| Objective: Intracerebral hemorraghe(ICH)is a nervous disease caused by cerebrovascular rupture,accounting for about 13% of the incidence of cerebral stroke.With the deeper research on the pathology and pathophysiology after cerebral hemorrhage,ICH injury mainly involves the destruction of the blood-brain barrier,cerebral edema,inflammatory autophagy,activation of microglia,proliferation of astrocytes,and neuronal cell apoptosis.Apoptosis caused by hypoxia,inflammation and oxidation products after intracerebral hemorrhage is considered to be the main mechanism of cell damage under ICH environment.Meanwhile,recent studies have shown that inhibiting neuronal apoptosis may improve the prognosis of ICH.There exit two distinct pathways of apoptosis.One is internal/mitochondrial pathway and the other one is external/death receptor pathway.Studies have shown that the PI3K/Akt signal transduction pathway is one of the signaling pathways for cell survival,which pathway is main for neuron survival promotion and apoptosis protection.Activated Akt maintains mitochondrial integrity through antagonizing the pro-apoptotic effects of Bad and Bax,which members of the bcl-2 family.Whereas,the precise anti-apoptotic pathway of neurons is still unknown.Therefore,further elucidation of the mechanism of ICH-induced apoptosis may help to provide important strategies for the treatment and prevention of ICH.Presently,some biomarkers that could affect protein expression in the apoptotic pathway have been testified.Micro RNA(mi RNAs)is one of the most important proved biomarkers,which can target specific m RNAs by base pairing to partially or completely complementary sites to promote or inhibit translation.Many increasing evidences show that mi RNAs are involved in the regulation of the pathophysiological processes of brain diseases.Recently,when researches on mi RNA expression patterns in human Alzheimer's disease brain samples,we found that mi R-206,a brain-derived neurotrophic factor,was significantly elevated in AD mice.Meanwhile,in the mouse stroke model,some mi RNAs were changed,which proved that the regulation of mi RNA had neuroprotective potential under the condition of hypoxia and glucose deprivation in vitro.Among the numerous mi RNAs,mi R-181 c is considered to be an important factor in a series of important biological processes,including mitochondrial apoptosis.These early findings suggest that mi R-181 c plays an important role in the growth of heart cells and the aggressive behavior of tumors.Some studies have reported its importance in ductal adenocarcinoma of the pancreas,while others have shown its anti-tumor effect in ovarian cancer cells.Meanwhile,recent studies have shown that mi R-181 c may have profound effects on the proliferation,differentiation and apoptosis of neurons and cells.Although mi R-181 c is an important regulatory factor involved in the regulation of apoptosis,whether it is involved in the apoptosis of cells in the early stage after intracerebral hemorrhage is still unclear.In this study,mi R-181 c was injected into the brains of ICH rat models to observe the effect of overexpression of mi R-181 c on the recovery of neurological defects in ICH rats,the functions on survival ability and the anti-apoptotic ability of neurocytes around ICH hematoma foci.Furthermore,the mechanism of mi R-181 c on apoptotic ability and its target were explore in vivo and in vitro simulated intracerebral hemorrhage environment.The new potential theoretical target is found by results,which may be a future treatment of ICH.Methods: 1.Clinical experiment: 22 cases of intracerebral hemorrhage in the department of neurology and 18 cases in the physical examination center from the first affiliated hospital of China medical university.Clinical data,peripheral venous blood of the case group and the physical examination subjects were all collected.Furthermore application of PCR detected the level of mi R-181 c after genomic RNAs were extracted.SPSS 20.0 was used for statistical analysis the differences in the levels between the intracerebral hemorrhage group and control group,and it was also evaluated the relationship between the amount of hemorrhage and the mi R-181 c expressions.p<0.05 was considered statistically significant.2.Animal