静脉注入胆红素对新生大鼠脾MyD88、磷酸化p38MAPK蛋白表达及凋亡的影响

发布时间：2018-09-10 09:34

【摘要】：目的：建立新生大鼠高胆红素血症模型，探讨静脉注入胆红素对脾髓样分化因子88（Myeloid differentiation factor88，MyD88）和磷酸化p38MAPK（phospho-p38MAP Kinase，p-p38MAPK）蛋白表达及凋亡的影响。方法：⒈实验分组：选用144只清洁级7～8d新生SD大鼠，雌雄不限，体重12.0～15.0g，随机分为空白对照组（Ⅰ）、脂多糖对照组（LPS，Ⅱ）、15mg/kg胆红素对照组（Ⅲ）、15mg/kg胆红素+LPS组（Ⅳa）、30mg/kg胆红素+LPS组（Ⅳb）和50mg/kg胆红素+LPS组（Ⅳc），共6组，每组24只，每组又分为2h、5h、24h三个时间点，每个时间点8只。⒉建立新生SD大鼠高胆红素血症模型：⑴乙醚麻醉新生SD大鼠，酒精消毒颈部，暴露一侧颈静脉，给予不同剂量胆红素（15mg/kg、30mg/kg和50mg/kg）颈静脉注入；空白对照组和LPS对照组给予生理盐水0.1ml颈静脉注入；缝合颈部切口；⑵在颈静脉注入1h后，空白对照组和15mg/kg胆红素对照组给予生理盐水0.05ml腹腔注入，不注射LPS；其余各组1h后给予LPS1mg/kg腹腔注入；⑶分别于颈静脉注入后2h、5h、24h采血，测血浆胆红素浓度；⑷处死动物，4％缓冲甲醛心脏灌注做内固定后快速取脾脏，固定、脱水、透明、包埋制成石蜡标本，采用免疫组织化学法检测脾MyD88和磷酸化p38MAPK蛋白的表达，TUNEL染色原位法检测脾细胞凋亡情况。结果：⒈新生SD大鼠在注入胆红素5～10min后皮肤出现不同程度的黄染，呈金黄色，1h后皮肤黄染减退；在注入1h时，15mg/kg、30mg/kg和50mg/kg胆红素组血浆总胆红素浓度95％的置信区间分别为：（106.31，123.49）μmol/L、（196.58，238.90）μmol/L和（325.15，349.07）μmol/L，且胆红素注入剂量与血浆总胆红素浓度呈正相关（rs=0.9452，P＜0.01）。⒉胆红素对脾MyD88蛋白表达的影响：⑴低浓度范围（106.31，123.49μmol/L）的胆红素和LPS单独作用可以促进脾细胞MyD88的表达（P＜0.01），但胆红素弱于LPS的促进作用（P＜0.01）；5h即可观察到胆红素的这种促进作用，24h作用消失；⑵低浓度范围的胆红素对LPS刺激MyD88表达的影响不明显（P＞0.05）；⑶中、高浓度范围[（196.58，238.90）和（325.15，349.07）μmol/L]的胆红素对LPS刺激MyD88表达有抑制作用（P＜0.01），，且随着浓度的升高，抑制作用增强；2h即可观察到这种抑制作用，5h作用明显，24h时中等浓度范围的胆红素的抑制作用消失，高浓度范围的抑制作用仍然存在（P＜0.01）。⒊胆红素对脾p-p38MAPK蛋白表达的影响：⑴低浓度范围的胆红素和LPS单独作用可促进脾细胞p38MAPK的磷酸化（P＜0.01），但弱于LPS的促进作用（P＜0.01）；2h即可观察到胆红素的这种促进作用，5h作用增强，24h作用仍存在；⑵中、低浓度范围的胆红素对LPS刺激p-p38MAPK表达有抑制作用（P＜0.01），且浓度越高，抑制作用越明显；2h即可观察到这种抑制作用，5h作用增强，24h作用消失；⑶高浓度范围的胆红素对LPS刺激p-p38MAPK表达有促进作用（P＜0.01）；5h时即可观察到这种促进作用，24h作用减弱。⒋MyD88和p-p38MAPK与胆红素浓度的相关分析结果：⑴2h、5h和24h的MyD88IOD（SUM）值与胆红素浓度呈负相关（r分别为：－0.856、－0.791和－0.875，P＜0.01），即随着胆红素浓度的升高，抑制脾细胞表达MyD88的作用越明显；⑵2h的p-p38MAPK IOD（SUM）值与胆红素浓度无相关关系（r=－0.123，P＞0.05）；5h的p-p38MAPK IOD（SUM）值与胆红素浓度呈正相关（r=0.897，P＜0.01）；24h的p-p38MAPK IOD（SUM）值与胆红素浓度呈正相关（r=0.827，P＜0.01）。表明在5h和24h时，随着胆红素浓度的升高，p-p38MAPK的表达有增加的趋势。⒌胆红素对脾细胞凋亡的影响：⑴低浓度范围的胆红素组凋亡率增加（P＜0.01）；⑵中、低浓度范围的胆红素与LPS共同作用可减少脾细胞凋亡（P＜0.01）；高浓度范围的胆红素与LPS共同作用可使细胞凋亡增多（P＜0.01）。结论：⒈低浓度胆红素对LPS刺激MyD88表达的影响不明显；中、高浓度的胆红素对MyD88表达有抑制作用，这种抑制作用呈浓度依赖性，随着浓度的升高，抑制作用增强，持续时间延长。⒉中、低浓度胆红素可抑制p38MAPK的磷酸化，高浓度则促进p38MAPK的磷酸化，且随着浓度的升高，作用时间延长。⒊高浓度胆红素对脾细胞有毒性作用，细胞凋亡增多。提示胆红素对免疫细胞的影响可能与调节TLRs信号通路中MyD88的表达和p38MAPK的磷酸化及诱导细胞的凋亡有关。
[Abstract]:AIM: To establish a hyperbilirubinemia model of neonatal rats and investigate the effects of intravenous bilirubin on the expression of myeloid differentiation factor 88 (MyD88) and phospho-p38MAP Kinase (p-p38MAPK) protein and apoptosis of spleen in neonatal rats. They were randomly divided into blank control group (I), lipopolysaccharide control group (LPS, II), 15 mg / kg bilirubin control group (III), 15 mg / kg bilirubin + LPS group (IVa), 30 mg / kg bilirubin + LPS group (IVb) and 50 mg / kg bilirubin + LPS group (IVc), totally 6 groups, 24 rats in each group. Each group was divided into 2 hours, 5 hours, 24 hours three time points, 8 rats at each time point. Establishment of hyperbilirubinemia model in neonatal SD rats: _Ether anesthesia neonatal SD rats, alcohol disinfection of the neck, exposure of one side of the jugular vein, given different doses of bilirubin (15mg/kg, 30mg/kg and 50mg/kg) jugular vein injection; blank control group and LPS control group given 0.1ml normal saline jugular vein injection; suture the neck incision; _in jugular vein One hour after injection, the blank control group and 15 mg/kg bilirubin control group were injected with 0.05 ml normal saline intraperitoneally, without LPS injection; the other groups were injected with LPS 1mg/kg intraperitoneally after 1 hour; _Blood was collected at 2 hours, 5 hours and 24 hours