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
OBJECTIVES@#Neuropathic pain (NP) is a chronic pain caused by somatosensory neuropathy or disease, and genistein (Gen) might be a potential drug for the treatment of NP. Therefore, this study aims to investigate the effect of Gen on lipopolysaccharide (LPS)-induced inflammatory injury of dorsal root ganglion neuron (DRGn) in rats and the possible molecular mechanism.@*METHODS@#The DRGn of 1-day-old juvenile rats were taken for isolation and culture. The DRGn in logarithmic growth phase were divided into a control group, a LPS group, a tubastatin hydrochloride (TSA)+LPS group, a Gen1+LPS group, a Gen2+LPS group, a Gen2+LPS+TSA group, a Gen2+pcDNA-histone deacetylase 6 (HDAC6)+LPS group, and a Gen2+pcDNA3.1+LPS group. The LPS group was treated with 1 μg/mL LPS for 24 h; the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group were treated with 5 μmol/L TSA, 5 μmol/L Gen, 10 μmol/L Gen respectively for 0.5 h, and then added 1 μg/mL LPS for 24 h; the Gen2+TSA+LPS group was treated with 10 μmol/L Gen and 5 μmol/L TSA for 0.5 h and then added 1 μg/mL LPS for 24 h; the Gen2+pcDNA-HDAC6+LPS group and the Gen2+pcDNA3.1+LPS group received 100 nmol/L pcDNA-HDAC6 and pcDNA3.1 plasmids respectively, and 24 h after transfection, 10 μmol/L Gen was pretreated for 0.5 h, and then added 1 μg/mL LPS for 24 h. Real-time RT-PCR was used to detect the HDAC6 mRNA expression in DRGn; CCK-8 method was used to detect cell viability of DRGn; flow cytometry was used to detect cell apoptosis of DRGn; ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in DRGn culture supernatant; Western blotting was used to detect the protein expression of HDAC6, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 in DRGn.@*RESULTS@#Compared with the control group, the expression levels of HDAC6 mRNA and protein, the expression levels of TLR4 and MyD88 protein in DRGn of LPS group rats were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, and the activity of DRGn was significantly decreased, the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). Compared with the LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group and the Gen2+TSA+LPS group were significantly down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly decreased, the activity of DRGn was significantly increased, the apoptosis rate was significantly decreased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly decreased (all P<0.05), and the above changes were most obvious in the Gen2+TSA+LPS group. Compared with the Gen2+LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the Gen2+pcDNA-HDAC6+LPS group were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, the activity of DRGn was significantly decreased, and the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05).@*CONCLUSIONS@#Gen can alleviate LPS-induced DRGn inflammatory injury in rats, which might be related to down-regulating the expression of HDAC6 and further inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.