experiments: Healthy male adult Sprague-Dawley rats(n=71,250-280 g,cleaned degree)according to the Rosenberg's method which injecting collagenase VII into the basal Anglia,were established cerebral hemorrhage rat models.However,the sham group was only treated by the equal volume of normal saline.Firstly,8 rats were constructed respectively in the intracerebral hemorrhage group and the sham operation group.After 24 hrs,the levels of mi R-181 c in the surrounding tissue around the hemorrhage and the same part in the sham group were detected using PCR when the rats were killed.Then the other 63 rats were randomly divided into(1)the blank group(n=9);(2)the sham group(n=9);(3)intracerebral hemorrhage group(n=9);(4)intracerebral hemorrhage + mi R-mimic NC group(n=9);(5)intracerebral hemorrhage + mi R-inhibitor NC group(n=9);(6)intracerebral hemorrhage + mi R-181 c mimic group(n=9);(7)intracerebral hemorrhage + mi R-181 c inhibitor group(n=9)respectively.The blank group was left untreated,while the remaining(3)-(7)groups were given in situ transfection mixtures including mimic NC,inhibitor NC and mir-181 c mimic/inhibitor 4 hrs after hemorrhage.Sham group and ICH group were given transfection agents of same volume in the cerebral same parts.Each group was evaluated at the 1st,7th,14 th after modeling of neurologic function defect evaluation(NSS score).14 days after the collagenase infusion,the rat brain tissue was dissected surgically.Tunnel was used to assess apoptosis and Western blot to detect the expression of bcl-2,Bax,PTEN,cleaved caspase-3/caspase-3 and p-Akt /Akt.TUNEL staining was applied in brain tissue sections to observe the apoptosis of nerve cells.The above results were statistically analyzed by SPSS 20.0.p<0.05 was representative the significant difference.3.In vitro cell experiment: PC12 cells cultured in medium,then add different micro RNAs mimics and inhibitors respectively with transfection reagents and randomly assigned to the treatment group:(1)NC group;(2)mimic NC group;(3)inhibitor NC group;(4)mi R-181 c mimic group;(5)the mi R-181 c inhibitor groups..After 24 hours of culture,the cells were treated with hemin for 4 hours to simulate intracerebral hemorrhage,then cell activity was detected by MTT test and apoptosis was assessed by TUNEL.Western blot detection was analyzed the levels of the expression of Bcl-2,Bax,PTEN,cleaved caspase 3 / caspase 3 and p-Akt/Akt.The potential target genes of mir-181 c were predicted online by the target gene prediction website.At the same time further dual luciferase reporter gene system was proved the PTEN and PI3k/Akt signal pathway of regulation the apoptosis through mi R-181 c expression.Results: 1.Clinical experiment: mi R-181 c was significantly decreased in peripheral blood of patients with intracerebral hemorrhage(p<0.05),and there was a negative correlation between mi R-181 c and the amount of intracerebral hemorrhage.(p < 0.01).2.Animal experiments:(1)The expression level of mi R-181 c in brain tissues of rats with intracerebral hemorrhage was lower than that in brain tissues without intracerebral hemorrhage(p<0.05).(2)Comparison of neurological injury in each group: Compared with the sham group,NSS score in the cerebral hemorrhage group was increased significantly on 1st,7th and 14 th.In comparison with ICH group,the mi R-181 c mimic group was able to reduce neurological deficits and improve neurological function scores in ICH rats on1 st,7th and 14 th.Although there was no statistical difference at 1st day,there was statistical significance at 7th day and 14 th day(p<0.01).Furthermore,compared with the ICH group and the sham operation group,the NSS score of the mi R-181 c inhibitor group was deteriorated at day 1st,7th and 14 th,and was also statistically significant at 7th and 14th(p<0.01).(3)Comparison of apoptosis in each group: paraffin sections of brain tissue which was surrounding ICH,were selected,and TUNEL assay was performed in each group.Compared with the sham group,the apoptosis rate of the ICH group increased significantly.However,the treatment group treated with mi R-181 c mimic significantly reduced the apoptosis rate(p<0.03),while the mi R-181 c inhibitor group increased the apoptosis rate(p<0.03).