The Protective Effects And Its Mechanism Of Probucol Pretreatment On Cerebral Ischemia Reperfusion Injury(CIRI) In High-fat-induced Obese Rats

CHAPTER I THE EFFECT OF PROBUCOL ON SYSTEMIC INFLAMMATION AND MICROGLIA ACTIVATION OF IN HIGH-FAT-INDUCED OBESE RATSObjective:To observe the effect of probucol on lipid metabolism, oxidative stress, systemic inflammation and microglia activation in high-fat-induced obese rats, and to explore the potential value for neuroprotection of it.Methods:Forty male SD rats were divided randomly into two groups:Normal diet group (NG, n=16), which were fed normal diet, eight rats which labeled NG8W were killed for experiment after8weeks, the remaining rats labeled NG16W were fed with normal diet for another8weeks. High-fat diet group (HG, n=24), which were fed with high-fat diet, eight rats which labeled HG8W were killed for experiment after8weeks, the remaining rats fed with high-fat diet for another8weeks, and divided randomly into two groups:HG16W (n=8) and PB(probucol) group (n=8). PB group were given a gavage of5%probucol suspension (100mg/d.kg)(5%probucol made with0.5%carboxy methyl cellulose(CMC))once a day for8weeks, while NG16W group and HG16W group were given a gavage of0.5%CMC of the same volume once a day for8weeks. At the end of16th week, the rats in all groups were fasted for12hours and weighed and measured before sacrificed. The specimen of plasma, liver, fat and brain were collected after anesthesia. Lee’s index, body fat index and liver index of rats were calculated. The level of plasmatic TC,TG, LDL, ox-LDL, HDL, FBS, FINS, ALT, AST, hs-CRP, TNF-α, IL-1β, IL-6, SOD, MDA, T-AOC were measured, HOMA-IR was calculated, the histopathology of liver and adipose tissue were analysed by HE stain, the expression of Iba-1in brain was determined by immunohistochemistry.Results:The weight of rats in all group were gradually increased as the age increases. The weight of rats in HG group were significantly higher than in NG group after12weeks(p<0.05).The lee’s index, liver index and body fat index of HG group were significantly higher than the NG group at the time of8,16week(p<0.05).The weight gain of rats in PB group slowed down after the treatment with probucol, the Lee’s index, liver index and body fat index decreased significantly in PB16W group compared with HG16W group at the time of16week (p<0.05).The level of FBS have no significant differences between NG and HG group at the end of8,16week (P>0.05), but FINS and HOMA-IR were higher in HG group than in NG group at the time of8,16week (p<0.05). FINS and HOMA-IR of PB group lower than HG16W group at the time of16th week (p<0.05).The levels of ALT, AST and the degree of liver steatosis were significantly higher in HG16W group than in the NG16W group at the time of16weeks(p<0.05). The diameter of fat cell increased and the inflammatory cell infiltrate obviously in HG16W group. The levels of ALT, AST and the degree of liver steatosis in PB16W group were decreased significantly after the treatment with probucol for8weeks(p<0.05).The diameter of adipocyte decreased significantly(p<0.05), and the infiltration of inflammatory cell reduced in PB16W group at the time of16th week.The levels of plasmatic TG, TC, LDL-C, ox-LDL were significantly higher in HG group than in NG group at the time of8,16weeks (p<0.05), but the levels of HDL-C was no significant difference (p>0.05).The levels of TG、TC、LDL-C、ox-LDL in PB16W group were decreased significantly in PB16W group compared with HG16W group (p<0.05), but higher than NG16W group(p<0.05). The levels of HDL-C were also reduced significantly in PB16W group (p<0.05).The levels of plasmatic hs-CRP、TNF-α、IL-1β、IL-6and MDA were significantly higher in HG16W group than in the NG16W group at the time of16th week (p<0.05), and the levels of SOD and T-AOC were lower in HG16W group than the NG16W group significantly(p<0.05).The levels of hs-CRP、TNF-α、IL-1β、IL-6and MDA were decreased significantly in PB16W group compared with HG16W group at the time of16week (p<0.05), and the levels of SOD and T-AOC were increased significantly in PB16W group compared with HG16W group(p<0.05).The expression levels of Iba-1in brain increased HG16W group after high fat diet for16weeks, but the expression levels