| Subarachnoid hemorrhage(SAH)is a relatively common hemorrhagic stroke disorder,and SAH is caused by aneurysm rupture in most cases.Although SAH accounts for only about 5%of the total number of stroke cases,it has a significant impact on individuals and society because of its high mortality and disability rates.With the development of medical technology,surgical clamping or embolization of aneurysms can effectively reduce mortality and improve patient prognosis.However,surgical treatment can only reduce their risk of rebleeding,and the damage to the brain from SAH remains.The main cause of prognosis was thought to be delayed cerebral vasospasm,yet treatment of cerebral vasospasm has not improved the prognosis of patients with SAH.This suggests that other etiologies influence the prognosis of SAH.Early brain injury(EBI),which refers to craniofacial injury from the onset of SAH to 72 hours,has received increasing attention from national and international experts.However,there are no drugs for early brain injury treatment.Therefore,exploring effective therapeutic drugs is the main task to solve the problem in this field.The Chinese medicine Fangqi has the effect of pungent to dispel and bitter to cool and drain.Its active ingredient,Tetrandrine,has been used in clinical practice.Some studies have pointed out the neuroprotective effect of powdered Tetrandrine,but the specific mechanism has not been fully elucidated.Autophagy is the process of eukaryotic cell degradation and recycling of its damaged proteins and organelles.It plays a crucial role in maintaining cellular homeostasis and energy metabolism.Autophagy is regulated by several signaling pathways,among which the AMPK/mOTR signaling pathway plays an important role in the regulation of autophagy.Does it exert a neuroprotective effect on EBI by affecting the level of autophagy?It has not been reported yet.SIRT3 is a member of the Sirtuins family.SIRT3 is abundantly expressed in brain tissues and mainly distributed in mitochondria.SIRT3 is involved in the regulation of cellular oxidative stress and energy metabolism.SIRT3 upregulation plays a role in reducing damage and improving prognosis in various diseases.SIRT3 upregulation has been a hot topic of research in reducing oxidative stress,ischemic injury,and inflammatory response.With the in-depth study of SIRT3,it was found that SIRT3 can regulate autophagy through AMPK/mOTR signaling pathway.However,in SAH,it is not yet known whethertetrandrine s can regulate AMPK/mOTR signaling pathway and thus affect autophagy through SIRT3.Therefore,this experiment was performed to simulate the clinical pathophysiology of EBI after SAH by intravascular puncture.To observe whether Tetrandrine can mediate autophagy through SIRT3/AMPK/mTOR signaling pathway to alleviate EBI after SAH.Part I.Tetrandrine ameliorate the neurological damage of EBI after SAH in rat brain by regulating autophagy.Objective:To observe the effect oftetrandrine s on autophagy after SAH through an animal model of SAH rats,and to investigate whethertetrandrine s could ameliorate the neurological injury of rat brain by EBI after SAH through regulating autophagy.Methods:Twenty-four 300-330 g male SD rats were randomly assigned into three groups:sham-operated group,SAH group,and tetrandrine group.All three groups used intravascular puncture to create the SAH model,except for the surgical group,and the other two groups punctured the blood vessel when they felt the vascular resistance.The surgical group withdrew the thread plugs when the vascular resistance was felt to avoid puncturing the vessels.The tetrandrine s group was injected intraperitoneally with tetrandrine(20 mg/kg)immediately after the model was made.The homogeneous stability of the SAH model was assessed by the SAH bleeding volume score.The degree of neurological damage was observed by the neurological function score and the determination of brain water content.TUNEL staining was used to observe the apoptosis in the cerebral cortical area of each group of rats.The cell morphology and the number of neuronal cells in each group of rats were observed by using NIR staining.The expression levels of LC3 and p62 protein were detected by Western Blot.Results:1 SAH hemorrhage score:the subarachnoid hemorrhage score in the SAH group and tetrandrine group was significantly higher than that in the sham-operated group(P<0.01).And there was no statistical difference between the SAH and tetrandrine groups.2 Brain water content and neurological deficit scores:The neurological deficit scores were significantly lower in the SAH compared with the sham-operated group(P<0.01),and the brain water content was significantly higher(P<0.01).At the same time,the neurological deficit score increased and brain water content decreased in the tetrandrine group compared with the SAH group(P<0.01).3 Apoptosis:TUNEL staining results showed that the apoptosis rate in the cerebral cortical area was significantly higher in the SAH group compared with the sham-operated group(P<0.01);the apoptosis rate in the cerebral cortical area was significantly lower in the tetrandrine group compared with the SAH group(P<0.01).4 Cell morphology observation:the results of nisin staining showed that the neuronal morphology of the sham-operated group was normal and the nisin bodies were abundant.the SAH group was severely damaged compared with the sham-operated group and accompanied by a large number of neuronal loss,and the difference was statistically significant(P<0.01).However,the injury was significantly relieved in tetrandrine group compared with the SAH group,and the number of neurons was increased(P<0.01).5 P62 and LC3Ⅱ/LC3Ⅰ protein expression:compared with the sham-operated group,LC3Ⅱ/LC3Ⅰ was significantly higher in the SAH group(P<0.01),and the expression level of P62 was significantly lower(P<0.01).compared with the SAH group,LC3Ⅱ/LC3Ⅰ was significantly lower(P<0.01)and P62 expression was significantly higher in the tetrandrine group(P<0.01).Summary:The neuroprotective effect of Tetrandrine on