| PART 1 Effectof mast cell stabilizer on pancreatic function of sreptozotocin-induced type 2 diabetic ratsObjective:As the socio-economic development and the improvement of living standards,the incidence of diabetes worldwide is an upward trend.In the large and medium-sized cities of China,the prevalence of diabetes is also rising.With recent years,some second-tier cities,the incidence of diabetes is also increasing.Number of diabetics’ people in China is about 40 million,the proportion of which more than 90% of Type 2 Diabetes Mellitus(T2DM).The long-term high sugar-fat diet induced insulin resistance(IR)precisely is the crux of T2 DM pathogenesis.By feeding the highsugar-fat diet and injecting STZ,it’s successful to establish a diabetic rat model.After Ketotifen treatment 8 weeks,to investigate the effect and mechanism ofmast cell stabilizer(Ketotifen)on pancreatic function of streptozotocin-induced type 2 diabetic rats.Methods: Male SD rats,which were 6-8 weeks old and weighted about 160-180 g,were housed in SPF level of the Experimental Animal Center of Wenzhou Medical.After the adaptive feeding one week,they were randomly divided into normal diet group which included 16 rats,and high sugar-fat diet group(HF)which consist of 20 rats.About 8 weeks feeding,HF group rats were injected STZ by abdominal cavity.If the fasting blood-glucose value was greater than 13mmol/L,it was successful to establish T2 DM rat model.There were 16 rats of HF group meeting the above criteria.The 16 rats were randomly divided into diabetic control group(DM)and ketotifen treatment of diabetic group(KTDM).And the normal diet group was randomly divided into blank control group(NC)and Ketotifen drug group(KT).The daily dosage of Ketotifen was 0.09g/kg.The same dose of physiological saline was given NC and DM group by intragastric administration.After lasting 8 weeks treatment,four groups were all sacrificed.We get blood samples to detect FBG and FINS for calculating ISI and HOME-IR,meantime,to measure serum IL-6 and TNF-a.According to Kit instructions,the activity of mitochondria SDH and CCO was measured.At the same time,it’s observed that morphological changes of pancreatic islets in the light microscope and electron microscope.Result:1.Compared with the NC and KT group,fasting glucose and insulin levels of DM ratssignificantly increased(P<0.01),the ISI obviously decreasedand HOMA-IR significantly rose(P<0.05).Contrasted with DM rats,KTDM rats blood glucose and insulin levels declined,ISI rebound,HOMA-IR decreased,there were statistically significant difference(P<0.05),but was higher than the NC group and KT group(P<0.05),which could show that: ketotifen can improve the state of the glucose metabolism and insulin resistanceof diabetic rats,but have not been able to recover normal.NC group and the KT group’s blood glucose and insulin levels was not statistically significant(P>0.05),which should be explained,ketotifen had no effect on normal rats blood sugar and insulin secretion.2.Compared with NC and KT group,DM rats the activity of mitochondria SDH and CCO decreased significantly(P<0.05),which showed diabetic rat pancreas mitochondrial fuction impaired.Given ketotifen treatment for 8 weeks,KTDM rats’ s activity rebounded significantly(P<0.05),which suggested that pancreatic mitochondrial function get some degree of improvement.Between the NC group and KT group,there had no statistically significant difference(P> 0.05),which illustrated ketotifen had no effect on normal rat mitochondrial function.3.Compared with NC and KT group,DM serum IL-6 and TNF –α expression levelswere significantly increased(P<0.01),which implied diabetic rats in the inflammatory state.After ketotifen treatment,serum IL-6 and TNF-α levels significantly declined(P<0.01,P<0.05),but is higher compared with the normal control group(P<0.01,P<0.05),which showed that ketotifen,to some extent,can improve diabetes rats inflammatory state.4.Light microscope: NCand KT group of islet displayed normal morphology.DM groupmajority of islet cell showed the degenerative and necroticchanges,islet irregularorientation and structural disorder.But the structure of KTDM groups appeared a good recovery.5.Transmission electron microscope: the NCand KT group of β-cell had ample endocrine particles and larger nucleolus,which was oval and karyotheca smooth.And DM group showed that secretory granules amount significantly reduced,dense core density decreased,the mitochondriaswelled,nuclei deformed,nuclear membrane was not smooth,and endoplasmic reticulumexpanded.The KTDM group displayed significantlyimproved and increased secretory granules and gradually restored dense core density.Conclusion:Ketotifen intervention decreased significantly IL-6 and TNF –α expression levels,alleviated diabetes inflammatory process,further lowered islet cellmitochondria injury from inflammatory cytokinesmediating damage,and lessened β-cell apoptosis and insulin resistance.Meanwhile,blood glucose value decreased and insulin sensitivity enhanced,which indicated islet function improved.Light microscopy and electron microscopy results also indirectly proved islet function had been restored to some extent,which manifested that anti-inflammatory effect was very favorable for islet cell repair process.PART 2 Effect and mechanism studies of mast cell stabilizer on pancreatic