Effects Of Garlicin On Oxidative Stress And Protection Of Organs Function In Chronic Intermittent Hypoxia Mice With Diabetes Mellitus

Research background and purpose:Obstructive Sleep Apnea Hypopnea Syndrom(OSAHS)is a common clinical sleep-related breathing disorder,refers to occur repeatedly in the process of sleep at night on airway complete or incomplete obstruction and leads to frequent apnea and(or)lower ventilation.Chronic intermittent hypoxia(CIH)is the most important physical characteristics of OSAHS,it may induce the occurrence of diabetes mellitus(DM),especially type 2 diabetes(T2DM),is one of the risk factors that affect patients with OSAHS.Many studies have found that OSAHS often coexist with DM.And oxidative stress is the common pathophysiological process of OSAHS and T2DM,which may play an important role in the occurrence and progression of OSAHS in T2DM.OSAHS can induces insulin resistance(IR),hyperinsulinemia,glucoseand lipid metabolism disorder,and diabetes through the mechanism of oxidative stress,if the oxidative stress reaction was not suppressed,it can further development of DM complications and even worse;in DM patients,abnormal glucose metabolism and glucose lipid metabolism aggravate oxidative stress,which leads to a vicious cycle.Therefore,we should pay attention to the early diagnosis,early prevention and intervention treatment of sleep apnea hypopnea in diabetic patients,so as to improve long-term intermittent hypoxia.Garlic is the underground bulb of Allium sativum L.Garlic bulb is both a favorite condiment and a common plant medicine.It is a kind of medicinal and eating plant.Modern research has shown that garlic and its extract has many pharmacological activities,such as reducing blood sugar,anti-hypertensive,anti-hyperlipidemia,heat-clearing and detoxifying,anti-inflammatory and sterilizational,antithrombotic,anti-tumor,enhancing immunlity and health care effect,et al,and many studies have found that garlic and its extract garlicin can effectively improve the antioxidant enzyme activity,enhance the ability of radical scavenging and inhibit oxidative stress injury.However,there is no relevant research on garlicin in the treatment of chronic intermittent hypoxia and diabetes mellitus.In this dissertation,garlicin are studied in CIH,DM and CIH-DM mice by intervention therapy,to observe the mice’s weight,blood glucose,serum insulin,insulin resistance index,the activity of oxidase and antioxidase,the change of lipid metabolism and related enzymes,to clear the effect of garlicin in islet β cell,oxidative stress and the protection of organ function in chronic intermittent hypoxia in mice with diabetes mellitus,to provide some experimental reference for clinical prevention and treatment.Method:A total of 32 rats were divided into 8 groups,with 4 rats/group.1.Control(C)group mice model:The health mice were untreated and given standard laboratory diet and free access to water.Meanwhile the mice were kept in the non-specific pathogen environment during the experiment.2.Chronic intermittent hypoxia(CIH)group mice model:The health mice were provided standard laboratory diet and unlimited water,were treated in a hypobaric hypoxia chamber with 5-min cycles of 90 sec hypoxia(5%02)and 210 sec normoxia(21%02),8 h/day for 6 weeks during light exposure.The experient was performed between 9:00 a.m.to 5:00p.m.3.Diabetes Mellitus(DM)group mice model:The health mice were injected intraperitoneally with a single dose of streptozotocin(STZ,50mg/kg).A high-fat diet was used as a daily diet.A total of 1 week following injection with streptozotocin,the blood glucose level was detected in the tail vein blood using a glucose meter.If the blood glucose level exceeded 250mg/dl,rats were considered diabetic.Non-diabetic rats were excluded from the study.At least 3 rats were included in each group.4.Chronic intermittent hypoxia-Diabetes Mellitus(CIH-DM)group mice model:The health mice were subjected to the same protocol as the DM group.Rats confirmed positive for diabetes were subsequently exposed to the same hypoxia conditions for 6 weeks as aforementioned for the CIH group.5.Control-Garlicin(C-G)group mice model:During the entire experiment,the health mice were provided standard laboratory diet and unlimited water in the context of no specific pathogen and injected 500mg/kg/day of garlicin in the abdominal cavity daily for 7 weeks.6.Chronic intermittent hypoxia-Garlicin(CIH-G)group mice model:The health mice were injected with 500mg/kg of garlicin per day in the abdominal cavity and then into the same experimental