EBI after SAH was reflected by:Tetrandrine improved the SAH-mediated neurological impairment and neuronal morphology,and reduced brain water content,apoptosis rate of neuronal cells and loss of neuronal cells.It also reduced the LC3Ⅱ/LC3Ⅰ ratio and increased the expression of P62 protein,suggesting that Tetrandrine can reduce EBI after SAH by inhibiting autophagy.Part II:Mechanisms by which Tetrandrine affect autophagy in EBI through SIRT3/AMPK/mTOR signaling pathwayObjective:To observe the effect of Tetrandrine on SIRT3/AMPK/mTOR signaling pathway through SAH model,and to investigate whether Tetrandrine can regulate autophagy through SIRT3/AMPK/mTOR signaling pathway to exert neuroprotective effects on EBI after SAH.Methods:Forty-eight male SD rats were randomly assigned into sham-operated group,SAH group,Tetrandrine group,and Tetrandrine+3-TYP group.The rats in the Tetrandrine+3-TYP group were pretreated with 3-TYP(30 mg/kg)given to the rats every two days for three times before the SAH surgery.Immediately after the surgery,rats in the Tetrandrine group and the Tetrandrine+3-TYP group were injected with Tetrandrine solution(20 mg/kg)intraperitoneally.The homogeneous stability of the SAH model was assessed by the SAH hemorrhage score.The degree of neurological damage was observed by the neurological function score and the determination of brain water content.HE staining was used to observe the morphology of neurons in the cortical area of the brain in each group of rats.Immunofluorescence and Western Blot were used to detect the protein expression level of SIRT3 in each group.The protein expression levels of LC3,p-AMPK,AMPK,p-mTOR and mTOR in each group were detected using Western Blot.Results:1 SAH bleeding score:There was a statistically significant difference in the scores between the sham-operated group and the SAH group,the tetrandrine group and the tetrandrine+3-TYP group(P<0.01).And there was no statistically significant difference in SAH scores between the SAH group,the tetrandrine group and the tetrandrine+3-TYP group(P>0.05).2 Comparison of the results of brain water content and neurological deficit scores in rats in each group:Compared with the sham-operated group,the brain water content of rats in the SAH group increased significantly and the neurological deficit score decreased significantly(P<0.01).However,compared with the SAH group,the brain water content of the tetrandrine group significantly decreased and the neurological deficit score significantly increased(P<0.01),and compared with the tetrandrine group,the brain water content of the tetrandrine+3-TYP group significantly increased and the neurological deficit score significantly decreased(P<0.05).3 Comparison of neural cell morphology among groups:HE staining results showed that the morphology of neurons in the cerebral cortex of the rats in the sham-operated group was normal;a large number of neurons in the cerebral cortex of the rats in the SAH group showed nuclear consolidation and deep staining;the above-mentioned situation in the brain tissue of the rats in the tetrandrine group was significantly alleviated compared with that in the SAH group.The neuronal damage in the group of tetrandrine+3-TYP was aggravated compared with the group of tetrandrine.4 The expression of SIRT3 in the brain tissue of rats in each group:The immunofluorescence results showed that the fluorescence intensity of SIRT3 was significantly reduced in the brain cortical area of rats in the SAH group compared with the sham-operated group(P<0.01).Compared with the SAH group,the fluorescence intensity was significantly increased in tetrandrine group(P<0.01).The fluorescence intensity was significantly decreased in tetrandrine+3-TYP group compared with the tetrandrine group(P<0.01).5 SIRT3 protein expression:Western blot results showed that SIRT3 protein expression was reduced in the SAH group compared with the sham-operated group(P<0.05).SIRT3 protein expression was upregulated in tetrandrine group compared with SAH(P<0.05).Compared with the tetrandrine group,the SIRT3 protein expression was decreased in the SAH+3-TYP group(P<0.05).6 Protein expression of LC3Ⅱ/LC3Ⅰ in rat brain tissue of each group:Western blot results showed that the ratio of LC3Ⅱ/LC3Ⅰ in SAH group was significantly increased compared with Sham(P<0.01).Compared with SAH,the ratio of LC3Ⅱ/LC3Ⅰ was significantly decreased in tetrandrine group(P<0.01).The ratio of LC3Ⅱ/LC3Ⅰ was significantly increased in the tetrandrine+3-TYP group compared with the tetrandrine group(P<0.01).7 Protein expression of p-AMPK/AMPK and p-mTOR/mTOR in rat brain tissue of each group:Western blot results showed that the ratio of p-AMPK/AMPK was significantly increased in the SAH group,but the ratio of p-mTOR/mTOR was significantly decreased compared with the sham-operated group(P<0.01).Compared with SAH,the p-AMPK/AMPK ratio was significantly decreased in the tetrandrine group,but the ratio of p-mTOR/mTOR was significantly increased(P<0.01).The ratio of p-AMPK/AMPK was significantly increased but the ratio of p-mTOR/mTOR was significantly decreased(P<0.01)in the tetrandrine+3-TYP group compared to the tetrandrine group.Summary.3-TYP partially reversed the neuroprotective effect of powdered alkaloids in rats after SAH.This effect may be related to the fact that 3-TYP inhibited the upregulation of SIRT3 protein in rats after SAH and activated the AMPK-mTOR signaling pathway,which in turn relieved the inhibition of autophagy in the brain of rats after SAH.It was suggested that powdered Fang Fangqi base could play a protective role against EBI by inhibiting autophagy through SIRT3-AMPK-mTOR signaling pathway.Conclusion:The neuroprotective effect of Tetrandrine on EBI after SAH may be related to the upregulation of SIRT3 protein expression and the inhibition of autophagy in brain tissue after SAH through the SIRT3-AMPK-mTOR signaling pathway.In addition,SIRT3 can negatively regulate AMPK-mTOR signaling pathway and thus affect autophagy. |
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