of Iba-1in PB16W group decreased significantly compared with HG16W group.Conclusion:The high-fat-induced obese rats showed the characteristics of metabolic syndrome, such as dyslipidemia, insulin resistance, non-alcoholic fatty liver, along with the decrease of systemic antioxidant capacity, the state of chronic low-grade systemic inflammation, the activation of microglia in brain. The treatment with probucol can improve systemic insulin resistances and antioxidant reserves, reduce systemic inflammation and microglia activation in high-fat-induced obese rats. CHAPTER Ⅱ THE PROTECTIVE EFFECTS OF PROBUCOL PRETREATMENT ON CEREBRAL ISCHEMIA REPERFUSION INJURY (CIRI) IN HIGH-FAT-INDUCED OBESE RATSObjective:To observe the effect of probucol on antioxidant, anti-inflammatory and the activation of microglia in brain tissue after ischemia reperfusion injury in high-fat-induced obese rats, and to explore the neuroprotective mechanisms of probucol on cerebral ischemia reperfusion injury.Methods:160male SD rats were divided randomly into five groups:sham operation group(Sham, n=32), normal diet control group(NG, n=32), normal intervention group(NG+PB, n=32), high-fat diet control group(HG, n=32), high fat intervention group(HG+PB, n=32). NG+PB group, which were fed normal diet, were given a gavage of5%probucol suspension (100mg/d.kg)(5%probucol made with0.5%CMC) once a day for8weeks from the9th week. Sham and NG group, which were fed normal diet, were given a gavage of0.5%CMC of the same volume once a day for8weeks from the9th week. HG+PB group, which were fed with high-fat diet, were given a gavage of5%probucol suspension (100mg/d.kg)(5%probucol made with0.5%CMC) once a day for8weeks from the9th week. HG group, which were fed with high-fat diet, were given a gavage of0.5%CMC of the same volume once a day for8weeks from the9th week. At the end of16th week, the animal model of left MAC ischemia-reperfusion injury was made by suture-occluded method in NG, NG+PB, HG, HG+PB group, Sham group only perform vascular separation operation. After ischemia for2hours and reperfusion for24hours, all the rats were killed to collect the brain after neurological function scoring. The infarction volume was indicated by TTC stain, cerebral water content were measured by wet-dry weighting method, the expression of IL-1β, TNF-a, Iba-1was investigated by immunohistochemistry, the levels of IL-1β、TNF-α、SOD、MDA、NO、NOS、iNOS were measured in ischemia cortex.Results:Sham group without neurological deficit, the neurological function score was significantly higher in HG group than in NG group (P<0.05), the score was slightly lower in NG+PB group than in NG group(P>0.05), the score was significantly lower in HG+PB group than in HG group (P<0.05).For cerebral water content, NG+PB group was slightly higher than Sham group (P>0.05), but NG, HG, HG+PB group was significantly higher than Sham group (P<0.05), HG group was significantly higher than in NG group (P<0.05), and two intervention groups was significantly lower than the corresponding contral group (P0.05), the HG+PB group was higher than NG+PB group (P<0.05).Sham group have no infarct, The cerebral infarct volume in HG group was significantly higher than in NG group (P<0.05), The two intervention groups was significantly lower than corresponding contral group (P<0.05), The HG+PB group was higher than NG+PB group (P<0.05).The expression level of Iba-1, IL-1, TNF-a in ischemic cortex were higher than Sham group (P<0.05), HG group were higher than NG group(P<0.05), two intervention groups was lower than the corresponding contral group (P<0.05), HG+PB group was still higher than the NG+PB group (P<0.05).The levels of SOD in ischemia cortex after CIRI were decreased significantly compared with Sham group, the levels of MDA, IL-1β, TNF-α were increased significantly in ischemia cortex (P<0.05). The level of SOD in HG group was decreased than in NG group, and the levels of MDA, IL-1β, TNF-α were increased significantly in HG group (P<0.05). The levels of SOD in two intervention groups were higher than the corresponding contral group, while the levels of MDA, IL-1β, TNF-α were decreased significantly in two intervention groups (P<0.05). The levels of NO、NOS、iNOS in model group were higher than the Sham