procedure as the CIH group.7.Diabetes Mellitus-Garlicin(DM-G)group mice model:The health mice were Injected with 500mg/kg of garlicin per day in the abdominal cavity and then into the same experimental procedure as the DM group.8.Chronic intermittent hypoxia-Diabetesa Mellitus--Garlicin(CIH-DM-G)group mice model:The health mice were injected with 500mg/kg of garlicin per day in the abdominal cavity and then into the same experimental procedure as the DM group.A week later,after the mice were diagnosed with diabetes,they were exposed to the same low-oxygen environment as CIH for six weeks.This study was divided into three parts.Part One The effect of garlicin on the function of islet beta-cell in chronic intermittent hypoxia mice with diabetes mellitus.Objective:To research the effect of garlicin on blood glucose,serum insulin,and homeostasis model assessment of insulin resistance(HOMA-IR)in chronic intermittent hypoxia mice with diabetes mellitus,it is mainly to determine the influence of garlicin on the function of islet beta-cell in chronic intermittent hypoxiamice with diabetes mellitus.Methods:Weight,blood glucose,insulin levels and HOMA-IR in mice were measured 1 day prior to the experiment(day 0)and then weekly on days 7,14,21,28,35,42 and 49 of the experiment.Results:Rats’ weight increased gradually in C group,but slower in CIH,DM and CIH-DM than healthy rats(P<0.05).Blood glucose increased in the DM and CIH-DM groups compared with the healthy control group,while serum insulin levels increased in CIH group,but decreased in the DM and CIH-DM groups,resulting in increased HOMA-IR value in the CIH group,compared with healthy controls(P<0.05).Garlicin treatment improved weight gain,blood insulin and HOMA-IR in the DM-G and CIH-DM-G groups,reduced blood glucose in the DM-G and CIH-DM-G groups,and glood insulin and HOMA-IR in the CIH-G group,compared with the respective garlicin-untreated groups(P<0.05).Conclusions:① CIH,DM and CIH-DM were important factors affecting the weight gain of mice.Garlicin treatment can improved the weight of DM-G and CIH-DM-G mice,especially in mice induced by DM.② Blood glucose levels in DM or CIH-DM mice were increased significantly,and garlicin could helped to reduce blood glucose levels in mice induced by DM or CIH-DM,but the blood glucose still did not return to normal levels at the end of the experiment.③ CIH could increased serum insulin levels in mice,while DM or CIH-DM reduced insulin levels in mice.Garlicin treatment could reduced serum insulin levels in mice induced by CIH,and increased the levels in mice induced by DM or CIH-DM.④ Insulin resistance was more obviously in mice induced by CIH.Garlicin treatment could significantly improved insulin resistance in CIH-G,DM-G and CIH-DM-G mice.Part Two The effect of garlicin on systemic oxidative stress and organ function in chronic intermittent hypoxia mice with diabetes mellitus.Objective:Application of garlicin to treat chronic intermittent hypoxia diabetic mice,and further monitoring serology related indicators,including oxidase,antioxidant enzyme activity,protein,lipid,serum liver transaminase,and urine protein in the urine,the aim is to research the effects of garlicin on oxidative stress and protection of organs function in chronic intermittent hypoxia mice with diabetes mellitus,it provides experimental reference for disease prevention and treatment.Methods:The blood and 24 hours urine of the mice were collected after the experiment,then the serum indexes thiobarbituric acid reactive substances(TBARS),Nitric oxide(NO),Superoxide dismutase(SOD),glutathione S-transferase(GST),glutathione peroxidase(GST-Px),glutathione reductase(GR),uric acid(UA),albumin(ALB),total lipids(TL),aspartate aminotransferase(AST),alanine aminotransferase(ALT),lactate dehydrogenase(LDH),alkaline phosphatases(ALP),acid phosphatases(ACP),total cholesterol(TC),triglycerides(TG)and Urine protein(UP)were determined respectively.Results:Serum TBARS,GST,UA,TC,TG,TL,AST,ALT,LDH,ALP,ACP,and UP increased in the CIH,DM and CIH-DM groups,while serum SOD in the CIH,DM and CIH-DM groups,serum ALB in the DM,CIH-DM groups decreased compared with healthy controls(P<0.05).NO increased in the DM and CIH-DM groups but decreased in CIH group,compared with the control group(P<0.05).GSH-Px increased in the CIH group but decreased in the DM and CIH-DM groups,compared with the control group(P<0.05).GR increased in the DM group but decreased in the CIH and CIH-DM groups(P<0.05).Garlicin treatment increased serum SOD in CIH-G,DM-G and CIH-DM-G groups,GSH-Px and ALB in the DM-G and CIH-DM-G groups,compared with the respective Garlicin-untreated groups.Garlicin treatment decreased serum TBARS,UA,TC,TG,TL,AST,ALT,LDH,ACP and UP in the CIH-G,DM-G and CIH-DM-G groups;as well as to NO in the DM-G group,GST in the CIH-G and CIH-DM-G groups,GR in DM-G group,ALP in the DM-G and CIH-DM-G groups,compared with the respective Garlicin-untreated groups(P<0.05).Conclusions:① CIH,DM and CIH-DM could caused higher serum lipid peroxides TBARS,the same elevated indicators included serum transaminase,UA,blood lipids,UP levels.After garlicin treatment,the above indexes all decreased significantly.