1.Protective Effect of Shentong Zhuyutang-containing Serum on Chondrocytes in Rats with Knee Osteoarthritis Based on AMPK/Sirt1 Signaling Pathway
Zhengzhen WANG ; Junqing HUANG ; Bin YANG ; Lin LI ; Jiaan DONG ; Shun YANG
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(16):33-42
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.