| Stroke, which is an acute blood circulation disorder in central nervous system, is recognized as the third fatal disease, with high morbidity, mortality and long-term disability. In our country, stroke is the first cause of death and the incidence of stroke ranks first. In the patients with stroke, ischemic stroke accounts for 60%-70% of the total patients, and ischemic stroke is the main type of stroke. The incidence of ischemic stroke is so high, but its treatment is limited to intravascular thrombolytic therapy of recombinant tissue plasminogen activator(rt-PA), confirmed by Food and Drug administration of America(FDA). Despite the time window of thrombolytic therapy has been expanded to 4.5h, only 22% patients enter into emergency room within 3h even in the United States with developed economy and health care. In these patients, only 8% patients are suitable for rt-PA thrombolytic therapy. rt-PA thrombolytic therapy has serious blood risk of side effect, especially it is limited to the treatment time window. Hence, it is not satisfied to prevent and treat ischemic stroke in the clinic. So, researchers gradually focused on endogenous neuroprotective mechanisms of brain itself(interventions) and bio-molecular markers. Cerebral ischemic injury is a complex pathophysiologic process, and the degree of neuronal damage and its clinic therapy directly decide the outcome of ischemic stroke. Evidence has suggested that neurons in the ischemic core first lose viability due to the acute block in cerebral blood flow and the lack of cellular energy metabolism, and then die by irreversible necrosis. Ischemic core area is surrounded by a zone of peri-infarct area, termed the penumbra, in which the energy metabolism and intracellular signal transduction are partly preserved by collateral circulation. It is well documented that the programmed cell death pathways are activated in penumbra, and it is reversible to rescue neurons in penumbra. The doom of reversible neurons in penumbra directly decides the outcome of cerebral ischemic stroke. Hence, it is important for investigators of cerebral ischemia to focus on rescuing neurons in penumbra, reducing programmed cell death to explore valid treatment on ischemic stroke.We used MCAO-induced ischemic stroke models to find that the protein expression level of endogenous c PKC? was negatively related with the infarct volume. As the protein expression of endogenous c PKC? was increasing, the neurological score was decreasing significantly. These results showed that the endogenous c PKC? protein expression level can affect the infarct volume and neurological deficit of mice with ischemic stroke. We found that c PKC? played neuroprotective role in the brain tissue of mice following MCAO-induced ischemic injury. To explore the effect of c PKC? on the autophage and its signal transduction, we used C57BL/6J mice with c PKC? gene wild type(c PKC?+/+, WT) and knock out(c PKC?-/-, KO) in vivo to establish 1h MCAO/Reperfusion(R) 1d-7d ischemic stroke models;We used postnatal 24 h C57BL/6J c PKC?+/+ and c PKC?-/- mice to establish the primary cultured cortical neurons after 1h oxygen-glucose deprivation(OGD)/Reoxgenation(R) 24h-induced ischemic injury models to further confirm the effect of c PKC? on the autophage and its signal transduction in vitro.The experimental groups included: Na?ve, Sham, 1h MCAO/R 1d, 3d and 7d, 1h MCAO/R 3d(injection Baf A1 into lateral ventricle 5min before MCAO) and DMSO. We evaluated the effect of c PKC? on the neurological deficit, neural cell loss and infarct volume of mice with ischemic stroke by using neurological score, Nissl staining and TTC staining; We used Western blot technology to observe the protein expression of Beclin-1 and the ratio of LC3-II/(LC3-I+LC3-II) to explore the effect of c PKC? on autophagy in peri-infarct region of mice after 1h MCAO/R 1d, 3d and 7d; We observed the phosphorylation level of Akt(Ser473), m TOR(Ser2448) and S6(Ser235/236) to further explore the effect of c PKC? on Akt-m TOR pathway in peri-infarct region of mice after 1h MCAO/R 3d;We explored the effect of autophagy on 1h MCAO/R 3d-induced ischemic injury of mice with the pretreatment