Importance Of Riboflavin Kinase In The Pathogenesis Of Stroke

According to the report of WHO, 15 million people suffered form stroke each year, in which 5 million people died and 5 million people became disabled. In China, there are 2 million people suffered from stroke including 1.2 million peoples died. The consequences of stroke are severe. Patients die or become disabling, and the rate of disability among the survival patients is high to 75%. China government has to cost 37.4 billion Yuan on stroke per year.While, except decreasing blood pressure, there is no effective method to prevent stroke. Hypertension has been confirmed to be the first risk factor to induce stroke. More than 60% stroke patients had the history of hypertension. But, not all the patients with hypertension suffered stroke and not all the patients with stroke had the history of hypertension. It is to say that hypertension isn't the only risk to induce stroke. Therefore, is it possible that some protective factors exist in patients with hypertension who didn't suffer from stroke or that some other risk factors exist except hypertension to induce strokeIt's an urgent demand to find out more protective or risk factors related to stroke except hypertension.Stroke-Prone Spontaneously Hypertensive Rats (SHR-SP) is the most frequently used animal model on stroke study. SHR-SP is preferential mating from Spontaneously Hypertensive Rats (SHR) and endogamy through generations. Adult SHR-SP spontaneously suffers from stroke. Therefore, SHR-SP is believed to be ideal animal model on stroke study.Both SHR and SHR-SP are hypetensive animals, whereas all SHR-SP are prone to suffer from stroke. Therefore, we compared the gene expression profiling of brain from SHR-SP with brain form SHR, through genechips, on the purpose of finding the other factors related to stroke except hypertension.In our experiment, genechips were used to comparatively analyse the gene expression spectrum of brain from SHR-SP and SHR. The result showed that compared with the level of riboflavin kinase (rfk) in SHR, it is down regulated in SHR-SP. Till now, no relationship between rfk and stroke has been reported.Therefore, our study was designed as follows:The gene expression spectrums of brain from SHR-SP and SHR were comparatively analysed by genechips;The differently expressed genes detected by genechips between SHR-SP and SHR were analysed by bioinformatics;The rfk level in SHR-SP and SHR were analysed by real-time PCR and Western Blot (WB);The relationship between riboflavin kinase and stroke were studied.【PURPOSE】: Our purpose was to find the risk factors inducing stroke excluding hypertension and to explore the relationship of riboflavin kinase with stroke.【METHODS and RESULTS】:1,Comparative analysis of gene expression spectrums of SHR-SP and SHR by genechipsGene expression profiling: Genechips were used to analyse the different expressed genes between SHR-SP and SHR. These genes may be the factors inhibit or prompt the occurrence of stroke. All the brain samples were qualified after quality monitoring. The gray scales of genechips were scaned and transformed to digital signals. The digital signals were used to analyse. With the condition of P≤0.05 and Ratio≥2 or≤-2, 106 genes were screened, in which 55 genes highly expressed in SHR-SP and 51 genes highly expressed in SHR.Bioinformatics analysis: The raw data obtained from genechips were performed bioinformatics analysis, to screen the important genes or pathways related to stroke. The data were uniformly treated and screened with the condition P≤0.05 and Fold Change≥2 or≤-2. The result showed 76 genes differently expressed in SHR-SP and SHR, in which 41genes highly expressed in SHR-SP and 35 genes highly expressed in SHR. 17 Pathways and 252 Gene ontology (GO) significantly changed (P≤0.05). According to the GO analysis, we deduced the down regulation of rfk (P<0.01) might highly related to the occurrence of stroke.2,The rfk level in SHR-SP and SHR was comparatively analysedThe level of rfk, which was doubted to down regulation in SHR-SP based on the genechip analysis and bioinformatics analysis, was comparatively analysed by real-time PCR and WB.Real-time PCR: compared with the mRNA level of rfk in SHR, it was obviously decreased to 