② The levels of antioxidant enzyme GSH-Px,GR and SOD in the serum of CIH,DM and CIH-DM groups were generally decreased.Garlicin could effectively improve the activity.Part Three The effect of garlicin on organs oxidative stress and protection in chronic intermittent hypoxia mice with diabetes mellitus.Objective:CIH,DM and CIH-DM groups of laboratory mice were treated with garlicin,after the expertment,the transaminase,related oxidase and antioxidant activity were detected in the experimental groups liver,kidney,and brain tissue,the aim is to research the effects of garlicin on organ oxidative stress and protection of organs function in CIH,DM and CIH-DM.Therefore,it provides experimental basis and experimental reference for the prevention and treatment of OSAHS,diabetes mellitus and complications.Methods:At the end of the experiment,all rats were sacrificed using sodium pentobarbital anesthesia according to the Guide for the Care and Use of Laboratory Animals.Blood and urine samples were collected,then th serum was separated.The liver,kidney,and brain were removed immediately and weighed.The organs were washed,cut into pieces,homogenized and centrifuged respectively.The supernatant was separated for subsequent enzyme assays.Liver TBARS,GST,GSH-Px,AST,ALT,LDH,ALP,ACP,catalase activity(CAT)and SOD levels were determined respectively;Determination of renal TBARS,GST,CAT,SOD levels and cerebral TBARS,GST and LDH levels.Results:Liver TBARS and GST increased,while AST,ALT,LDH,ALP,ACP,CAT and SOD decreased in the CIH,DM and CIH-DM groups,compared with the control group,liver GSH-Px decreased in the DM and CIH-DM groups,compared with the control group(P<0.05).Renal TBARS in the DM and CIH-DM groups increased compared with the control group,renal GST increased while CAT and SOD decreased in the CIH,DM and CIH-DM groups,compared with the control group(P<0.05).Cerebral TBARS increased in the CIH,DM and CIH-DM groups and LDH increased in the DM and CIH-DM groups,compared with the control group,cerebral LDH in CIH decreased compared with the control group(P<0.05).After garlicin treatment,liver AST,ALT,LDH,ALP,CAT,SOD in the CIH-G,DM-G and CIH-DM-G groups increased,GSH-Px increased in the DM-G and CIH-DM-G groups,ACP in the CIH-G and DM-G groups,renal CAT in the CIH-DM-G group,and renal SOD in the CIH-G and CIH-DM-G groups,compared with the respective garlicin-untreated groups(P<0.05).Liver and cerebral TBARS decreased in all garlicin-treated experimental groups,liver and renal GST,and cerebral LDH decreased in the DM-G and CIH-DM-G groups,compared with the respective Garlicin-untreated groups(P<0.05).Conclusions:① CIH,DM and CIH-DM could caused higher organs lipid peroxides,after garlicin treatment,the levels of lipid peroxides decreased significantly.② The levels of antioxidase GSH-Px,GR,SOD and CAT in the organs of CIH,DM and CIH-DM groups were generally decreased,while garlicin can effectively improved the activity.Summary,this study successfully used garlicin to treat mice with CIH,DM or CIH-DM.Through the analysis,CIH,DM and CIH-DM will induce mice islet Pcell dysfunction,insulin resistance,resulting in abnormal blood glucose,at the same time,it can induce liver cell damage,lipid metabolic disorders,kidney damage and brain metabolic disorders.But garlicin has the ability to improve antioxidase activity,remove superoxide anion,inhibit oxidative stress damage,and it has regulation effect in protecting organs from damage and metabolic disorders.Therefore,this study found that garlicin has antioxidant properties,detoxification and protect the cells and organs from damage,it also found that garlicin can effectively regulate and improve the CIH,DM,or CIH-DM mice body weight,blood glucose,serum insulin,insulin resistance and lipid metabolism imbalance.According to the analysis,it believes that garlicin has a significant effect in treating CIH,DM or CIH-DM,and it can prevent the occurrence and development of CIH,DM or CIH-DM to some extent.