of autophagic inhibitor Baf A1(Bafilomycin A1)(2.5g/kg) by using Nissl staining. We used c PKCγ+/+ and c PKCγ-/- mice to inhibit autophage flux by injection Baf A1(2.5g/kg) into lateral ventricle 5min before MCAO, and then we examined the number of neural cells to evaluate the effect of autophage on the outcome of mice with ischemic stroke.In vitro, we used the primary cortical neurons that obtained from postnatal 24 h C57BL/6J mice to culture for 7 days, and then cultured neurons were treated with 1h OGD/R 24 h. c PKCγ+/+ and c PKCγ-/- neurons were treated with Baf A1(100n M) to inhibit autophage flux. c PKC? was restored using lentivirus-mediated gene transfer in c PKCγ-/- neurons. The experimental groups included: Normoxia and 1h OGD/R 24 h; Baf A1(100n M) and DMSO; LV-c PKC?(+) and LV-c PKC?(-). The colorimetric method of thiazolyl blue tetrazolium bromide(MTT) and the leakage rate of lactate dehydrogenase(LDH) were applied to assess neurons survival rate and death rate. To explore the effect of c PKC? on the autophage and Akt-m TOR pathway, we used Western blot to detect the protein expression of Beclin-1 and the ratio of LC3-II/(LC3-I+LC3-II), and to determine the phosphorylation level of Akt(Ser473), m TOR(Ser2448) and S6(Ser235/236). Then c PKC? was restored using lentivirus-mediated gene transfer in vitro in c PKCγ-/- neurons following 1h OGD/R 24 h. Then MTT and LDH assays were used to evaluate neurons survival rate and death rate. Beclin-1 protein expression and the ratio of LC3-II/(LC3-I+LC3-II) were measured to further confirm the effect of c PKC? on the autophage of neurons following 1h OGD/R 24 h. The experimental data were analysed using SPSS 11.5 statistical software. Statistic analysis was conducted by using One-Way ANOVA which is followed by all pairwise multiple comparison procedures using Bonferroni test. Significance was regarded as p<0.05,p<0.01,p<0.001. The data were presented as X±S.E. The results were reported as follows: 1. c PKC? knockout increased the infarct volume of mice with ischemic strokeTo explore the effect of c PKC? on the regional(cerebral blood flow,CBF), we examine the regional CBF of mice during MCAO operation. The results of CBF showed thatthe regional CBF of mice was 100% under normal circumstances, and regional CBF decreased by 80% after 1h MCAO, and then recovered 100% completely after the occlusion was removed 1h later both in c PKCγ+/+ and c PKCγ-/-mice. These results suggested that c PKCγ could not affect the regional CBF of mice during the procedure of 1h MCAO. To examine the effect of c PKC? on infarct volume of mice with ischemic stroke, TTC staining results showed that c PKC? knockout enhanced the infarct volume when compared with that of c PKCγ+/+ mice after 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P=0.004),(n=5 per group). These results showed that c PKC? knockout enhanced the infarct volume, and it was not related with CBF variation. 2. c PKC? knockout aggravated the neurological deficit of mice with ischemic strokeTo examine the effect of c PKC? on the neurological deficit of mice with ischemic stroke,we evaluated mice by using neurobehavioral tests. Results of neurological score showed that the knockout of c PKCγ could cause higher neurological score in mice following 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P=0.039),(n=6 per group) when compared with c PKCγ+/+ mice after the same treatment,. Similarly, the pole test assays indicated that the knockout of c PKCγ may cause longer time to turn the head downwards of mice following 1h MCAO/R 1d(P=0.007), 3d(P<0.001) and 7d(P<0.001),(n=6 per group), and to reach the ground of mice following 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P=0.002),(n=6 per group) when compared with c PKCγ+/+ mice after the same treatment. The Wire Hanging test assays indieated that the knockout of c PKCγ may cause shorter time to stay hanging on the wire of mice following 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P<0.001),(n=6 per group) when compared with c PKCγ+/+ mice after the same treatment. The Cylinder test assays indicated that the knockout of c PKCγ may cause higher left forepaw contact