A Preliminary Study On Glycopathological Mechanisms Of Pancreatic Damage And Effects Of Chinese Herbal Compounds In Diabetic Rats

Backgrounds and aimsDiabetes mellitus is a very common endocrine and metabolic disease,and is one of the three difficult diseases.Its high incidence of complications induces a high fatality rate and high rate of deformity.China has the largest number of diabetics in the world,and the incidence of the disease is increasing all the time.Nowadays,we can control the high blood sugar efficiently through oral hypoglycemic drugs and insulin therapy,but we lack the prevention of chronic complications and treatment.At the same time,using oral hypoglycemic drugs continuously will lead to hyperinsulinemia,insulin resistance,drug failure,and so on.Therefore,we need a further study on the molecular mechanisms of diabetes to find more effective drugs.Using traditional Chinese medicine to prevent and cure diabetes has a long history and experience,there is a close relationship between the pancreas and spleen.Many scholars believe that hypo-function of the spleen is an important pathogenesis of Diabetes.Buzhong Yiqi decoction(BZYQD) is a very commonly used formula in traditional Chinese medicine to reinforce qi and invigorate spleen,BZYQD has received good curative effect to treat the gastric ulcer,diabetic gastroparesis,diabetic diarrhea and other diseases.It also has the effect of regulating immune system,body temperature,gastrointestinal function and anti-ulcer activity.Many studies showed that the composing prescriptions of BZYQD were reasonable,and its pharmacological actions were complex.It deserves our thorough study and will be used better to treat the diseases.Traditional Chinese medicine has unique advantage on treating diabetes, so therapy researches of diabetes on traditional Chinese medicine can not be ignored.The animal experiment is an important and indispensable aspect among the studies.At present,researchers always use STZ or alloxan to set up animal model of diabetes.In the TCM-based researches,yin-deficiency models or yang-deficiency models have been established,but model researches on diabetes with deficiency of both yin and yang were barely reported.Some scholars made type 2 diabetes with deficiency of both yin and yang by traditional Chinese herb,in the same way,we build the both yin and yang-deficiency model in type 1 diabetes mellitus.Deficiency of both yin and yang are almost all forms of end-stage diabetes.Golden Chamber Pill(GCKQ) has effects of nourishing the kidney-yin and Warming Yang and Invigorating Qi.It has received good curative effect in clinical application,and is especially suitable for the kidney Yin -Yang deficiency patients in end-stage diabetes.Thus GCKQ was used as counterevidence Chinese herbal compound in this study.In 1985,a striking finding was reported by Akira Kobata,i.e.the N-glycans in the IgG from patients with Rheumatoid arthritis were prominently deficient in galactose.Agalactosyl glycoforms of IgG may be considered to be more directly involved in Rheumatoid arthritis pathogenesis.This glycopathologic topic is expected to provide new insights into Rheumatoid arthritis pathogenesis.Now,the pathology of the sugar chains of protein is becoming a big research focus of life sciences.Many related research results have revealed much important life phenomenon that we could not explain in the past.Researchers have found that defects ofα-mannosidase and N-glycosylation inhibitor could induce cell pathological damage,and N-glycosylation was closely related with cell survival,proliferation, differentiation and endoplasmic reticulum stress.Hence,it is very important to carrying out researches on diabetes pathogenesis and TCM therapy in the research area.According to these theories,in this article we analyzed amylase activity, lectin-binding ratios of amylase,mannosidase activity,pancreas pathology, death mode of pancreas cells and effects of Buzhong Yiqi Pill(BZYQP) and Golden Chamber Pill(GCKQ),and had a preliminary study on glycopathological mechanisms of pancreatic damage and effects of Chinese herbal compounds in streptozotocin diabetic rats.Methods and Contents1.Diabetic models were induced by administrating streptozocin(STZ) and effects of BZYQPThe diabetic models were induced by twice intraperitoneal injection of 55mg/kg streptozotocin(STZ).We calculated the incidence and mortality, investigated the pathological changes of pancreas and multiple non-pancreas organs,and studied the value of oral glucose tolerance test(OGTT).The rats of BZYQP group were gavaged with Buzhong Yiqi aqueous solution for four weeks. We observed whether BZYQP could improve symptoms.2.Establishment of diabetes with deficiency of both yin and yang,and the effect of GCKQ and BZYQPDiabetes was induced by a twice middle doses intraperitoneal injection of streptozocin.Diabetes with deficiency of both yin and yang(YY) group were induced by traditional Chinese medicine decoction(Pericarpium Citri Reticulatae Viride 18g,ructus aurantii 18g,Rhizoma Zingiberis 18g and phelloden-dri cortex 18g) for four weeks to make the diabetic model with deficiency of both yin and yang after the diabetic model were established, the diagnosis was confirmed by analysis of defined signs and symptoms,After four weeks,yin and yang deficiency-golden chamber pill(YY-G) group were gavaged with GCKQ decoction.yin and yang deficiency-Buzhong Yiqi Pill(YY-B) group YY-B group were gavaged with BZYQP decoction.We detected the cortisol, FT3,insulin,thyroxin B₂ and 6-Keto-PGF_1α,and observed the pathological changes of pancreas and multiple non-pancre- as organs,and the effect of GCKQ and BZYQP.3.The pancreas pathological damage of diabetic rats-amylase activity, lectin-binding ratios of amylase andα-mannosidase activity,and the effect of GCKQ and BZYQPDiabetes was induced by a twice middle doses intraperitoneal injection of streptozocin.Diabetes with deficiency of both yin and yang were induced by Chinese medicine after diabetic model were established,the diagnosis was