生酮饮食对癫痫大鼠海马的保护作用及机制研究

Ketogenic diet attenuates hippocampus injury in pentylenetetrazol-kindled seizure and its relation to mammalian target of rapamycin pathway suppression in rats

目的探讨生酮饮食(KD)对戊四氮(PTZ)点燃大鼠海马损伤及哺乳动物雷帕霉素钯蛋白(mTOR)通路表达的作用机制。方法将55只雄性SD大鼠隔日腹腔注射PTZ制作癫痫动物模型直至完全点燃,另5只大鼠(空白对照组)腹腔注射0.9%氯化钠溶液。完全点燃后的44只大鼠随机分成4组,即正常食物(ND)+PTZ组、ND+PTZ+雷帕霉素(RAP)组、KD+PTZ组、KD+PTZ+RAP组,每组11只,均予侧脑室置管并再次给予隔日腹腔注射PTZ,共5次;其中ND+PTZ+RAP组、KD+PTZ+RAP组给药前30min侧脑室注射RAP,ND+PTZ组、KD+PTZ组予以等量人工脑脊液。观察并比较5组大鼠癫痫发作等级,海马区神经元密度,海马区p S6、S6、pAkt、Akt、pAMPK、AMPK的表达(采用Western blot法检测)。结果 KD+PTZ组大鼠癫痫发作等级明显低于ND+PTZ组(P<0.01);KD+PTZ+RAP组发作等级明显低于ND+PTZ组及ND+PTZ+RAP组(均P<0.05)。海马CA1区神经元密度:KD+PTZ组明显大于ND+PTZ组(P<0.05),KD+PTZ+RAP组大于ND+PTZ+RAP组(P<0.05),ND+PTZ+RAP组大于ND+PTZ组(P<0.05)。与空白对照组相比,ND+PTZ组p S6/S6、pAkt/Akt蛋白表达增加(均P<0.05),其余4组pAMPK/AMPK蛋白表达均增高(均P<0.05);与ND+PTZ组相比,KD+PTZ组p S6/S6、pAkt/Ak蛋白表达均降低(均P<0.05),pAMPK/AMPK蛋白表达增高(P<0.05);与ND+PTZ组相比,ND+PTZ+RAP组p S6/S6、pAkt/Akt蛋白表达均明显降低(均P<0.05);与KD+PTZ组相比,KD+PTZ+RAP组p S6/S6、pAkt/Akt蛋白表达明显降低(均P<0.05);与ND+PTZ+RAP组相比,KD+PTZ+RAP组p S6/S6蛋白表达明显下降(P<0.05),pAMPK/AMPK增高(P<0.05)。结论 KD具有抗癫痫发作、减少神经元丢失的作用,这种保护作用可能与其对m TOR通路的抑制有关,且其上游可能通过抑制Akt通路的磷酸化、激活AMPK通路的磷酸化来调节m TOR通路。

Objective To investigate the effect of ketogenic diet on hippocampus injury in pentylenetetrazol-kindled seizure and its relation to mammalian target of rapamycine（mTOR） pathway in rats. Methods The PTZ-kindled seizure model was induced by intra-ventricular infusion of 35mg/kg pentylenetetrazol（PTZ） in male Sprague-Dawley（SD） rats. Forty four rats, in which the seizure was fully kindled, were divided into 4 groups with 11 rats in each group. Rats in ND＋PTZ and ND＋PTZ＋RAP groups were fed with normal diet, while those in KD＋PTZ and KD＋PTZ＋RAP were fed with ketogenic diet. The m TOR pathway inhibitor rapamycin（RAP） was given to rats in ND＋PTZ ＋RAP and KD ＋PTZ ＋RAP groups by intra-ventricular infusion 30 min before PTZ injection. The seizure stages and the neuron in hippocampal CA1, CA3 and DG regions were counted. And the expressions of p S6, S6, pAkt, Akt, pAMPK and AMPK were detected by Western blot. Results The seizure stage of the kindled rats in the KD＋PTZ group was lower than that of ND＋PTZ group significantly（P〈0.05）. The stages of KD＋PTZ＋RAP group were lower than those of ND＋PTZ and ND＋PTZ＋RAP groups significantly（P〈0.05）. The density of neuron in hippocampal CA1 subarea in KD＋PTZ group was higher than ND＋PTZ group significantly（P〈0.05）, and the density of KD＋PTZ＋RAP was higher than ND＋PTZ＋RAP group（P〈0.05）. Additionally, the density of ND＋PTZ＋RAP was higher than ND＋PTZ（P〈0.05）. Western blot result shows that the expression of phosphor-S6 to total S6（p S6/S6） and phosphor-Akt to total Akt（pAkt/Akt） was increased in ND＋PTZ group compared to control（P〈0.05）. The expression levels of phosphor-AMPK to total AMPK（pAMPK/AMPK） in all kindled groups were higher than those of non-kindled control groups（P〈0.05）. Compared to the ND＋PTZ group, the expression levels of p S6/S6 and pAkt/Akt were decreased（P〈0.05）, while the pAMPK/AMPK was increased in KD＋PTZ group（P〈0.05）. Compared to the KD＋PTZ group, the expression of p S6/S6 and pAkt/Akt in KD＋PTZ＋RAP group were decreased（P〈0.05）. Similarly, compared to the ND＋PTZ group, the expression of p S6/S6 and pAkt/Akt in ND＋PTZ＋RAP group was also significantly decreased（P〈0.05）. Moreover, compared to ND＋PTZ＋RAP, the expression level of p S6/S6 in KD＋PTZ＋RAP group was decreased significantly（P〈0.05）, while the expression level of pAMPK/AMPK was increased（P〈0.05）. Conclusion Our study indicated that ketogenic diet has the anti-seizure and neuronal protective effect in PTZ kindled rat model,which may be related to the mTOR pathway suppression. Moreover, the m TOR pathway is modulated by both the inhibition of Akt and activation of AMPK pathway.