after jugular vein injection, and plasma bilirubin concentration was measured; _Animals were sacrificed and spleen was quickly taken after 4% buffered formaldehyde cardiac perfusion and internal fixation. The expression of MyD88 and phosphorylated p38 MAPK protein in spleen was detected by immunohistochemistry, and the apoptosis of spleen cells was detected by TUNEL staining in situ. The confidence intervals of 95% of plasma total bilirubin concentration in 15 mg/kg, 30 mg/kg and 50 mg/kg bilirubin groups were (106.31, 123.49) micromol/L, (196.58, 238.90) micromol/L and (325.15, 349.07) micromol/L, respectively, and the dose of bilirubin was positively correlated with plasma total bilirubin concentration (rs = 0.9452, P < 0.01). The effect of low concentration of bilirubin and LPS on myD88 expression in spleen cells (P < 0.01), but the effect of bilirubin was weaker than that of LPS (P < 0.01); the effect of bilirubin on myD88 expression was observed in 5 hours, and disappeared in 24 hours; and the effect of low concentration of bilirubin on LPS-stimulated myD88 expression in spleen cells (P < 0.01). _Bilirubin in high concentration range [(196.58,238.90) and (325.15,349.07) micromol/L] inhibited the expression of MyD88 stimulated by LPS (P _The effect of bilirubin on the expression of p-p38 MAPK protein in spleen: _Bilirubin and LPS at low concentration alone could promote the phosphorylation of p38 MAPK in spleen cells (P < 0.01), but was weaker than that of LPS (P < 0.01); _Bilirubin at low concentration inhibited the expression of p-p38 MAPK stimulated by LPS (P < 0.01), and the higher the concentration was, the more obvious the inhibition was; the inhibition was observed at 2 h, the effect was enhanced at 5 h, and disappeared at 24 h; _Bilirubin at high concentration range inhibited the expression of p-p38 MAPK stimulated by LPS. _MyD88 and p-p38 MAPK were negatively correlated with bilirubin concentration (r: - 0.856, - 0.791, and - 0.875, respectively) at 2h, 5h and 24h (P < 0.01). The expression of MyD88 in spleen cells was more obvious; _2 h p-p38 MAPK IOD (SUM) value had no correlation with bilirubin concentration (r = - 0.123, P > 0.05); 5 h p-p38 MAPK IOD (SUM) value was positively correlated with bilirubin concentration (r = 0.897, P < 0.01); 24 h p-p38 MAPK IOD (SUM) value was positively correlated with bilirubin concentration (r = 0.827, P < 0.01). With the increase of bilirubin concentration, the expression of p-p38 MAPK increased. _The effect of bilirubin on apoptosis of spleen cells: _Apoptosis rate of low concentration of bilirubin group increased (P < 0.01); _In low concentration of bilirubin combined with LPS can reduce the apoptosis of spleen cells (P < 0.01); in high concentration range of bilirubin and LPS can reduce the apoptosis of spleen cells (P < 0.01); CONCLUSION: Low concentration of bilirubin has no obvious effect on LPS-induced MyD88 expression, and high concentration of bilirubin inhibits MyD88 expression in a concentration-dependent manner. With the increase of concentration, the inhibitory effect increases and the duration prolongs. The phosphorylation of p38 MAPK was inhibited, and the phosphorylation of p38 MAPK was promoted at high concentration, and the action time was prolonged with the increase of concentration. _The toxic effect of high concentration bilirubin on spleen cells and the increase of apoptosis were observed. Apoptosis is related to cell apoptosis.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.17

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