| It is well known that activation of dorsal horn microglia play a critical role in neuropathic pain. Recent evidences indicate P2Y13 receptor may be involved in the development of pain behavior induced by peripheral nerve injury, and the receptors also expressed in dorsal horn microglia. However, we do not clear whether P2Y13 receptors cause the activation of microglia resulting in a sensation of pain in diabetic neuropathic pain model. In thus, this proposal will use some methods include morphology, ethology, molecular biology to investigate the behavioral responses and the relationships between the expression of P2Y13 receptor and the activation of microglia cells and the change in the expression of IL-6 in dorsal horn in diabetic rats; to speculate the role of P2Y13 receptor in microglia activation and IL-6 release from microglia in vitro. The research works will provide some evidence for developing effective analgesics beses on microglia as the new therapeutic targets, and provide new ideas and methods for clinical treatment of pathological pain.Objective: 1. To explore whether dorsal horn microglia P2Y13 receptor is involved in STZ-induced pain neuropathy in diabetic rats. 2. To explore whether P2Y13 receptor can affect ADP-evoked function change of the spinal cord microglia In vitro condition.Methods: The experimental animals were provided by the Experimental Animal Center of The Third Military Medical University. 1. All rats were randomized into four groups(n=8): normal group(intrathecal normal saline), MRS2211+normal group(intrathecal injection of MRS2211 100pmol/L), diabetes mellitus group(DM, intrathecal saline), diabetes mellitus + MRS2211 group(intrathecal 100pmol/L MRS2211). Diabetes mellitus was induced by a single intraperitoneal injection of STZ at a dose of 60 mg/kg. After two weeks, diabetic neuropathic pain model was confirmed when mechanical pain threshold decreased significantly. Then, MRS2211(100pmol/L) or normal saline were intrathecally administered twice per day for 4 weeks. All rats were tested for mechanical withdrawl threshold(MWT) at the plantar surface of the hindpaw 1 day before and 2th, 4th and 6th week after STZ injection. The expression of Iba-1 and IL-6 in dorsal horn was examined by western blot on the 2th, 4th and 6th weekeed after STZ injection, respectively. The level of glucose in the blood and urine in different groups were observed. 2. Primary dissociated culture of dorsal spinal cord microglia cells was prepared from Sprague–Dawley rats. The expression of Iba-1 and IL-6 in m RNA levels were detected via RTFQ-PCR analysis. In addition, ADP-induced IL-6 release from cultured spinal dorsal horn microglia was detected by using enzyme-linked immuno sorbent assay.Results: 1. In diabetie rats, after STZ adiministration, the fasting blood glucose and urine glucose were significantly increased than that of control rats at 2, 4 and 6 weeks(P<0.01). The body weight of diabetie rats was signifieantly reduced than that of control rats at 2, 4 and 6 weeks(P<0.01). Compare with normal rats, the drinking intake of diabetie rats was also obvious increased than that of control rats at 2, 4 and 6 weeks(P<0.01). Compared with DM group, the fasting blood glucose, urine glucose, body weight and drinking intake were produced no obvious changes after MRS2211(100pmol/L) administration on 4th weekend(P<0.01). 2. MWT in DM group were significantly lower than that in normal group(P<0.01). Compared with normal group, the expression of Iba-1 and IL-6 was significantly increased in dorsal horn on 2th(P<0.01), 4th(P<0.01) and 6th(P<0.01) weekend after STZ injection. Compared with DM group, the MWT were significantly increased after MRS2211(100pmol/L) administration on 4th weekend(P<0.01). Intrathecal administration of MRS2211 markedly suppressed the increased expression of Iba-1 and IL-6 at the 4th weekend after STZ injection(P<0. 01) as compared with DM. However, intrathecal administration of MRS2211 produced no obvious changes in the increased Iba-1 and IL-6 expression on the 6th weekend after STZ injection. 3. In cultured spinal dorsal horn microglia cells, RTFQ-PCR assay revealed that ADP(100μmol/L) induced the increased gene expression of Iba-1(P<0. 01) and IL-6(P<0. 01). Administration of MRS2211 partially and significantly suppressed the increased expression of expression of Iba-1 m RNA, which indicats that P2Y13 receptor partially involved in this process. In addition, administration of MRS2211 significantly blocked the increased expression of of expression of IL-6 m RNA, which indicats that P2Y13 receptor is involved in this process. Subsequently, ADP(100μmol/L) induced IL-6 release from cultured spinal dorsal horn microglia cells by using ELISA technique(P<0.01). Administration of MRS2211 partially and significantly suppressed IL-6 release from microglia cells(P<0.05), which indicats that P2Y13 receptor maybe partially involved in this process.Conclusion: 1. Intrathecal injection of the specific P2Y13 antagonist MRS2211 attenated tactile allodynia and the increased expression of Iba-1 and IL-6 in diabetic rats, suggesting that MRS2211 may attenuate diabetic neuropathic pain by inhibition of dorsal horn microglia activation. 2. In cultured dorsal spinal cord microglia cells, P2Y13 receptor activation evoked the increased increased gene expression of Iba-1 and IL-6 expression and the subsequent IL-6 release. |