group (P<0.05), HG group was higher than in the NG group (P<0.05), two intervention groups was lower than the corresponding contral group (P<0.05), There were no significantly difference between NG+PB group and HG+PB group(P>0.05).Conclusion:The level of inflammatory factors and oxidative stress in ischemia cortex after CIRI were significantly higher in high-fat-induced obese rats than normal diet rats. Probucol pretreatment can increase antioxidative capacity, reduce the production of free radicals and inflammatory factors, and reduce the activation of microglia in high-fat-induced obese rats after CIRI. Probucol pretreatment can decrease the cerebral infarction volume and water content, and improve neurological function scor in high-fat-induced obese rats after CIRI. CHAPTER ⅢTHE EFFECTS OF PROBUCOL PRETREATMENT ON CELL APOPTOSIS AND RELATED PROTEIN EXPRESSION OF ISCHEMIA CORTEX IN OBESE RATS AFTER CIRIObjective:To observe the effect of probucol on cell apoptosis and related protein expression of ischemia cortex in high-fat-induced obese rats after CIRI, to explore further the protective mechanisms of probucol on cerebral ischemia-reperfusion injury.Methods:80male SD rats were divided randomly into five groups:sham operation group(Sham, n=16), normal diet control group(NG, n=16), normal intervention group(NG+PB, n=16), high-fat diet control group(HG, n=16), high fat intervention group(HG+PB, n=16). NG+PB group, which were fed normal diet, were given a gavage of5%probucol suspension (100mg/d.kg)(5%probucol made with0.5%CMC) once a day for8weeks from the9th week. Sham and NG group, which were fed normal diet, were given a gavage of0.5%CMC of the same volume once a day for8weeks from the9th week. HG+PB group, which were fed with high-fat diet, were given a gavage of5%probucol suspension (100mg/d.kg)(5%probucol made with0.5%CMC) once a day for8weeks from the9th week. HG group, which were fed with high-fat diet, were given a gavage of0.5%CMC of the same volume once a day for8weeks from the9th week. At the end of16th week, the A animal model of left MAC ischemia-reperfusion injury was made by suture-occluded method in NG, NG+PB, HG, HG+PB group, Sham group only perform vascular separation operation. After ischemia for2hours and reperfusion for24hours, all the rats were killed to collect the brain. The cell apoptosis was detected by TUNEL, and the expression of Bcl-2, caspase-3, Akt, p-Akt protein of ischemia cortex were measured by Western Blot.Results:Sham group showed apoptotic cells occasionally in cerebral cortex. The number of apoptotic cells of ischemic cortex increased significantly in HG group compared with HG group(P<0.05). The number of apoptosis cells decreased remarkably in NG+PB group and HG+PB group by the pretreatment of probucl (P<0.05), while the number of apoptotic cells in HG+PB group is still higher than in NG+PB group(P<0.05).The expression levels of Bcl-2of ischemia cortex have no significant change in NG group compared with the sham group (P>0.05), the expression levels of Bcl-2increased significantly in HG group compared with NG group (P<0.05). The expression levels of Bcl-2increased significantly in NG+PB group HG+PB group by the pretreatment of probucl (P<0.05). The expression of caspase-3protein of ischemic cortex was higher in NG, HG, NG+PB, HG+PB group than in Sham group (P<0.05). The expression decreased significantly in NG+PB group and HG+PB group by the pretreatment of probucl (P<0.05). The expression level of caspase-3in HG+PB group is still higher than NG+PB group(P<0.05). The expression levels of total Akt protein in cerebral cortex have no significant change between each groups(P>0.05). The levels of p-Akt (Ser473) protein in NG group increased slightly than Sham group (P>0.05), increased significantly in the other groups(P<0.05).Conclusion:Obesity can increase the expression levels of caspase-3protein, aggravate cell apoptosis after CIRI. Probucol pretreatment can increase the expression levels of Bcl-2, inhibit the activation of Caspase-dependent apoptotic pathway, promote Akt phosphorylation, activate PI3K/Akt signaling pathway, reduce cell apoptosis induced by ischemia-reperfusion. Probucol pretreatment has a neuroprotective effect on CIRI in high-fat-induced obese rats.