| Objective:1.A cohort study evaluated the therapeutic efficacy and prognostic implications of Nao-chu-xue Prescription(NCXP),a traditional Chinese medicine formula,in patients with intracerebral hemorrhage(ICH)of different courses.Guided by the principles of "breaking blood stasis,removing stagnant blood,replenishing essence,and nourishing marrow," this study provides an evidence-based rationale for the clinical application of NCXP in ICH management.2.By using in vivo and in vitro experiments to explore the mechanism of Nao-chu-xue Prescription’s neuroprotective effect through the regulation of PI3K/AKT/mTOR signaling pathway to intervene in autophagy and to provide an experimental basis for the clinical application of this method for the treatment of intracerebral hemorrhage.Methods:1.Clinical Study: A cohort study was conducted,enrolling patients with intracerebral hemorrhage(ICH)who met the inclusion and exclusion criteria from January 2020 to January 2024.Participants were divided into treatment and control groups based on whether they received the Nao-chu-xue Prescription(NCXP)as an exposure factor.Each group was further stratified into three subgroups according to the disease stage: acute,recovery,and sequelae.Baseline data were collected and statistically described.The National Institute of Stroke Scale(NIHSS),Barthel Index(BI),and modified Rankin Scale(mRS)scores were assessed pre-and post-treatment to compare the clinical efficacy and prognosis between groups.Relative risk(RR),attributable risk(AR),and one-way logistic regression analyses were employed to evaluate the association between NCXP treatment and favorable/unfavorable prognosis.Biochemical indices were monitored,and adverse events were recorded to assess the safety profile of NCXP.2.In Vivo Experiments: Rats were randomly assigned to Sham,Model,and NCXP low-,medium-,and high-dose groups.The autologous blood injection method was used to construct the ICH rat model.The administration groups were given doses of NCXP of0.13g/100 g,0.26g/100 g,and 0.52g/100 g,respectively,while the sham and model groups received an equal volume of saline once daily for ten days.The Zea Longa5 method was used to evaluate the neurological function damage at 24 h,3d,7d,and 10d;The ratio of the maximum area of the coronal hematoma in each group was observed and calculated;Hematoxylin-eosin(HE)staining was performed to assess brain tissue pathology.Western blotting detected the expression of autophagy-related proteins LC3Ⅱ/Ⅰ,Beclin1,ULK1,and p62.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)analyzed the m RNA levels of PI3 K,AKT,and mTOR.3.In vitro experiments:(1)Microglia BV-2 was used to establish an in vitro model of ICH induced by Hemin,and the CCK8 method was used to explore the optimal intervention concentration of Hemin and NCXP before the medicine was administered.Apoptosis was evaluated by flow cytometry and Western blotting of apoptosis-related proteins to evaluate the establishment of an in vitro model of ICH and the protective effect of NCXP.(2)Autophagy expression was assessed by Western blotting and RT-qPCR.The CCK8 assay identified the optimal intervention concentration of the autophagy inhibitor 3-MA.We used immunofluorescence,Western Blot,and RT-qPCR to detect autophagy-related genes and proteins after 3-MA intervention and verified the regulation of autophagy by cerebral hemorrhage formula.(3)Western blotting and RT-qPCR analyzed the m RNA and phosphorylation protein levels of PI3 K,AKT,and mTOR.The CCK8 method explored the optimal intervention concentration of LY294002(the PI3 K pathway inhibitor)and Western Blot detection of autophagy protein expression after LY294002 intervention to verify that cerebral hemorrhage parties play a neuroprotective role through the regulation of cellular autophagy after ICH via the PI3K/AKT/mTOR pathway.Results:1.Clinical study:(1)No significant differences were observed between the two groups in baseline National Institutes of NIHSS,BI,and mRS scores during the acute,recovery,and sequelae periods(P> 0.05).Both groups could effectively reduce the NIHSS,induce BI,and reduce mRS scores after treatment.Both groups effectively reduced NIHSS scores,improved BI scores,and decreased mRS scores post-treatment.Regarding the enhancement of neurological function and activities of daily living,significant intergroup differences were noted in the acute stages(P < 0.05)but no significance between the two groups in the recovery and sequelae stage(P> 0.05).In reducing the degree of disability,there was no significance in comparing the groups in the acute,recovery,and sequelae stages(P> 0.05).