异丙酚通过PKC途径抑制氯化铵处理的大鼠脑星形胶质细胞AQP-4表达上调
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摘要
目的:
肝性脑病系严重的肝功能失调或障碍所致、以代谢紊乱为主要特征的中枢神经系统功能失调综合症。除肝移植之外,目前尚无有效的治疗手段。脑水肿是肝性脑病的一个重要病理变化,其中以星形胶质细胞水肿最为明显。氨中毒仍处于HE发病机制的中心地位,但其导致星形胶质细胞水肿的机制尚未完全清楚,有研究显示水通道蛋白-4参与了氨导致的星形胶质细胞水肿。
研究表明异丙酚通过活化PKC、磷酸化底物蛋白对缺血再灌注损伤引起的心肌、神经元损伤及脑水肿具有保护作用。为探讨异丙酚对肝性脑病脑水肿的保护作用,本实验拟通过氯化铵处理原代培养的星形胶质细胞建立肝性脑病的离体模型,检测不同时间点AQP-4的表达,观察异丙酚对氯化铵处理的星形胶质形态、细胞活性及AQP-4表达的影响,探讨异丙酚对肝性脑病脑水肿的保护作用及可能的机制。
方法:
取出生2-3天的Sprague-Dawley大鼠脑皮质分离培养星形胶质细胞。将原代培养的星形胶质细胞随机分成6组,分别为对照组(正常组),氯化铵处理6h、12h、24h、48h组(NH_4Cl组),0.1%二甲基亚砜预处理组(DMSO组),PKC激动剂TPA预处理组(TPA组),异丙酚预处理组(Pro组)及异丙酚与PKC拮抗剂Ro31-8220共同预处理组(PR组)。免疫荧光法鉴定星形胶质细胞的纯度,Western-blot法检测各组星形胶质细胞AQP-4的表达情况,MTT法测定细胞活性,光学显微镜观察星形胶质细胞形态学变化。
结果:
1)与对照组相比,氯化铵处理12 h后星形胶质细胞AQP-4的表达明显增加(P<0.05),在24 h达到高峰,到48 h开始下降。2)氯化铵处理的星形胶质细胞活性较对照组明显下降(P<0.05)。3)与NH_4Cl组相比,异丙酚明显降低氯化铵处理后星形胶质细胞AQP-4的表达上调(P<0.05),减轻细胞水肿,抑制细胞活性的下降(P<0.05)。4)氯化铵处理的星形胶质细经PKC激动剂TPA预处理后,与NH_4CL组相比,AQP-4表达明显下调(P<0.05),细胞活性提高(P<0.05),细胞水肿减轻。5)氯化铵处理的星形胶质细胞经PKC拮抗剂Ro31-8220与异丙酚共同预处理后,AQP-4表达呈可逆性上调,与Pro组相比,差异具有统计学意义(P<0.05),细胞水肿加重。
结论:
氯化铵处理后星形胶质细胞AQP-4的表达上调,细胞活性下降,细胞水肿,而异丙酚预处理抑制氯化铵处理的星形胶质细胞AQP-4的上调,减轻细胞水肿。异丙酚的此作用部分是通过激活PKC途径实现的。
Objective:
Hepatic encephalopathy(HE) is a neurological disorder that occurs in patients with severe liver failure.The only effective treatment for this condition is an emergency liver transplantation.Brain edema is the principal neurological complication in patients with the acute form of HE.Astroglial swelling is generally believed to represent a major component of the brain edema in HE,ammonia has been strongly implicated as a major toxin in liver failure.While the precise mechanism by which ammonia brings about such swelling remains poorly understood,recent studies have invoked aquaporin-4 (AQP-4) in the mechanism of ammonia-induced astrocyte swelling.
Previous studies have suggested that propofol may be implicated in brain edema and neuroprotective activities through protein kinase C pathway.The purpose of this study was to examine the effects of propofol on edema and assess neuroprotection in an ammonia-treated astrocytes model of HE,We assessed propofol effects on AQP-4 expression and activation of the protein kinase C pathway.
