关键词: 原花青素, 骨骼肌卫星细胞, 缺血缺氧, 增殖, 分化

Abstract:

BACKGROUND: Limb ischemic injury is mainly caused by peripheral arterial disease. Surgical treatments can restore blood supply to the limbs, but ischemia-reperfusion can still aggravate tissue damage. Reducing oxidative stress and promoting regeneration of impaired skeletal muscle may offer a novel treatment opportunity for the ischemic limb.

OBJECTIVE: To investigate the effect of proanthocyanidins on proliferation and differentiation of skeletal muscle satellite cells under hypoxic-ischemic condition.
METHODS: The primary cultured skeletal muscle cells were purchased and the passage 2 cells were used for subsequent experiment. There was a control group treated with normal saline and two experimental groups treated with 2.5 or 5 mg/L procyanidins. The cells in each groups were cultured in an environment of low oxygen (1% O2) and low serum (1% fetal bovine serum), and were observed at different time points. Cell proliferation was detected by cell counting kit-8 method at 1, 2, 3 and 4 days of culture. The cells were collected at 1 day of culture. Cell cycle was detected by flow cytometry. Total proteins were extracted and the expression of proliferation-related proteins, myogenic marker genes and pathway-related proteins were detected by western blot assay. The morphology of myotubes was detected using laser scanning confocal microscope. To further clarify the role of the P38-MAPK signaling pathway, four groups were set up, namely a normal saline group (control group), a proanthocyanidin group, a normal saline+P38-MAPK pathway inhibitor group, and a proanthocyanidin+P38-MAPK pathway inhibitor group. The expressions of myogenic marker genes and related pathway proteins in the cells were detected. The animal experiment was approved by the Animal Experiment Ethics Committee of Southwest Medical University. The mice were randomly treated with saline (n=12) or 20 mg/kg proanthocyanidin (n=12) via intraperitoneal injection after hind limb ischemia modeling. Gastrocnemius was taken from the mice at 7 days after intervention, and muscle regeneration was observed by hematoxylin-eosin staining. 
RESULTS AND CONCLUSION: (1) Cell experiment: Under hypoxic-ischemic condition for 1, 2, 3 and 4 days, the proliferation ability of cells in the proanthocyanidin groups were significantly higher than that of the control group (P < 0.05).Under hypoxic-ischemic condition for 1 day, the expression levels of proliferating cell nuclear antigen protein in the proanthocyanidin groups were significantly higher than that in the control group (P < 0.05).Under hypoxic-ischemic condition for 1 day, the number of S-phase cells in the proanthocyanidin groups was significantly higher than that in the control group (P < 0.05).Under hypoxic-ischemic condition for 3 or 5 days, the expression of myogenic marker gene and the formation of myotubes in the proanthocyanidin groups were significantly higher than those in the control group (P < 0.05). The above-mentioned indexes showed no significant difference between the two proanthocyanidin groups (P > 0.05). Under hypoxic-ischemic condition for 3 days, p-P38-MAPK signaling pathway-related proteins were significantly highly expressed in the proanthocyanidin groups compared with the control group (P < 0.05). However, there was no significant difference between saline+p38-mapk pathway inhibitor group and proanthocyanidin+p38-mapk pathway inhibitor group (P > 0.05). (2) Animal experiment: After lower extremity ischemia for 7 days, proanthocyanidin-treated mice contained a higher number of regenerating myofibers than that in the control group (P < 0.05). To conclude, proanthocyanidin can promotes the proliferation of skeletal muscle satellite cells under hypoxic-ischemic condition, and meanwhile induces the expression of myogenic marker genes and formation of muscle tube by activating p38-MAPK signaling pathway, so as to promote regeneration of ischemic damaged skeletal muscle and repair damaged myofibers, exerting an important protective role.

Key words: proanthocyanidins, skeletal muscle satellite cells, ischemia-hypoxia, proliferation, differentiation