score of mice following 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P<0.001),(n=6 per group) when compared with c PKCγ+/+ mice after the same treatment. The Handedness test assays indicated that the knockout of c PKCγ may cause higher laterality index of mice following 1h MCAO/ R 1d(P<0.001), 3d(P<0.001) and 7d(P=0.005),(n=6 per group) when compared with c PKCγ+/+ mice after the same treatment. The Foot Fault test assays indicated that the knock out of c PKCγ may cause the more number of contralateral foot faults of mice following 1h MCAO/R 1d(P<0.001), 3d(P=0.001) and 7d(P=0.009),(n=6 per group) when compared with c PKCγ+/+ mice after the same treatment. The results showed that c PKC? knockout aggravated the neurological deficit of mice with ischemic stroke. 3. c PKC? knockout aggravated the neural cell loss in peri-infarct region of mice with ischemic strokeTo examine the effect of c PKC? on the neural cell loss in peri-infarct region of mice with ischemic stroke, we used brain frozen section for Nissl staining. Results of Nissl staining showed that c PKC? knockout enhanced neural cell loss in peri-infarct region of mice after 1h MCAO/R 3d(P<0.001),(n=5 per group) when compared with c PKCγ+/+ mice after the same treatment. The results showed that c PKC? knockout aggravated the neural loss in peri-infarct region of mice with ischemic stroke. 4. The effect of c PKC? on autophage in peri-infarct region of mice with ischemic stroke via Akt-m TOR pathwayWe used Western blot to examine the effect of c PKC? on autophage in peri-infarct region of mice with ischemic stroke. Results showed that c PKC? knockout increased the protein expression of Beclin-1 in in peri-infarct region of mice after 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P=0.005), when compared with c PKCγ+/+ mice after the same treatment(n=7 per group). Similarly, Western blot results showed that c PKC? knockout increased the ratio of LC3-II/(LC3-I+LC3-II) in in peri-infarct region of mice after 1h MCAO/R 1d(P<0.001), 3d(P<0.001) and 7d(P<0.001), when compared with c PKCγ+/+ mice after the same treatment(n=7 per group). To explore whether c PKCγ was involved in autophage through Akt-m TOR pathway, the results of Western blot showed that c PKC? knockout aggravated significantly the decreasing of P-Akt(Ser473), P-m TOR(Ser2448) and P-S6(Ser235/236) in in peri-infarct region of mice following 1h MCAO/R 3d(P<0.001, n=5 per group). These results indicated that c PKC? knockout increased the protein expression of Beclin-1 and the ratio of LC3-II/(LC3-I+LC3-II) in peri-infarct region of mice with ischemic stroke through downregulating phosphorylation levels of Akt-m TOR pathway 5. The effect of Baf A1 autophage inhibition on neural cell loss in peri-infarct region of mice with ischemic strokeWe used Baf A1 to inhibit autophage flux successfully in peri-infarct region of mice with ischemic stroke. Autophagic inhibitor, Baf A1, can inhibit the fusion of autophage and lysosome. Western Blot analysis showed that Baf A1(0.25mg/kg) could not induce LC3-II accumulation in peri-infarct region of both c PKCγ+/+ and c PKCγ-/-mice after 1h MCAO/R 3d compared with DMSO group(n=5 per group). Baf A1(2.5mg/kg and 25mg/kg) induced LC3-II accumulation in peri-infarct region of both c PKCγ+/+ and c PKCγ-/- mice after 1h MCAO/R 3d(P<0.001, n=5 per group) compared with DMSO group. Therefore, we choosed Baf A1(2.5 mg/kg) as the next experiment dose. To examine the effect of Baf A1 autophage inhibition on neural cell loss in peri-infarct region of mice with ischemic stroke, the results of Nissl staining showed that Baf A1(2.5 mg/kg) enhanced neural cell loss in c PKCγ+/+ mice after 1h MCAO/R 3d(P<0.001, n=5 per group),while Baf A1(2.5 mg/kg) could attenuate neural cell loss in c PKCγ-/-mice after 1h MCAO/R 3d(P<0.001, n=5 per group). Taken together, these results showed that c PKCγ can affect ischemic injury by modulating autophage level in peri-infarct region of mice with ischemic stroke. 