Method:
Astrocytes isolated and purified from Sprague Dawley rats(1～2 days) were cultured for 3 weeks.The cells were randomly divided into six groups:the normal group(Group C) cultured with normal medium;Incubated in culture medium with 5mmol/L ammonia for 6h、12h、24h、48h groups(Group NH_4Cl);Pretreated with 0.1% dimethyl sulfoxide group(Group DMSO);Pretreated with 12-o-tetradecanoylphorbol 13-acetate(TPA) group(Group TPA);Pertreated with propofol group(Group Pro); Pretreated with propofol and Ro31-8220 group(Group PR).Cell morphology was assessed by light microscopy.Cell viability was assessed by measuring MTT absorbency(A value) and AQP-4 expression was evaluated with Western blot analysis.
Results:
1) The expression of AQP-4 was first observed increase significantly at 12h,the concentration reached to peak at 24 h,remained elevated at 48 h,compared with the control group,they increase significantly respectively(P＜0.05).2) In Group NH_4Cl and Group DMSO,the cell viability of astrocytes decrease significantly respectively (P＜0.05 and P＜0.05) compared to Group C.3) Propofol increases cell viability and attenuates aquaporin-4 over-expression of astrocytes which treated with ammonia significantly(P＜0.05 and P＜0.05),it can also decreases the degree of astrocytes swelling.4)Pertreated with 12-o-tetradecanoylphorbol 13-acetate,a PKC activator,the expression of AQP-4 of astrocytes treated with ammonia decreases significantly (P＜0.05),and the cell viability of astrocytes decrease significantly also(P＜0.05).5) Pretreated with propofol and Ro31-8220 can increases the expression of AQP-4 significantly(P＜0.05) compared to Group Pro,but there is no differences between Group NH_4Cl and Group PR(P＞0.05).
Conclusion:
1) The expression of AQP-4 is up-regulated in ammonia-treated asrtocytes,the cell viability decrease significantly,and astrocytes swelling notely.2) Propofol applied before ammonia provided neuroprotective effects and down-regulated AQP-4 expression in the HE model of cultured rat astrocytes,and protein kinase C pathway participant in this progress.
肝性脑病系严重的肝功能失调或障碍所致、以代谢紊乱为主要特征的中枢神经系统功能失调综合症。除肝移植之外,目前尚无有效的治疗手段。脑水肿是肝性脑病的一个重要病理变化,其中以星形胶质细胞水肿最为明显。氨中毒仍处于HE发病机制的中心地位,但其导致星形胶质细胞水肿的机制尚未完全清楚,有研究显示水通道蛋白-4参与了氨导致的星形胶质细胞水肿。
研究表明异丙酚通过活化PKC、磷酸化底物蛋白对缺血再灌注损伤引起的心肌、神经元损伤及脑水肿具有保护作用。为探讨异丙酚对肝性脑病脑水肿的保护作用,本实验拟通过氯化铵处理原代培养的星形胶质细胞建立肝性脑病的离体模型,检测不同时间点AQP-4的表达,观察异丙酚对氯化铵处理的星形胶质形态、细胞活性及AQP-4表达的影响,探讨异丙酚对肝性脑病脑水肿的保护作用及可能的机制。
方法:
取出生2-3天的Sprague-Dawley大鼠脑皮质分离培养星形胶质细胞。将原代培养的星形胶质细胞随机分成6组,分别为对照组(正常组),氯化铵处理6h、12h、24h、48h组(NH_4Cl组),0.1%二甲基亚砜预处理组(DMSO组),PKC激动剂TPA预处理组(TPA组),异丙酚预处理组(Pro组)及异丙酚与PKC拮抗剂Ro31-8220共同预处理组(PR组)。免疫荧光法鉴定星形胶质细胞的纯度,Western-blot法检测各组星形胶质细胞AQP-4的表达情况,MTT法测定细胞活性,光学显微镜观察星形胶质细胞形态学变化。
结果:
1)与对照组相比,氯化铵处理12 h后星形胶质细胞AQP-4的表达明显增加(P<0.05),在24 h达到高峰,到48 h开始下降。2)氯化铵处理的星形胶质细胞活性较对照组明显下降(P<0.05)。3)与NH_4Cl组相比,异丙酚明显降低氯化铵处理后星形胶质细胞AQP-4的表达上调(P<0.05),减轻细胞水肿,抑制细胞活性的下降(P<0.05)。4)氯化铵处理的星形胶质细经PKC激动剂TPA预处理后,与NH_4CL组相比,AQP-4表达明显下调(P<0.05),细胞活性提高(P<0.05),细胞水肿减轻。5)氯化铵处理的星形胶质细胞经PKC拮抗剂Ro31-8220与异丙酚共同预处理后,AQP-4表达呈可逆性上调,与Pro组相比,差异具有统计学意义(P<0.05),细胞水肿加重。
结论:
氯化铵处理后星形胶质细胞AQP-4的表达上调,细胞活性下降,细胞水肿,而异丙酚预处理抑制氯化铵处理的星形胶质细胞AQP-4的上调,减轻细胞水肿。异丙酚的此作用部分是通过激活PKC途径实现的。
Objective:
Hepatic encephalopathy(HE) is a neurological disorder that occurs in patients with severe liver failure.The only effective treatment for this condition is an emergency liver transplantation.Brain edema is the principal neurological complication in patients with the acute form of HE.Astroglial swelling is generally believed to represent a major component of the brain edema in HE,ammonia has been strongly implicated as a major toxin in liver failure.While the precise mechanism by which ammonia brings about such swelling remains poorly understood,recent studies have invoked aquaporin-4 (AQP-4) in the mechanism of ammonia-induced astrocyte swelling.
Previous studies have suggested that propofol may be implicated in brain edema and neuroprotective activities through protein kinase C pathway.The purpose of this study was to examine the effects of propofol on edema and assess neuroprotection in an ammonia-treated astrocytes model of HE,We assessed propofol effects on AQP-4 expression and activation of the protein kinase C pathway.
Method:
Astrocytes isolated and purified from Sprague Dawley rats(1～2 days) were cultured for 3 weeks.The cells were randomly divided into six groups:the normal group(Group C) cultured with normal medium;Incubated in culture medium with 5mmol/L ammonia for 6h、12h、24h、48h groups(Group NH_4Cl);Pretreated with 0.1% dimethyl sulfoxide group(Group DMSO);Pretreated with 12-o-tetradecanoylphorbol 13-acetate(TPA) group(Group TPA);Pertreated with propofol group(Group Pro); Pretreated with propofol and Ro31-8220 group(Group PR).Cell morphology was assessed by light microscopy.Cell viability was assessed by measuring MTT absorbency(A value) and AQP-4 expression was evaluated with Western blot analysis.
Results:
1) The expression of AQP-4 was first observed increase significantly at 12h,the concentration reached to peak at 24 h,remained elevated at 48 h,compared with the control group,they increase significantly respectively(P＜0.05).2) In Group NH_4Cl and Group DMSO,the cell viability of astrocytes decrease significantly respectively (P＜0.05 and P＜0.05) compared to Group C.3) Propofol increases cell viability and attenuates aquaporin-4 over-expression of astrocytes which treated with ammonia significantly(P＜0.05 and P＜0.05),it can also decreases the degree of astrocytes swelling.4)Pertreated with 12-o-tetradecanoylphorbol 13-acetate,a PKC activator,the expression of AQP-4 of astrocytes treated with ammonia decreases significantly (P＜0.05),and the cell viability of astrocytes decrease significantly also(P＜0.05).5) Pretreated with propofol and Ro31-8220 can increases the expression of AQP-4 significantly(P＜0.05) compared to Group Pro,but there is no differences between Group NH_4Cl and Group PR(P＞0.05).
Conclusion:
1) The expression of AQP-4 is up-regulated in ammonia-treated asrtocytes,the cell viability decrease significantly,and astrocytes swelling notely.2) Propofol applied before ammonia provided neuroprotective effects and down-regulated AQP-4 expression in the HE model of cultured rat astrocytes,and protein kinase C pathway participant in this progress.
引文
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