The Study Of Neuroprotective Mechanism Of Tetrandrine In Traumatic Brain Injury

Objective: Traumatic brain injury(TBI)is a major cause of health loss and disability worldwide.Globally,the annual incidence of TBI varies,with an estimated 27 to 69 million cases.Many survivors are left with significant disabilities,resulting in a significant socio-economic burden.Trauma leads to both immediate mechanical tissue damage(i.e.primary damage)and biochemical changes that lead to delayed or progressive cell loss(i.e.secondary damage).Therefore,the current clinical treatment of TBI is carried out around the principles of the above two aspects.Surgical treatment is the core of primary brain injury treatment for patients with TBI,especially severe TBI.Similarly,the identification,prevention and treatment of secondary brain injury are also the focus of TBI patient care.Cell damage and death from secondary injury to TBI often overlap with the recovery phase,during which brain tissue reshapes itself to compensate for tissue damage,thus providing an opportunity for treatment.These repair mechanisms include proliferation and differentiation of endogenous neural precursors,stimulation of neurite growth and myelin regeneration,and angiogenesis.In addition to the mechanisms mentioned above,autophagy has also been observed in secondary injuries of TBI.It has been found in previous autopsies and TBI mouse model studies that autophagosome accumulation is an early event of secondary injury of TBI,which appears within hours after injury.Moreover,autophagy markers remained elevated for weeks to months after injury.The continuous up-regulation of autophagy function further indicates its important role.Therefore,how to induce autophagy and repair autophagy flow are potential therapeutic methods for treating secondary injuries of TBI.Tetrandrine(Tet)is a bibenzyl isoquinoline alkaloid extracted from the root tubule of Stephamia tetrandra S.Moore.It belongs to bibenzyl isoquinoline compounds and is a novel calcium antagonist.Numerous studies have shown that Tet has a broad spectrum of pharmacological activities,including anti-tumor,anti-inflammatory,analgesic,antidepressant,anti-rheumatoid arthritis,anti-microbial,and memory improvement functions.Tet regulates apoptosis and autophagy of tumor cells through AMPK/m TOR,PI3K/AKT/m TOR and caspase/Becline I/LC3-I/II signaling pathways,and exerts significant anticancer activity.In the field of neurological diseases,Lv and other studies have shown the neuroprotective effect of Tet on vascular dementia;Wang et al. showed that Tet can reduce ischemia/reperfusion induced neuronal injury in the subacute phase;Lin et al.found that Tet could alleviate neurological defects,improve brain water content and reduce infarct volume in MCAO model rats.More recently,Liu H et al.showed that Tet reduces iron death and improves TBI symptoms by regulating autophagy during the acute phase(within 7 days),and reduces inflammation and neuronal apoptosis in experimental traumatic brain injury by regulating IRE1α/JNK/CHOP signaling.In conclusion,Tet has great potential in the treatment of neurological diseases by providing protection of neurological function through multiple mechanisms.The relevance of this study will further provide important experimental basis for the specific mechanism of Tet treatment of TBI.Methods: 1.Network pharmacology and molecular docking,98 targets of Tet,2022 TBIrelated targets and 65 common targets were identified,suggesting AKT(Protein kinase B,Protein kinase B)and m TOR(mammalian target of rapamycin,Mammalian target protein of rapamycin)is the core action site.Molecular docking suggests that Tet is well connected with AKT and m TOR,and is a potential site of action.2.Cell experiments suggested that the autophagy of SH-SY5 Y cell lines was up-regulated by the OGD(oxygen glucose deprivation,oxygen glucose deprivation)treatment by Tet by inhibiting m TOR phosphorylation.(1)The IC50 of SH-SY5 Y cell line was 16.09μmol/L and the optimal drug concentration of Tet was 5μmol/L under OGD.(2)The autophagy function of SHSY5 Y cells was up-regulated after OGD treatment.After addition of Tet,autophagy function was significantly up-regulated.The phosphorylation of AKT was not significantly affected by Tet,and the phosphorylation of m TOR was statistically significant,suggesting that TET up-regulated autophagy by inhibiting m TOR phosphorylation.3.Animal experiments suggest that Tet plays a neuronal protective role by inhibiting m TOR phosphorylation and up-regulating neuronal autophagy:(1)Starting from the 7th day of successful modeling,the 14-day and 28-day m NSS(modified Neurological Severity Score)showed that Tet had a definite effect on improving the neurological dysfunction of TBI model rats between the medium-dose group and the high-dose group,while there was no statistically significant difference in the therapeutic effect of Tet in the low-dose group,indicating that TET had a definite effect on the neurological dysfunction of TBI model rats.But it is time-dependent and dose-dependent.(2)HE(hematoxylin eosin)staining in braintissue sections showed that the damaged area of the experimental group was significantly smaller than that of the model group and the proportion of intact neurons in the experimental group was significantly better than that of the model group,indicating that Tet had a protective effect on neurons.(3)Western Blot and LC3(light chain 3,microtubule-associated protein light chain 3)were performed on traumatic brain tissues in the hippocampus.After addition of Tet,autophagy was more significantly up-regulated in the experimental group than in the model group,indicating that Tet can significantly promote the up-regulation of autophagy in animal models.The number of autophagy bodies in the experimental group was significantly higher than that in the model group and the sham group after the repair of autophagy flow and LC3 fluorescence.Conclusions: Upregulation of autophagy is a very important response of the body to external pressure such as TBI.However,impaired autophagy flux of neurons is a common phenomenon after TBI,which eventually leads to neuronal apoptosis,necrosis or iron death.Tet can play a protective role in neurons by inhibiting m TOR phosphorylation,upregulating autophagy function and repairing autophagy flux.The treatment of TBI by Tet is time-dependent and dose-dependent.Tet showed neuroprotective effects in the acute,subacute and chronic stages of TBI and improved cognitive function in the chronic stages of TBI.