["Shugan Tiaoshen" acupuncture improves chronic inflammatory pain and depression comorbidity by inhibiting ferroptosis of hippocampal neurons].

OBJECTIVES: To observe the effect of "Shugan Tiaoshen" (regulating mental activities by relieving depressed liver) acupuncture on hippocampal iron metabolism, antioxidant system and lipid peroxidation related factors in chronic inflammatory pain and depression comorbidity (CIPDC) rats and its protective effect on hippocampal neurons, so as to explore its mechanisms underlying improvement of CIPDC.

METHODS: A total of 48 male SD rats were randomly divided into control, model, electroacupuncture (EA) and medication (paroxetine) groups, with 12 rats in each group. The CIPDC model was established by injection of complete Freund's adjuvant into the toe surface of the left hind paw. From 15 to 28 days after modeling, EA was applied to "Yintang" (GV24+), "Baihui" (GV20), "Hegu" (LI4) and "Taichong" (LR3) for 30 min, once a day for 14 days. Rats in the medication group were treated with intragastric administration of paroxetine (10 mg/kg), once a day for 14 days. Paw withdrawal mechanical threshold (PWMT) and paw thermal withdrawal latency (PTWL) were detected for evaluating the rats' pain response behavior in each group at 0, 7, 14, 21 and 28 days after modeling. The sucrose preference test, open field test (OFT) and forced swimming test (FST) were conducted to calculate the sucrose preference value, and to record the total distance traveled in 30 min and the time spent in the central zone of the open field during the first 5 min, and the immobility (floating) time during the last 4 min for evaluating the rats' depression-like behavior at 0, 14 and 28 days after modeling. H.E. staining was used to observe the histopathological changes of hippocampal neurons. Prussian blue staining was used to observe the iron deposition in the hippocampus tissue. A transmission electron microscopy was used to observe the ultrastructure of mitochondria of hippocampal neurons. The content of Fe2+ in the hippocampus tissue was detected by colorimetry method. ELISA method was used to measure the contents of glutathione (GSH) and malondialdehyde (MDA) in the hippocampus tissue. The expression levels of ferritin heavy chain 1 (FTH1), ferritin light chain (FTL), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-coa synthetase long chain family member 4 (ACSL4), and lipoxygenase (LOX) proteins and mRNAs of hippocampus were detected by Western blot and real-time fluorescence quantitative PCR, separately.

RESULTS: Compared with the control group, the PWMT and PTWL at 7, 14, 21 and 28 days after modeling, sucrose preference value (SPV), total motion distance of OFT, time spent in the central zone, GSH content, and expression levels of FTH1, FTL, SLC7A11 and GPX4 proteins and mRNAs were significantly decreased (P<0.01), while immobility time of FST, the contents of Fe2+ and MDA, and the expression levels of ACSL4 and LOX protein and mRNAs were considerably increased (P<0.01) in the model group. In contrast to the model group, both EA and medication group had an obvious increase in the PWMT and PTWL at 21 and 28 days after modeling, SPV, total motion distance, time spent in the central zone, GSH content, and the expression levels of FTH1, FTL, SLC7A11 and GPX4 proteins and mRNAs (P<0.01, P<0.05), and a striking decrease in the immobility time, contents of Fe2+ and MDA, and the expression levels of ACSL4 and LOX protein and mRNAs (P<0.01, P<0.05). No significant differences were found between the EA and medication groups in all the indexes mentioned above. Morphological observation showed a large number of yellow-brown iron deposition in the hippocampus, neuronal degeneration, disordered arrangement and necrosis of neurons, shrinkage and deformation of mitochondria, increased membrane density, blurred boundary, and decrease and disappearance of mitochondrial cribriae in the model group, which was obviously milder in the injury state in both EA and medication groups, including reduction of yellow-brown iron deposition, regular arrangement of neurons, obvious nucleolus and complete structure of mitochondria, etc.

CONCLUSIONS: "Shugan Tiaoshen" acupuncture can improve the inflammatory pain and depression in rats with CIPDC , which may be related to its functions in activating antioxidant activity and promoting iron metabolism balance, reducing lipid peroxidation, and inhibiting ferroptosis, thereby promoting the repair and remodeling of hippocampal neurons and ameliorating the disease process of CIPDC.