| Objective Opioids are the first choice for surgical anesthesia and the treatment of moderate and severe pain. However, their use is plagued by side effects, such as hyperalgesia, analgesic tolerance and addiction. These side effects are mediated by inflammatory cytokines released by the activated brain glial cells. The opioids combine with the μ receptor and the κ receptor on memberane of neurons cells and then play analgesic role. While, the activation of microglia and astrocytes in the generation of opioid tolerance is known to be mediated by the μ receptor. The effects of different opioids on the activation of glial cells and the mechanisms remain to be elucidated. On the other hand, Studies have found that the activation of glial cells and the high expression of TSPO(Translocator) are found in a variety of inflammatory and degenerative neurological disease. The application of TSPO ligands can inhibit the activation of glial cells. But whether TSPO is involved in opioid activation and induce opioid tolerance of glial cells is unknown. This study aims: 1) To studythe effects of different opioids on the activation of microglia and to explore whether this process is mediated by the κ receptor. 2) To explore whether the TSPO is involved in the process of glial cells activation and mediate opioid tolerance. And whether the midazolam can relieve the activation of glial cells and the side effects of opioid tolerance. This study will provide a more scientific and reasonable solution for clinical anesthesia drug combination through cellular and animal level research.Methods 1 Analyze the effect of opioids, which activate both μ receptors and κ receptors, on the activation of mouse microglia cell line BV2 cells. The BV2 cells were treated with clinical equivalent dose of morphine 5 μM, oxycodone 5 μM, hydromorphine 1 μM, fentanyl 0.03 μM, sufentanil 0.003 μM in the medium, respectively. After 24 h, the total RNA in each group was extracted and the RT-q PCR was used to detect the m RNA level of inflammatory cytokine such as TNF-α(Tumor necrosis factor-α), IL-6(Interleukin-6) and IL-1β(Interleukin-1β). 2 Analyze the effect of dezocine, which activates κ receptors but antagonizes μ receptors, on the activation of BV2 cells. And then to analyze the effect of non-selective opioid receptor antagonist naloxone, which antagonizes μ receptors and κ receptors, on the antagonism to dezocine. BV2 cells was treated with dezocine(4 μM, 40 μM, 400 μM), naloxone(0.1 μM, 1 μM, 10μM), dezocine + naloxone(4 μM+0.1 μM, 40 μM+1 μM, 400 μM+10 μM). After 24 h, the total RNA in each group was extracted and the RT-q PCR was used to detect the m RNA level of TNF-α, IL-6, IL-1β. 3 BV2 cells were treated with different concentrations of morphine(1 μM, 5 μM, 25 μM, 100 μM) for 24 h. On the other hand, BV2 cells were treated with 25 μM morphine for(6 h, 12 h, 24 h, 48 h, 72 h) respectively. After that, the total RNA in each group was extracted and the RT-q PCR was used to detect the m RNA level of TNF-α, IL-6, IL-1β. 4 Analyze the effects of midazolam on the activation of BV2 cells by morphine. 5 μM midazolam, 25 μM morphine, 5 μM midazolam+25 μM morphine in the medium were given to BV2 cells respectively. After 24 h, the total RNA in each group was extracted and the RT-q PCR was used to detect the m RNA level of TNF-α, IL-6, IL-1β. And the total protein in each group was extracted and the Western blot was used to detect the protein level of and TSPO and the Iba1(Ion calcium adaptor protein 1), which was the marker of microglia activation. 5 The BV2 cells were transfected with si RNA of TSPO after 24 h, and were treated with 5 μM midazolam, 25 μM morphine, 5 μM midazolam+25 μM morphine in the medium for 24 h. The total RNA in each group was extracted and the RT-q PCR was used to detect the m RNA level of TNF-α, IL-6 and IL-1β. And the total protein in each group was extracted and the Western blot was used to detect the protein level of Iba1. 6 Normal male SD rats for acute and chronic morphine tolerance model were divided into four groups, each group had 6 rats. The rats were treated with morphine 10 mg/kg, midazolam 0.5 mg/kg, midazolam 0.5 mg/kg morphine 10 mg/kg and equal volume of normal saline, respectively. Acute morphine tolerance model was administered once every two hours, 7 times in all. And chronic morphine tolerance model was administered two times a day, 7 days in all. Every time after treatment for 15 min by tail flick method was used to detect the changes of pain threshold of rats. Midbrain tissue samples in acute and chronic morphine tolerance rats were taken for extracting total RNA after administration of eighth days in the morning. And the RT-q PCR method was used to detect the m RNA level of TSPO. The changes in protein level of TSPO, Iba1 and GFAP(glial fibrillary acidic protein) in the periaqueductal gray of each rats was detected by immunohistochemical method.Results 1 The m RNA level of TNF-α, IL-6, IL-1β in BV2 cells were significantly increased by clinical equivalent dose of morphine, oxycodone, hydromorphone, fentanyl and sufentanil(P<0.05). 2 The m RNA level of TNF-α, IL-6, IL-1β were significantly increased by 10 times of the clinical dose of dezocine(P<0.05). The opioid receptors antagonist- naloxone had no effect on the m RNA expression of TNF-α, IL-6, IL-1β(P>0.05), but it decreased the up-regulated expression induced by dezocine(P<0.05). 3 The m RNA levels of TNF-α, IL-6 and IL-1β were upregulated by morphine in a time-and dose-dependent manner. 4 The m RNA levels of TNF-α, IL-6, IL-1β and TSPO were not affected by the equivalent clinical dose of midazolam(P>0.05), but the m RNA levels of TNF-α, IL-6, IL-1β and TSPO and the protein level of Iba1 and TSPO were significantly decreased in mor+mida group comparing with mor group(P<0.05). 5 The activation of microglia mediated by morphine was not affected in TSPO knocked-down BV2 cells. But the m RNA levels of TNF-α, IL-6, IL-1β and the protein level of Iba1 were significantly decreased in mor+mida group comparing with mor group in TSPO knocked-down BV2 cells(P<0.05). 6 Withdrawal threshold measured by tail-flick baseline latencies were significantly increased in mor+mida group after the fifth injections(P<0.05) and midazolam rescued the increase of TSPO m RNA level(P<0.05). The protein levels of Iba1 and TSPO were not affected by midazolam, but midazolam rescued the increase by morphine in acute morphine tolerance rats(P<0.05). In chronic morphine tolerance rats, the withdrawal threshold measured by tail-flick baseline latencies were significantly increased in mor+mida group at days 4-7(P<0.05). And the m RNA levels of TSPO not been affected by midazolam(P>0.05), but midazolam rescued the increase of TSPO m RNA level(P<0.05). The protein levels of GFAP and TSPO were not been affected by midazolam, but midazolam rescued the increase by morphine(P<0.05).Conclusions 1 Equivalent clinical dose of opioids can induce the activation of microglia and promote the expression of inflammatory cytokines. 2 Opioids activated microglia not only mediated by μ receptors, but also mediated by κ receptors. 3 Morphine activates microglia in both time- and dose- dependent manner. 4 Midazolam has no effect on the activation of microglia, but it can inhibit the activation of microglia induced by morphine and can down-regulates TSPO expression. 5 The TSPO does not mediate the activation of microglia induced by morphine, but it is one of the factors that mediate the inhibition of midazolam on the activation of microglia induced by morphine. 6 In vivo experiments also confirmed that the combination of midazolam and morphine can inhibit the activation of microglia and astrocytes in periaqueductal gray and decrease the up-regulation expression of TSPO. |
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