| Epilepsy is a common neurological disorder caused by the abnormal firing of neurons in the brain.This abnormal electrical discharge can occur anywhere,including the cortex and hippocampus,and travel quickly to other areas of the brain,causing many forms of seizures.Autophagy is a biological process within cells that allows cells to remove damaged organelles,proteins and other cellular components through autophagy.Autophagy is a cellular self-protection mechanism that helps cells adapt to different environments and maintain intracellular balance,while also providing energy and nutrients when metabolism and nutrient supply are inadequate.In recent years,many studies have revealed the regulatory mechanisms,signaling pathways and the relationship between autophagy and diseases,providing ideas for the development of new therapies for a variety of diseases.Metformin is an oral hypoglycemic drug used primarily in the treatment of type 2 diabetes.In addition to its hypoglycemic effect,metformin also affects the autophagy process of cells.Studies have shown that metformin promotes autophagy through a variety of mechanisms.First,metformin activates AMP-activated protein kinase(AMPK),a key signaling pathway related to energy metabolism,which promotes the initiation of autophagy.Secondly,AMPK can inhibit downstream mammalian target of rapamycin(mTOR)to promote autophagy.In this study,Kainic acid(KA)-ignited amygdala epileptic rat model was used to explore the temporal variation trend of autophagy level after epileptic seizure.Secondly,the protective effect and mechanism of metformin on hippocampal neurons through autophagy after gavage of metformin were studied.This study is divided into two parts.Experiment 1:Temporal and spatial expression of autophagy associated proteins LC3B-Ⅱ/Ⅰ and Beclin-1 in epileptic rat modelsPurpose:To investigate the expression changes of autophagy related proteins LC3B-Ⅱ/Ⅰ and Beclin-1 in amygdala lit epileptic rat models at different time points.Methods:(1)The experimental rats were randomly divided into control group(Control)、3h epileptic seizure group(3h)、6h epileptic seizure group(6h)、12h epileptic seizure group(12h)、24h epileptic seizure group(24h)and 48h epileptic seizure group(48h).The behavioral changes of epileptic rats were observed after modeling.(2)HE and Nissl staining were used to observe the pathological changes of hippocampal tissue at different time points after epileptic seizure.(3)Immunohistochemistry and Western blot were used to locate and semi-quantitatively analyze the expressions of autophagy related proteins LC3B-II/I and Beclin-1 in the hippocampal tissues of rats at different time points after epileptic seizure.Results:(1)KA amygdala kindled epileptic rat model had high seizure rate and low mortality;(3)HE and Nissl staining showed that,compared with Shanm group,the number of neurons in hippocampus CA1 had no significant change at different time points,while the number of neurons in hippocampus CA3 decreased significantly with the increase of seizure time,and the number of neurons in CA3 was the least in 48h group.(3)Western blot showed that the levels of autophagy related proteins LC3B-Ⅱ/Ⅰ and Beclin-1 in the total proteins of hippocampus decreased gradually with the increase of seizure time,and reached the lowest point at 24h.The immunohistochemical results showed that,The number of positive cells expressing LC3B-II/I and Beclin-1 in CA3 hippocampal neurons decreased significantly.Conclusion:In the epileptic rat model,the autophagy level decreased with the increase of seizure time,and reached the lowest level at 24h.Experiment 2:Metformin promotes autophagy and protects hippocampal neurons in epileptic rats through AMPK/mTOR pathwayPurpose:To investigate the effect and mechanism of metformin on autophagy protection of hippocampal neurons in epileptic rats.Method:(1)Experimental rats were randomly divided into no gavage group(0d),metformin gavage group for 3 days(3d)、metformin gavage group for 5 days(5d)、metformin gavage group for 7 days(7d)and metformin gavage group for 14 days(14d).After gavage of metformin,amygdala KA model was established.The behavioral manifestations of epileptic rats were observed after modeling,and the changes of blood glucose before and after gavage were recorded by convenient glucose meter.(2)Pathological changes of hippocampal tissue in epileptic rats were observed by HE and Nissl staining.(3)Immunohistochemistry and Western blot were used to locate and semi-quantitatively analyze the AMPK/mTOR pathway and the expression of autophagy related proteins LC3B-Ⅱ/Ⅰ and Beclin-1.Results:(1)After metformin administration,the blood sugar of rats decreased significantly;(2)HE,Nissl staining showed that,compared with the 0d group,the number of neurons in the hippocampal CA1 region did not change significantly in epileptic rats given metformin,the damage of neurons in the CA3 region was significantly reduced with the extension of gavage time,and the number of dead neurons was significantly reduced with the extension of gavage time.(3)Western blot showed that the level of autophagy related proteins in the total proteins of hippocampal tissue increased gradually with the increase of the time of gavage,and the expression of AMPK/mTOR pathway protein increased.Immunohistochemical results showed that the number of positive cells expressing LC3B-Ⅱ/Ⅰand Beclin-1 in hippocampal neurons in CA3 region increased significantly.Conclusion:Metformin promotes autophagy and protects hippocampal neurons in epileptic rats through AMPK/mTOR pathway. |