(2)RR = 1.054,the probability of the treatment group occurring in the excellent prognosis is1.278 times higher than that of the control group;AR = 0.17,AR% = 59%,59% of the excellent prognosis occurring in the treatment group is attributable to the administration of the cerebral hemorrhage formula;one-way Logistic regression analysis showed that the ORs for the acute,recovery,and sequelae phases were 1.95,0.84,and 0.29 respectively,indicating that the probability of good prognosis in the acute,recovery,and sequelae periods increased by 95%,decreased by 16%,and decreased by 71%,respectively,when taking NCXP compared with not taking cerebral hemorrhage formula.(3)No adverse events occurred during the treatment period in either group,and the safety of both treatments was plausible.2.In vivo experiments:(1)Rats with intracerebral hemorrhage(ICH)exhibited higher neurological function scores than the control group.The hemorrhage foci in the brain tissue were apparent,accompanied by many necrotic neuronal cells and inflammatory cell infiltration.The nuclear cells were solidified and deeply stained.After the intervention of the NCXP,the Zea Longa5 score of ICH rats was reduced,the hematoma area was reduced,and the histopathological morphology was improved.(2)In ICH rats,autophagy proteins LC3 Ⅱ/Ⅰ,Beclin1,and ULK1 were upregulated,while p62 expression was downregulated in the perihematoma tissues.The expression of PI3 K,AKT,and mTOR genes was down-regulated.After the intervention of the NCXP,autophagy activity was inhibited by decreased expression of autophagy proteins LC3 Ⅱ/Ⅰ,Beclin1,and ULK1 and increased expression of p62.Additionally,the m RNA expression of PI3 K,AKT,and mTOR was upregulated,indicating activation of the PI3K/AKT/mTOR pathway.3.In vitro experiments:(1)CCK8 screened 12.5μmol/L Hemin as the optimal safe concentration for inducing BV-2 cell injury,and 10 μg/m L,20 μg/m L,40 μg/m L NCXP as the low,medium,and high doses for intervention.Increased apoptosis rate after ICH and up-regulate expression of Bax and Csapase3 indicated that the vitro model of ICH was successfully established.NCXP decreased apoptosis rates,reduced Bax and Csapase3 expression,and increased Bcl-2 expression,significantly inhibiting apoptosis following ICH.(2)Cell autophagy was activated after Hemin induction and autophagy activity was inhibited after the intervention of NCXP;0.5 mmol/L 3-MA was screened as the optimal intervening concentration,and cell viability was significantly rebounded after the intervention of 3-MA compared to the model group.After the addition of 3-MA in the model and NCXP-administration groups,the expression of LC3,Beclin1,and ULK1 decreased,and the expression of p62 increased.(3)The expression of PI3 K,AKT,and mTOR m RNA and proteins was down-regulated after Hemin induction,and their expression increased after the intervention of the NCXP.LY294002 at 50 μmol/L was screened as the optimal intervention concentration.Cell viability was further reduced in the model group after the LY294002 intervention,but no significant change was observed in the NCXP group.After the LY294002 intervention,the expression of LC3 Ⅱ/Ⅰ and Beclin1 proteins increased,p62 expression decreased,and the inhibitory effect of NCXP on cellular autophagy was counteracted.Conclusion:1.There is an advantage in using NCXP in patients in the acute and recovery phases in improving their neurological deficits and ability to perform daily life.Regarding prognostic outcomes,the probability of a good prognosis occurring with NCXP was higher.However,the advantage in terms of overall prognostic impact was not significant.The overall safety of both methods during the clinical study was favorable.2.NCXP can reduce the neurological impairment of ICH rats,promote hematoma resorption,and improve the histopathological morphology of perihematomal.In the Hemin-induced ICH in vitro model,the NCXP can improve cell viability and inhibit cell apoptosis.3.NCXP inhibits cellular autophagy and exerts neuroprotective effects by activating the PI3K/AKT/mTOR signaling pathway. |
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