73% in SHR-SP (P<0.01). This confirmed the result of genechips.WB: compared with the protein level of rfk in SHR, it was obviously decreased in SHR-SP. This confirmed the result of both genechips and real-time PCR. The results further indicated that the down regulation of rfk might highly relate to the occurrence of stroke.3,Effect of flavins on strokeVB2, FMN and FAD are called flavins together. Based on the above results, we believe the down regulation of rfk may highly relate to the occurrence of stroke. Flavokinase is an enzyme that catalyzes the phosphorylation of riboflavin (VB2) to formFMN, which is an obligatory step in VB2 utilization and flavin cofactor synthesis. Down regulation of rfk will affect the bioavailability of riboflavin in vivo and therefore affect the function of riboflavin. To study whether flavins avoid or alleviate stroke, we designed the experiment as follows:MCAO: Forty C57BL mouse were randomly assigned into four groups: the controls, VB2 group, FMN group and FAD group. All animals were intraperitoneally injected 3.5 mmol/kg corresponding falvins. The controls were injected the same volume of physiological saline. After a week administration, the operation of Middle Cerebral Artery Occlusion (MCAO) was performed on animals on day 8. Additional injections of corresponding flavins were performed both 0h and 12h after MCAO. 24h after MCAO,brains of all the animals were dissected and proceeded TTC stain. From the result, compared with the infarction rate of the controls with 16.6±3.3%, VB2, FMN and FAD all decreased the infarction rate with 11.5±3.9% (P<0.01), 9.5±3.2% (P<0.01) and 11.2±4.4% (P<0.01), respectively. This indicated that VB2, FMN and FAD could obviously protect brain from stroke.Flow cytometry: Fetuses of SHR-SP were used to cultivate primary neurons of cerebral cortex. Between d7 ~ 10 after mother blank, neurons could be used to perform experiment. The model of Oxygen and glucose deprivation (OGD) on neurons was established to simulate the ischemia and anerobic environment of stroke. Neurons were treated with high glucose Krebs solution, in which flavins were added respectively, for 2h before OGD. Then neurons were treated with no glucose Krebs solution, in which flavins were added respectively, and were moved into incubator with 94% N2, 5%CO2 and 1% O2 for 1.5h. After1.5h OGD, neurons were immediately digested and collected into eppendorf tubes and the flow cytometry related operating was performed. The results showed VB2, FMN and FAD decreased the apoptosis rate and the death rate statisticly on the concentration of 0.5μM, but these protective effect diappeared when the concentration was up to 2.5μM. This suggesting that flavins protect neurons against OGD on the concentration of 0.5μM.WB: to explore the accurate mechnisums of protective effect of flavins on neurons against apoptosis, we designed the experiment as follows. The neurons cultivation, OGD model establishment and the flavins treating were the same as described in Folw cytometry. After OGD for 1.5h, the neurons were immediately treated with protein extraction. Protein level of bcl-2 and bax among groups were detected by WB. The results showed the level of bcl-2 did not alter among different doses of flavins. Whereas, the level of bax had the lowest expression at the concentration of 1.5μM VB2 and FMN, but not FAD. These suggest that VB2 and FMN at the concentration of 1.5μM displayed the strongest protection of neurons against apoptosis induced by OGD.【CONCLUSIONS】:Between SHR-SP and SHR, 76 genes differently expressed, of which 35 genes highly expressed in SHR and 41 genes highly expressed in SHR-SP.Down regulation of rfk might highly relate to the occurrence of stroke. The level of rfk may affect the absorbance and utility of riboflavin. That's the reason why brain loses the protection from riboflavin and increases the risk of stroke.Brain infarction induced by MCAO could be obviously inhibited by flavins. The apoptosis rates and death rates of neurons induced by OGD could be obviously inhibited by flavins. Flavins protect brain from MCAO through anti-apoptosis. The mechenisum of flavins anti-apoptosis may mediated by depressing the expression of bax protein, who paly an important role in inducing mitochondrial apoptosis.