ABSTRACT
Objective:To explore the effect of silent information regulator 1 (SIRT1) activator SRT1720 on inflammatory response in chronic periodontal disease mice and whether its mechanism is related to the toll like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway.Methods:Forty 8-week-old male C57BL/6 mice were selected and divided into a blank control group ( n=8) and an experimental group ( n=32). The experimental group mice were ligated with periodontal pockets and fed with high sugar drinking water. The experimental group was randomly divided into a model group ( n=8) and an SRT1720 group ( n=24). The blank control group and the model group were given physiological saline orally every day. The SRT1720 group was further divided into a low dose group [20 mg/(kg·d), n=8], a medium dose group [50 mg/(kg·d), n=8], and a high dose group [100 mg/(kg·d), n=8] based on the different doses of SRT1720. Four weeks later, the expression levels of interleukin-6 (IL-6), interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in gingival crevicular fluid of mice in each group were detected by enzyme linked immunosorbent assay (ELISA); The real-time quantitative polymerase chain reaction (RT-qPCR) method was used to detect the mRNA expression levels of IL-6, IL-1β, MCP-1, SIRT1, TLR4, NF-kB p65 in the gingival tissue of mice in each group; Western blot was used to determine the expression levels of SIRT1, TLR4, and NF-κB p65 proteins in mouse gingival tissue. Results:Compared with the blank control group, the expression levels of IL-6, IL-1β, and MCP-1 inflammatory factors in the gingival crevicular fluid of experimental group mice increased, while the expression levels of IL-6, IL-1β, MCP-1, TLR4, NF-κB p65 mRNA in gingival tissue increased. The expression levels of TLR4, NF-κB p65 protein in gingival tissue increased, while the expression levels of SIRT1 mRNA and protein in gingival tissue decreased, with statistical significance (all P<0.05). Compared with the model group, the expression levels of IL-6, IL-1β, and MCP-1 inflammatory factors in the gingival crevicular fluid, IL-6, IL-1β, MCP-1, TLR4, NF-κB p65 mRNA expression levels in gingival tissue, and TLR4, NF-κB p65 protein expression levels in the gingival tissue of SRT1720 group mice showed a dose-dependent decrease. The expression levels of SIRT1 mRNA and protein in gingival tissue showed a dose-dependent increase, and the differences were statistically significant (all P<0.05). Conclusions:SIRT1 activator SRT1720 can improve the inflammatory response of chronic periodontal disease mice, which may be related to the inhibition of TLR4/NF-kB signaling pathway.
ABSTRACT
ObjectiveTo investigate the protective effect of Shentong Zhuyutang-containing serum against oxidative stress and apoptosis in chondrocytes of rats with knee osteoarthritis （KOA）. MethodFifty male rats were orally administered with normal saline， low-， medium-， and high-dose Shentong Zhuyutang （1.73， 3.46， 6.92 g·kg-1）， and glucosamine sulfate （0.3 g·kg-1） for two weeks. Serum samples were collected after the treatment period. The KOA model was established， and chondrocytes were isolated and randomly divided into normal group， model group， low-， medium-， and high-dose Shentong Zhuyutang-containing serum groups， and glucosamine sulfate group. During the chondrocyte culture， adenosine monophosphate （AMP）-activated protein kinase （AMPK） inhibitor Compound C （10 μmol·L-1） was added， and the cells were divided into normal group， model group， Shentong Zhuyutang-containing serum group， and Compound C + Shentong Zhuyutang-containing serum group. Cell proliferation was detected using 5-ethynyl-2'-deoxyuridine （EdU） staining. Apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）. Reactive oxygen species （ROS） levels were measured using DCFH-DA probe. Glutathione （GSH） and malondialdehyde （MDA） levels were determined using the colorimetric method. Real-time polymerase chain reaction （PCR） was used to measure the mRNA expression levels of matrix metalloproteinase-3 （MMP-3）， MMP-13， type Ⅱ collagen （Col Ⅱ）， and Aggrecan. Western blot was performed to measure the protein expression of phosphorylated （p）-AMPK and silent information regulator factor 1 （Sirt1）. ResultCompared with the normal group， the model group showed a significant decrease in chondrocyte proliferation rate， GSH activity， Col Ⅱ and Aggrecan mRNA expression， p-AMPK and Sirt1 protein levels （P<0.01）， and increased ROS levels， MDA content， TUNEL-positive cell rate， and MMP-3 and MMP-13 mRNA expression （P<0.01）. Compared with the model group， Shentong Zhuyutang-containing serum increased the number of EdU-positive cells， GSH activity， Col Ⅱ and Aggrecan mRNA expression， p-AMPK and Sirt1 protein levels in KOA rat chondrocytes （P<0.05， P<0.01）， and decreased the TUNEL-positive cell rate， ROS levels， MDA content， MMP-3 and MMP-13 mRNA expression （P<0.05， P<0.01）. Compared with the Shentong Zhuyutang-containing serum group， the Compound C + Shentong Zhuyutang-containing serum group showed significantly reduced p-AMPK and Sirt1 protein expression， GSH activity， Col Ⅱ and Aggrecan mRNA levels （P<0.01）， and increased TUNEL-positive cell rate， ROS levels， MDA content， MMP-3 and MMP-13 mRNA levels （P<0.01）. ConclusionShentong Zhuyutang-containing serum attenuates oxidative damage and reduces apoptosis in chondrocytes of rats with KOA， and its protective effect may be associated with the activation of the AMPK/Sirt1 signaling pathway.
ABSTRACT
Objective To observe the influence of silent information regulator factor 1(SIRT1)on TNF-αinduced intestinal epi-thelial Caco-2 cell barrier function destroy and to investigate its molecular machenism.Methods Caco-2 cells were randomly divided into three groups:normal control group (control),TNF-αgroup (TNF-α,100 ng/mL for 24 h)and 100 ng/mL plus 40μm resvera-trol group (TNF-α+Res).Transepithelial electrical resistance (TER)was determined.SIRT1 and the protein expressions of ZO-1 , occludin were examined by using Western blot.Results The relative expression amounts of SIRT1 protein were 0.81 ± 0.02, 0.43±0.04 and 0.60±0.03 respectively.TER of three groups were (154.00±5.00),(97.00±4.00)and(128.00±6.00)Ohm/cm2 respectively.Compared with the control group,the expression of SIRT1 protein was reduced by 47% and TER was decreased by 37.00% in the TNF-αgroup.After resveratrol precondition,TER was increased by 32.00% compared with the TNF-αgroup. The relative expression amounts of ZO-1 and occludin protein in the control group,TNF-αgroup and TNF-α+Res group were (0.62±0.06,0.57±0.03),(0.23±0.05,0.33±0.04)and(0.41±0.03,0.50±0.02)respectively.After TNF-αtreatment,the ex-pressions of ZO-1 and occludin protein were significantly deceased(P<0.05),but the resveratrol precondition could attenuate this phenomenon,compared with TNF-αgroup,the protein expression was increased by 78.00% and 51.00% respectively (P<0.05). Conclusion Under the condition of TNF-αtreatment,the SIRT1 level is decreased,but increasing SIRT1 level could increase the in-testinal tight j unction protein ZO-1 and occludin protein expression,thus alleviate the damage of TNF-αon the epithelial barrier function constituted by Caco-2 cells.