-?B/p65 and TNF-? mRNA in the experimental group were detected by qRT-PCR.(7)The expression of SIRT1,NF-?B/p65,Acetyl-NF-?B/p65(Lys310)and TNF-? in the total protein of the experimental group was detected by Western Blotting technique.(8)Western Blotting was used to detect the expression of SIRT1 and NF-?B/p65 in cytoplasm and nuclear proteins.Results: 1.HAG cells treated with Polygonum capitatum concentration ?80?g/mL for 48 h had no obvious inhibitory effect on the proliferation of HAG cells.2.The surface of the HAG cells was not smooth under the inverted microscope,the particulate matter was significantly increased.Spotted or flaky material adhered on the cell surface in the scanning electron microscope.Under the transmission electronmicroscope,the decrease of autophagosomes in the villus and cytoplasm of the cell surface.After treatment with HAG cells,the granular material of the cells was reduced under inverted microscope.Cell adhesion on the cell surface was reduced by scanning electron microscopy,a large number of autophagosomes were observed in the cytoplasm by transmission electron microscopy.3.Results of SIRT1 activity showed that: the model group decreased 1.653±0.259 fold compared with the normal control group,the drug group increased 6.458±0.477 fold compared with the model group(p<0.05).4.The results of qRT-PCR showed that compared with the normal control group,the expression level of SIRT1 mRNA in the model group was decreased,while that of NF-?B/p65 and TNF-? was increased(p<0.05);Compared with the model group,SIRT1 mRNA in the drug group increased,NF-?B/p65 and TNF-? mRNA decreased(p<0.05),the inhibitor group the SIRT1 m RNA decreased,the NF-?B/p65 and TNF-? mRNA increased,(p<0.05).The SIRT1 m RNA increased in the inhibitor+drug group compared with the inhibitor group,mRNA of NF-?B/p65 and TNF-? decreased(p<0.05).;Compared with the drug group,SIRT1 and TNF-? mRNA decreased in the inhibitor + drug group(p<0.05).5.Western Blotting results showed that compared with the normal control group,the SIRT1 in the model group decreased,the NF-?B/p65,Acetyl-NF-?B/p65(Lys310)and TNF-? increased(p<0.05);compared with the model group,SIRT1 in the drug group increased,NF-?B/p65 and TNF-? decreased.In particular,Acetyl-NF-?B/p65(Lys310)decreased,(p<0.05);SIRT1 and TNF-? decrea sed,Acetyl-NF-?B/p65(Lys310)increased in the inhibitor group compared with the model group(p<0.05).Compared with the inhibitor group,Acetyl-NF-?B/p65(Lys310)and TNF-? decreased in the inhibitor+drug group(p<0.05);Compared with the drug group,SIRT1 and TNF-? decreased in the inhibitor+ drug group(p<0.05).6.The expression of SIRT1 and NF-?B/p65 in cytoplasm and nucleus: compared with the normal control group,the expression of SIRT1 in the cytoplasm of the model group decreased,the expression of NF-?B/p65 in the nucleus increased(p<0.05).Compared with the model group,the expression of SIRT1 in the nucleus of the drug group increased,the expression in the NF-?B/p65 cytoplasm increased,the expression in the nucleus decreased(p<0.05).Conclusions:1.Polygonum capitatum can play its therapeutic role by activating SIRT1 and deacetylating the p65Lys310 locus.2.Polygonum capitatum increased the expression of SIRT1 in the nucleus,transferred the activated p65 in the nucleus to the cytoplasm,inhibited the secretion of TNF-?,and antagonized the H.pylori-induced NF-?B pathway.