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Objective:To evaluate the role of ferroptosis in the dorsal root gangions in neuropathic pain (NP) in rats.Methods:Thirty-two healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 180-220 g, were randomized into 4 groups ( n=8 each) using a random number table method: sham operation group (Sham group), NP group, NP+ solvent control group (NP+ Veh group), and NP+ liproxstatin-1 (Lip-1) group (NP+ Lip group). NP was induced by chronic constrictive injury (CCI) to sciatic nerve in anesthetized animals.In NP+ Lip group, liproxstatin-1 (diluted to 10 μg/μl in DMSO) 30 μl was intrathecally injected for 3 consecutive days after surgery.NP+ Veh group received intrathecal injection of DMSO 30 μl for 3 consecutive days after surgery.Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 3 days before surgery and on days 1, 3, 5, 7 and 10 after surgery.Rats were sacrificed after the end of pain threshold measurement on day 10 after surgery, and DRGs of the lumbar segment (L 3-5) on the left side were removed for determination of the levels of iron ion, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD), expression of glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) (by Western blot), and expression of ACSL4 in each nerve cells of DRGs (by immunofluorescence) and for microscopic examination of the ultrastructure of mitochondria in DRGs (by transmission electron microscopy). Results:Compared with Sham group, MWT was significantly decreased and TWL was shortened at T 2-6, levels of iron ions, ROS and MDA in DGRs were increased, activities of SOD and GSH-Px were decreased, ACSL4 expression was up-regulated, GPX4 expression was down-regulated, and ACSL4 expression in astrocytes and Schwann cells of DRGs was up-regulated in NP group ( P<0.05). Compared with NP group, MWT was significantly increased and TWL was prolonged at T 3-6, levels of iron ions, ROS and MDA in DGRs were decreased, activities of SOD and GSH-Px were increased, ACSL4 expression was down-regulated, GPX4 expression was up-regulated, and ACSL4 expression in astrocytes and Schwann cells of DRGs was down-regulated ( P<0.05), and no significant change was found in the parameters mentioned above in NP+ Veh group ( P>0.05). The results of electron microscopy showed that collapsed mitochondrial cristae and membrane rupture were found in astrocytes and Schwann cells of DRGs in NP group, and the number of collapsed mitochondrial cristae and membrane rupture was significantly decreased in NP + Lip group when compared with NP group. Conclusions:The ferroptosis in DRGs is involved in NP in rats.
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Objective@#To investigate the impact of n-3 polyunsaturated fatty acid (n-3 PUFA) on function and expression of store-operated calcium channels (SOCC) in coronary artery smooth muscle cells (SMC) derived from diabetic rat.@*Methods@#A total of 180 healthy male Sprague-Dawley (SD) rats were randomly divided into normal group (N, n=45), placebo-treated diabetic group (D, n=45), lose dose n-3 PUFA treated diabetic group (DL, n=45) and high dose n-3 PUFAs treated diabetic group (DH, n=45). Streptozotocin-induced diabetic rat animal model was established by two consecutive intraperitoneal injections. After modeling, rats in group DL and DH were treated with 10 mg·kg-1·d-1 and 50 mg·kg-1·d-1 n-3 PUFAs respectively per gavage for eight weeks. After eight weeks, rat coronary artery SMC was isolated by enzyme digestion. Changes of cytosolic calcium concentration in coronary artery SMC were examined by calcium fluorescence imaging technique, coronary artery tension was detected by myograph system, and protein expressions of SOCC on coronary artery SMC were measured by Western blot.@*Results@#SOCC induced ΔF340/F380 of group N, D, DL and DH were 0.425±0.023, 0.838±0.037, 0.342±0.052 and 0.364±0.045 respectively, which was significantly lower in group N, DL, DH than in group D (P<0.05). SOCC induced changes of tensions were 0.94±0.09, 1.95±0.18, 1.35±0.24 and 1.01±0.18 in the group N, D, DL and DH, respectively, which was significantly lower in group N and DH than in group D (P<0.05). Protein expressions of STIM1, Orai1 and TRPC1 were significantly higher in diabetic rat coronary SMC than in group N (P<0.05). STIM1 protein expressions were significantly lower in group DL and DH than in group D, and Orai1 and TRPC1 protein expressions were similar among group.@*Conclusions@#Coronary artery tension, cytosolic calcium concentration and protein expressions of SOCC are higher in diabetic rat coronary artery SMC when compared with normal rats. n-3 PUFA intervention could downregulate the protein expression of SOCC, reduce cytosolic calcium concentration and coronary artery tension, and is protective to the diabetic injury in coronary artery.
