Abstract:

Objective To investigate the effects of Xiaozhong Zhitong Mixture （XZZT） on oxidative stress and inflammatory injury in rats with diabetic ulcers through the AMPK/SIRT1/NF-κB signaling pathway. Methods A total of one hundred specific-pathogen-free （SPF）-grade male sprague-dawley （SD） rats were randomly allocated into five groups： a control group， a model group， an XZZT group， an AMP-activated protein kinase （AMPK） inhibitor （Compound C， CC） group， and an XZZT + CC group. Except for the rats in the control group， diabetic ulcer models were established through a high-fat and high-sugar diet， intraperitoneal injection of streptozotocin， and full-thickness skin excision. After successful model establishment， the rats in the XZZT and XZZT + CC groups were administered XZZT via gavage at a dosage of 1 mL/（100 g·d）， whereas the rats in the control， model， and CC groups received an equal volume of 0.9% sodium chloride solution once daily. The rats in the CC and XZZT + CC groups were intraperitoneally injected with 0.2 mg/kg of the AMPK inhibitor Compound C once. Tissue samples were collected on the 7th and 14th days after the intervention. The wound conditions were observed， the wound area was measured， and the healing rate was calculated. Hematoxylin-eosin （HE） staining was employed to examine the pathological changes in the granulation tissue. Enzyme-linked immunosorbent assay （ELISA） was conducted to detect the levels of interleukin-6 （IL-6）， interleukin-10 （IL-10）， and tumor necrosis factor-α （TNF-α） in the wound tissues. The malondialdehyde （MDA） content was measured using the thiobarbituric acid method， and the superoxide dismutase （SOD） activity was determined by the hydroxylamine method. Western blot analysis was utilized to assess the protein expression of AMPK， phosphorylated AMPK （p-AMPK）， sirtuin 1 （SIRT1）， nuclear factor-kappa B （NF-κB）， and phosphorylated NF-κB （p-NF-κB）. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） was applied to measure the mRNA expression levels of AMPK， SIRT1， IL-6， and TNF-α. Results When compared to the control group， the model group demonstrated elevated levels of IL-6， TNF-α， MDA， p-NF-κB protein， as well as IL-6 and TNF-α mRNA， accompanied by a decrease in the IL-10 level， SOD activity， p-AMPK/AMPK ratio， and SIRT1 mRNA and protein expression on days 7 and 14 （P < 0.01）. In comparison with the model group， the XZZT group presented a higher wound healing rate， IL-10 level， SOD activity， p-AMPK/AMPK ratio， and SIRT1 mRNA and protein expression， along with lower levels of IL-6， TNF-α， MDA， p-NF-κB protein， and IL-6 and TNF-α mRNA （P < 0.01）. Conversely， the CC group exhibited opposite trends （P < 0.05）. When compared to the XZZT group， the XZZT + CC group showed increased levels of IL-6， TNF-α， MDA， p-NF-κB protein， and IL-6 and TNF-α mRNA， and a reduced wound healing rate， IL-10 level， SOD activity， p-AMPK/AMPK ratio， and SIRT1 mRNA and protein expression （P < 0.01）. Conclusion Xiaozhong Zhitong Mixture may alleviate oxidative stress and inflammatory injury， thereby accelerating diabetic ulcer healing in rats. This effect is possibly achieved by activating the AMPK/SIRT1 signaling pathway and suppressing NF-κB phosphorylation.

Key words: Xiaozhong Zhitong Mixture, AMPK/SIRT1/NF-κB signaling pathway, diabetic ulcer, oxidative stress, inflammation