confirmed by analysis of defined signs and symptoms,the pancreas pathological damage was confirmed by histopathological examination,amylase activity andα-mannosidase activity were determined by colorimetric method,the N-glycan patterns of amylase were determined by lectin(ConA,PSA,PNA,LCA) affinity precipitation method.The aim of this study was to examine the relationship between pancreas pathological damage and above indexes,and had a preliminary study on glycopathological mechanisms of pancreatic damage and effects of Chinese herbal compounds(GCKQ and BZYQP) in streptozotocin diabetic rats.4.The pancreas pathological damage of diabetic rats-The study on the mode of pancreas cells death and the effect of GCKQ and BZYQPDiabetes was induced by a twice middle doses intraperitoneal injection of streptozocin.Diabetes with deficiency of both yin and yang were induced by Chinese medicine after diabetic model were established,the diagnosis was confirmed by analysis of defined signs and symptoms,the pancreas pathological damage was confirmed by histopathological examination.The isolation of DNA fragments were electrophoretically separated on agarose gel to detect pancreas cells was necrotic or apoptosis.The aim of this study was to examine the mode of pancreas cells death in streptozotocin diabetes rats,and had a preliminary study on glycopathological mechanisms of pancreatic damage and effects of Chinese herbal compounds(GCKQ and BZYQP) in streptozotocin diabetic rats.Results1.Injecting twice could increase incidence and reduce mortality.The animal models could maintain three months,there were pathological changes of pancreas and multiple non-pancreas organs,and they accorded with manifestations of diabetic animal models.After treating by BZYQD,the pathological sections showed that individual islet cells had regeneration phenomenon.Pancreatic acini and angiopathy were better than model group.2.The signs and symptoms of YY group were accord with the characteristic of yin and yang deficiency.The FT3 of YY group had a decreasing trend,no significant difference was seen between YY group and YY-B group,YY-G group. The cortisol of YY group were obviously higher than normal group(P<0.01), the cortisol of YY-B groups markedly lower than YY group(P<0.01),the cortisol of YY-G groups were decreased at different degrees.3.â‘ Amylase activity:The model group was significantly lower than normal group(P<0.01).The BZYQ group was markedly higher than model group(P<0.01). Obvious differences could be seen between YY group,model group and normal group(P<0.01,P<0.05).The YY-G group and YY-B group were markedly higher than YY group(P<0.01).â‘¡Lectin-binding ratios of amylase:The model group' s LCA,PNA-binding ratios of amylase were markedly higher than normal group(P<0.05,P<0.01),PSA, ConA-binding ratios of amylase showed a rising trend.The YY group' s LCA were markedly higher than normal group,The YY group' s PSA,PNA,ConA-binding ratios of amylase showed a rising trend.After treatment,the lectin-binding ratios of amylase of three treated groups were decreased at different degrees.③α- mannosidase activity:There was a decreasing tendency of model group, the BZ-YQ group had a increasing trend.The YY group was markedly lower than normal group(P<0.05).Theα-mannosidase activity of YY-G group,YY-B group showed a increasing trend.â‘£The pancreas pathological section results suggested that pancreatic acini and islet cells occurred pathological changes.Three treated groups were better than non-treatment group.4.The DNA fragments of model group and YY group were smear,no obvious DNA ladder was detected.The DNA smear of BZYQ group,YY-G group and YY-B group were weakened.Conclusion1.The diabetes animal models induced by streptozocin were steady and reliable.Using SD rats,moderate dose STZ and injecting twice could increase the incidence of diabetes model and reduce the mortality.There were pathological changes of pancreas and multiple non-pancreas organs,and they accorded with manifestations of diabetic animal models.OGTT had a better value than fasting blood glucose for making an estimate of the incidence of diabetes mellitus.Using OGTT could avoid missing diagnosis.BZYQD could improve symptom and pathological changes of pancreas.2.The signs and symptoms of YY group were accord with the characteristic of yin and yang deficiency.FT3 and cortisol reflected that cold syndrome and yin deficiency syndrome was primary double syndrome in YY group,the combination of traditional diabetes mellitus model with TCM herb was available, but the method still needs further research.Golden chamber pill(GCKQ) and Buzhong Yiqi Pill(BZYQP) could promote the secretion of insulin,while the effect of GCKQ was significantly better than BZYQP.Thus we could conjecture that GCKQ was suitable for yin and yang deficiency.The pathological sections of pancreas showed that islet cells of treatment group were better than YY group at different degree.Above the general state,indexes and pathological changes of rats,this method and the counterevidence were available.3.The result showed that amylase andα-mannosidase activities of model group and YY group had decreasing tendency,and amylase N-linked sugar chains were defected in model group and YY group.After treatment,some detection indexes had improved,which suggested GCKQ and BZYQP could improve the defects of N-linked sugar chains andα-mannosidase.With the pancreas pathological section results,we considered pancreas pathological damage related to defects of N-linked sugar chains,α-mannosidase activities and amylase activities. GCKQ and BZYQP may alleviate pancreas lesion through improvingα-mannosidase activities and N-linked sugar chain defects.4.According to the DNA fragments,we conjectured the pancreatic cells of model group and YY group occurring obvious DNA damnification,no obvious DNA ladder was detected,and they were necrotic but not apoptosis.BZYQP and GCKQ had functions of protecting DNA and treating necrosis by improving the defect N-linked sugar chain andα-mannosidase activities.