6. The effect of c PKC? on autophage of neurons after 1h OGD/R 24 h through Akt-m TOR pathwayTo examine the effect of c PKC? on the survival rate of neurons after 1h OGD/R, MTT assays showed that neurons survival rate was decreased significantly from c PKCγ-/- neurons when compared with those from c PKCγ+/+ neurons after 1h OGD/R 24h(P<0.001, n=6 per group). LDH assays showed that neurons death was increased significantly in c PKCγ-/- neurons when compared with c PKCγ+/+ neurons after 1h OGD/R 24h(P<0.001, n=6 per group). To examine the effect of c PKCγ on autophage of neurons after 1h OGD/R 24 h, the ratio of LC3-II to LC3-I and Beclin 1 protein expression were examined in 1h OGD/R 24h-treated neurons. Western Blot analysis showed that knockout of c PKC? could increase the protein expressions of Beclin-1(P<0.001, n=6 per group) and the ratio of LC3-II/(LC3-I+LC3-II)(P<0.001, n=6 per group) in neurons after 1h OGD/R 24 h. Moreover, to explore whether c PKCγ affects autophage in neurons after 1h OGD/R 24 h through Akt-m TOR pathway, we used Western blot to examine the phosphorylation levels of Akt(Ser473), m TOR(Ser2448) and S6(Ser235/236). The knockout of c PKCγ could aggravate significantly 1h OGD/R 24h-induced the decreasing of P-Akt(P<0.001, n=6 per group), P-m TOR(P<0.001, n=6 per group) and P-S6(P<0.001, n=6 per group). These results supported the conclusion that c PKC? knockout decreased neurons survival rate by decreasing autophage of neurons through Akt-m TOR pathway. 7. The effect of Baf A1 autophagic inhibition on survival rate of neurons after 1h OGD/R 24 h We used Baf A1 to inhibit autophage flux successfully, Western blot analysis showed that Baf A1(100n M) induced LC3-II accumulation in neurons after 1h OGD/R 24h(P<0.001, n=6 per group). To examine the effect of Baf A1 autophagic inhibition on survival rate of neurons after 1h OGD/R 24 h, MTT assays showed that Baf A1 can further reduce neuron survival rate in c PKCγ+/+ neurons whereas Baf A1 can significantly improve neuron survival in c PKCγ-/- neurons after 1h OGD/R 24 h compared with DMSO group(P<0.001, n=6 per group). Similarly, LDH assays showed that Baf A1 can further increase neuron death rate in c PKCγ+/+ neurons(P<0.001, n=6 per group), while Baf A1 can further decrease neuron death rate in c PKCγ-/- neurons with 1h OGD/R 24h(P<0.001, n=6 per group) compared with DMSO group. These results demonstrated that c PKCγ can affect ischemic injury of neurons after 1h OGD/R 24 h through modulating autophage level. 8. Restoration of c PKC? protein expression alleviated autophage and improved survival rate of neurons after 1h OGD/R 24hTo confirm that the effect of c PKC? on neurons survival and neurons death, and beclin-1 protein expression and the ratio of LC3-II/LC3-I+LC3-II after 1h OGD/R 24 h, we transduced c PKC?-/- cortical neurons with lentivirus coding c PKC? and treated neurons with 1h OGD/R 24 h. Representative Western blot results showed that restoration of c PKC? decreased the expression of Beclin-1(P<0.001, n=6 per group) and the ratio of LC3-II/LC3-I+LC3-II after 1h OGD/R 24h(P<0.001, n=6 per group) compared with LV- c PKC?(-) group in neurons after 1h OGD/R 24 h. By using MTT and LDH methods, we have found that LV-c PKC? could improve neurons survival rate(P<0.001, n=6 per group) and decrease neurons death rate(P<0.001, n=6 per group) compared with LV- c PKC?(-) group in neurons after 1h OGD/R 24 h. The results confirmed that Restoration of c PKC? alleviated autophage and improved survival rate of neurons after 1h OGD/R 24 h, which is related with autophage level.Above all, using MCAO-induced ischemic stroke models in vivo and OGD-induced ischemic injury models in vitro, we found that c PKC? can modulate Akt-m TOR pathway mediated-autophage to alleviate ischemic injury and improve the outcome of mice with ischemic stroke. It is specific for neurons to c PKC?-modulated autophage, and c PKCγ played an important role in maiteinning Akt(Ser473) phosphorylation at certain level. But the detailed molecular mechanism need to be further explored in future. The results will further enrich the understanding of the underlying cerebral ischemic/hypoxic injury. Moreover, the results may provide the experimental evidence for the prevention and treatment of ischemic stroke in clinic practice. |
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