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Chinese Journal of Tissue Engineering Research
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2022, Vol. 26 ›› Issue (36): 5868-5874
Antioxidative stress of trihydroxyethyl rutin on cervical spinal cord injury in rats
Liu Yapu1, 2, Su Yuanyuan1, 2, Liu Qi1, Yang Zhou1, Li Rong1, Huang Zucheng1, Huang Zhiping1, Wu Xiaoliang1, Zhu Qingan1
1Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China; 2Second Affiliated Hospital of Luohe Medical College, Luohe 462300, Henan Province, China
Contact: Zhu Qingan, MD, Professor, Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China
About author:Liu Yapu, MD, Associate chief physician, Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong Province, China; Second Affiliated Hospital of Luohe Medical College, Luohe 462300, Henan Province, China
the Key Scientific Research Project of Universities in Henan Province, No. 21B320004 (to LYP); Joint Co-construction Project of Henan Medical Science and Technology Research Plan, No. LHGJ20200887 (to LYP); Luohe Youth Talents Fund of China, No. 2018QNBJRC01004 (to LYP); Innovation and Entrepreneurship Promotion Project of Luohe Medical College, No. 2019-LYZKYZD004 & 2019-LYZKYYB016 (to LYP)
Abstract: BACKGROUND: Previous animal studies have found that trihydroxyethyl rutin can improve the neurological function of rats with cervical spinal cord injury, but there is a lack of relevant studies on the mechanism of action.
OBJECTIVE: To investigate the antioxidant stress effect of trihydroxyethyl rutin on spinal cord injury in vitro and in vivo, and to explore the possible mechanism.
METHODS: (1) Cell experiment: The rat adrenal gland PC12 cells were purchased from cell bank of American Type Culture Collection. MTT assay was used to detect the effects of gradient concentrations of tert-butyl hydroperoxide (0, 25, 75, 100, 150, 200, and 250 μmol/L) and trihydroxyethyl rutin (0, 50, 100, 200, and 400 μmol/L) on the activity of PC12 cells. Experimental concentrations of tert-butyl hydroperoxide and trihydroxyethyl rutin were determined. (2) Animal experiment: Male Sprague-Dawley rats were assigned to sham operation group, injury group, trihydroxyethyl rutin 50 mg/kg group, trihydroxyethyl rutin 100 mg/kg group, and STAT3 inhibitor group (n=12). In addition to the sham operation group, cervical spinal cord injury was made in rats. Rats in the trihydroxyethyl rutin 50 mg/kg group and trihydroxyethyl rutin 100 mg/kg group were intraperitoneally given trihydroxyethyl rutin 50 mg/kg (once a day) and 100 mg/kg (once a day) after model establishment, respectively. The rats in the inhibitor group were given STAT3 inhibitor 2 000 µg/kg (once a day) immediately after model establishment. Animals in the sham operation group were subjected to laminectomy, but no contusion operation was performed. Rats in the sham operation group and the injury group were injected intraperitoneally with normal saline for 3 consecutive days. DCFH probe was used to detect the effects of trihydroxyethyl rutin on reactive oxygen species production in tert-butyl hydroperoxide-induced PC12 cells. Western blot assay was used to detect the effects of trihydroxyethyl rutin and STAT3 inhibitor on the expression levels of oxidative stress related proteins Catalase and MnSOD in PC12 cells and cervical spinal cord injury tissues. Immunofluorescence staining was used to detect the effects of trihydroxyethyl rutin on STAT3 and MnSOD protein expression in cervical spinal cord injury tissue.
RESULTS AND CONCLUSION: (1) The reactive oxygen species formation in PC12 cells induced by tert-butyl hydroperoxide was significantly decreased by trihydroxyethyl rutin (P < 0.05). (2) Trihydroxyethyl rutin and STAT3 inhibitor significantly up-regulated the expression levels of Catalase protein and MnSOD protein in PC12 cells and cervical spinal cord injury tissue (P < 0.05), while significantly inhibited the expression levels of STAT3 and NCK1 protein (P < 0.05). (3) To conclude, trihydroxyethyl rutin exerts anti-oxidative stress effect and can reduce the damage of oxidative stress after spinal cord injury by regulating the expression of STAT3.
Key words: triehydroxyethyl rutin, cervical spinal cord injury, rat, oxidative stress, STAT3