function of newly diagnosed type 2 diabetesObjective: To investigate the effect and mechanism of mast cell stabilizer on pancreatic function of newly diagnosed type 2 diabetes.Methods: 32 newly diagnosed type 2 diabetes mellitus patients in outpatient of our department of Endocrinology were selected and were divided into experimental group and control group.The criteria were as follows: type 2 diabetes was defined according to the WHO 1999 criteria;fasting blood glucose(FBG)≥10mmol·L-1,glycated hemoglobin(Hb A1c)≥10%;aged 18-60 years,body mass index(BMI)between 25 and 30kg·m-2 among newly diagnosed type 2 diabetes mellitus.The experimental group was treated with humalog 25 and ketotifen while the control group was treated with humalog 25 alone to control blood glucose.16 patients with type 2 diabetes were selected as the initial diabetic control group.The criteria were as follows: type 2 diabetes was defined according to the WHO 1999 criteria;fasting blood glucose(FBG)between 7 and 10 mmol·L-1,glycosylated hemoglobin(Hb A1c)in 7-10%;aged 18-60 years,body mass index(BMI)between 25 and 30kg·m-2.In addition,16 cases of physical examination center in our hospital were selected as healthy control group.The time period(0,4,8,12 weeks)were detected: a)Evaluate islet function: Instant noodle meal test: Fasting for more than 8 hours,400 m L of boiling water was added to the content of a package of instant noodles(75 g)in the morning.Then,venous blood was taken at 5 time points(before meal,after meal,0.5,1,2 and 3h).Blood glucose,insulin,C peptide levels,and other peripheral blood parameters were measured,including IL-6,IL-1,FFA,blood lipids,Hb A1 c,histamine,adiponectin,leukotrienes,human trypsin,chymase,SOD,MDA,etc.Peripheral blood flow cytometry was used to detect changes in mast cells.HOMA-IR and HOMA-β,HOMA-IR =(FINS*FPG)/22.5,HOMA-β =(FINS * 20)/(FPG-3.5)were measured,blood pressure,waist circumference,height,weight,and BMI were measured and calculated.Results:1.Blood glucose,lipid profile,Hb A1 c level in newly diagnosed type 2 diabetes patients were significantly increased than that in healthy control group and the initial diabetic control group(P<0.05).After treatment,these items were improved significantly.Although the blood glucose levels of the experimental group and the experimental control group were similar,the Hb A1 c of the experimental group was significantly lower than that of the experimental control group(P<0.05).The change of blood lipid spectrum was not statistically significant.2.Inflammatory markers: IL-6,IL-1β,TNF-α,leukotriene hs-CRP in newly diagnosed type 2 diabetes patients were significantly increased than that in healthy control group and the initial diabetic control group(P<0.05).After treatment,these items were improved significantly(P<0.05).The improvement of the experimental group was more obvious than that of the experimental group(P<0.05).3.The experimental group and experimental control group was significantly lower than the normal control group(P<0.05)at the level of adiponectin,which has the effect of anti-inflammatory.Even compared with the initial diabetic control group,there was a marked reduction(P<0.05).After treatment the level increased,and the experimental group increased more amplitude at the beginning.4.Islet function: fasting insulin,C peptide levels in newly diagnosed type 2 diabetes patients was significantly increased than that in healthy control group(P<0.05).This reminds that newly diagnosed type 2 diabetes patients had hyperinsulinemia,but the level of postprandial insulin and C peptide release could not reach normal release rate.After treatment,the function of the islets was restored obviously,and the experimental group had better curative effect than the experimental control group.5.Oxidative stress index: compared with healthy control group and the initial diabetic control group,the experimental group with SOD level decreased significantly,and MDA level increased significantly(P<0.05).After treatment,all have obvious recovery.In the detection of mast cells,peripheral blood loss cell test indicated that peripheral blood mast cells increased in patients with type 2 diabetes mellitus,and decreased after treatment.6.The numbers mast cells and the strength of TLR4 expressed by mast cells in peripheral blood and blood glucose levels,inflammation,oxidative stress,etc,are closely related.Mast cell activity: tryptase and chymase in the experimental group were significantly increased than that in healthy control group and the initial diabetic control group.After 12 weeks of treatment,the levels of tryptase and chymase decreased significantly,and the experimental group was more significantly than the experimental control group(P<0.05).Conclusion:1.The numbers of mast cells and TLR4 level expressed by mast cells in peripheral blood are related to blood glucose,inflammatory biomarkers and oxidative stress.2.Islet function in patients with newly diagnosed type 2 diabetes mellitus improved with ketotifen and insulin treatment,while the inflammatory and oxidative stress indices decreased gradually.3.Ketotifen may protect against pancreatic islet dysfunction by inhibiting the expression of the TLR4 signaling system and achieving anti-inflammatory and antioxidant stress effects. |
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