Expression of Erk kinase, AMPA receptor subunits GluR1 and GluR2, and protection of chondroitin sulfate in brain of rats with chronic fluorosis

Objective To study the mechanism of central nervous system (CNS) injury in chronic fluorosis and the neuroprotective effect of chondroitin sulfate (CS). Methods Forty-eight female Sprague-Dawley rats weighting 90 - 120 g were divided into 8 groups according to body weight by random number table, 6 rats in each group: control group, drinking tap water freely; low dose and high dose fluoride groups, freely drinking tap water with fluoride content of 10 and 50 mg/L, respectively; control + normal saline (NS), low dose fluoride + NS, and high dose fluoride + NS groups, each group was fed for 180 d, and treated with intraperitoneal injection of 0.66 mg/kg NS for 5 d (once a day); low dose fluoride + CS and high dose fluoride + CS groups, each group was fed for 180 d, 0.66 mg/kg CS was injected intraperitoneally for 5 d (once a day). All groups were fed standard nutritive animal feed for 185 d and dissected for brain tissue. The pathologic change was observed after hematoxylin-eosin (HE) staining; the expression levels of phosphorylated extracellular signal-regulated protein kinase 1/2 (phospho-Erk1/2) and glutamate receptors 1, 2 (GluR1, GluR2) in the brain cortex were detected by immunohistochemistry; the protein levels of Erk1/2, phospho-Erk1/2, GluR1, and GluR2 in the brain cortex were detected by Western blotting. Results Brain cortex of all rats in the fluoride groups showed eosinophilic degeneration, loss and disordered arrangement of neurons, and the brain morphological changes in each fluoride + CS groups were significantly improved compared with those in the fluoride groups. Immunohistochemistry results showed that compared with the control group [(0.44 ± 0.09)%, (1.49 ± 0.05)%, (2.51 ± 0.54)%], the expression levels of phospho-Erk1/2 [(1.47 ± 0.09)%, (1.03 ± 0.05)%], and GluR2 [(2.37 ± 0.06)%, (3.38 ± 0.12)%] in the low dose and high dose fluoride groups were increased, and the expression levels of GluR1 [(1.49 ± 0.02)%, (0.99 ± 0.19)%] were decreased (P < 0.05). Western blotting results showed that compared with the control group (1.00 ± 0.12, 1.76 ± 0.33), the protein levels of Erk1/2 (3.10 ± 0.76, 1.99 ± 0.01) and phospho-Erk1/2 (3.27 ± 0.25, 2.67 ± 0.05) in low dose and high dose fluoride groups were significantly increased (P < 0.05); compared with low dose fluoride group, the protein levels of Erk1/2, and phospho-Erk1/2 (1.30 ± 0.31, 2.20 ± 0.34) in low dose fluoride + CS group decreased significantly (P < 0.05). Compared with control group (1.86 ± 0.47, 1.17 ± 0.27), the protein levels of GluR1 (1.09 ± 0.26, 0.61 ± 0.14) in low dose and high dose fluoride groups decreased significantly, while the protein level of GluR2 (1.99 ± 0.42, 3.38 ± 0.27) increased significantly (P < 0.05); compared with low dose and high dose fluoride groups, the protein levels of GluR2 in low dose fluoride + CS and high dose fluoride + CS groups (1.53 ± 0.41, 2.65 ± 0.32) decreased significantly (P < 0.05). The protein level of phospho-Erk1/2 was negatively correlated with GluR1 protein level (r = - 0.975, - 0.991, P < 0.05) in low dose and high dose fluoride groups, and it was positively correlated with the protein level of GluR2 (r = 0.986, 0.993, P < 0.05). Conclusion The CNS injury caused by chronic fluorosis may be related to GluR1 and GluR2 activated Erk1/2 signaling pathway, and CS has certain protection to the injury. Key words: Rats; Fluorosis; Erk1/2; Receptors, glutamate; Chondroitin sulfate