(4)Comparison of the expression of apoptotic proteins in the brain tissues surrounding intracerebral hemorrhage in each group: Western blot results indicated that the level of Bcl-2 in the brain tissues of ICH group was decreased,but the level of Bax and cleaved cappase-3 was significantly increased(p<0.05)which compared with the sham group,.Comparison with the intracerebral hemorrhage group,the level of Bcl-2 in the brain tissues of hematomas surrounding ICH was increased after the intervention with mi R-181 c mimic,but the expression levels of Bax and cleaved caspase-3 were significantly decreased(p<0.05).It was also found that the addition of mi R-181 c inhibitor could further reduce the content of Bcl-2 and increase the expression levels of Bax and cleaved caspase-3 compared with the ICH group(p<0.05).(5)Comparison of PTEN/PI3k/Akt expression in each group: Western blot results revealed that compared with the sham group,PTEN in the brain tissues of ICH group increased and p-Akt level decreased(p<0.05).Compared with ICH group,PTEN decreased and p-Akt level significantly increased in mi R-181 c mimic group(p<0.05).However,PTEN increased and p-Akt level decreased in brain tissues of rats with combined mi R-181 c inhibitor group,which was significantly different from mi R-181 c mimic group(p<0.03), but was not significantly different from expression in brain tissues of cerebral hemorrhage.3.In vitro experiment:(1)Comparison of cell activity and apoptosis in each group: under the simulated condition of hemin-induced intracerebral hemorrhage in vitro,the activity of PC12 cells in each group was detected by MTT method.Compared with the NC group,the ICH group showed decreased cell activity.While compared with the ICH group,mi R-181 c mimics could increase cell activity(p<0.03),and mi R-181 c inhibitors could reduce cell activity(p<0.03).TUNEL was also used to detect apoptosis.Compared with the NC group,apoptosis rate of ICH group was decreased.In comparison to the ICH group,mi R-181 c mimics could effectively reduce the apoptosis rate of PC12 cells(p<0.05),while mi R-181 c inhibitors could increase the hemin-induced apoptosis rate of PC12 cells compared with the mi R-181 c mimic group(p< 0.03).(2)Comparison of PTEN and p-Akt expression in each group: Western blot analysis showed that PTEN expression increased compared with the NC group.Compared with ICH group,the level of PTEN was decreased and the level of p-Akt significantly was increased in mi R-181 c mimic group(p<0.03).However,the expression of PTEN was increased and the expression of p-Akt was decreased in brain tissues of rats treated with mi R-181 c inhibitor,which was significantly different from that of mi R-181 c mimic group(p<0.03).(3)Dural luciferase reporter experiments showed that mi R-181 c could bind directly to the 3 '-UTR region of PTEN m RNA.(4)Comparison of apoptosis in each group with treated overexpression of PTEN: compared with mi R-181 c mimic group,application of PCR detection found that mi R-181 c was significantly decreased in the the group with overexpression of PTEN(p < 0.01).Then Western blot test further suggested that the expression of Bcl-2 expression level was lower(p < 0.05),while the expression of Bax and cleaved caspase-3 was increased after treated with overexpression PTEN compared with untreated.(p < 0.03)Conclusion: 1.The expression of mi R-181 c in the patient's serum was decreased,and there was a linear negative correlation with the amount of cerebral hemorrhage.2.The rat ICH model showed that the expression of mi R-181 c was down-regulated.At the same time,the recovery of neurological function in ICH rats could be effectively improved,which was achieved by improving the anti-apoptotic function of neurons after treated overexpression of mi R-181 c.3.Mi R-181 c can directly inhibit the expression of PTEN by binding to the 3'-UTR region of PTEN m RNA in neurons,further activating the PI3k/Akt pathway to achieve neuroprotection.In summary,mi R-181 c can influence the expression of bcl-2 /Bax through the mi R-181 c /PTEN/Akt pathway,enhance the anti-apoptotic ability of neurons,improve the neurological function after ICH,and provide potential targets for the prevention and treatment of cerebral hemorrhage in the future. |
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