A Study On The Effect Of Electroacupuncture On The Rat Models Of DM2 Combined With Renal Hypertension

Background:Diadetes mellitus is a kind of chronic metabolic disorder disease characterized by a chronically raised blood glucose level and it can be divided into type 1 and type 2 diabetes in clinical practice according to the types of disease.Type 2 diabetes mellitus(T2DM) which is also called the non insulin dependent diabetes mellitusis (NIDDM)almost occupied for 90% of diabetic.Persistent high blood sugar (glucose) levels and long-term metabolic disorders result in the damage to the tissues and organs of the whole body especially eyes, kidney, cardiovascular,nervous system as well as its dysfunction and failure,among them the most serious phenomenon is the atherosclerosis which will block the major blood vessels by the blood clot which will stay in the renal artery to narrow or block the main renal artery or its branches,causing renal hypertension by decrease the renal blood flow.At present there are so many methods on clinical treatment for the diabetes and renal hypertension but the therapeutic effects is not so satisfactory,such as lower blood sugar levels by injecting the insulin and lower the blood pressure by taking anti-hypertensive drug.They cannot reach the goal of treatment because forgetting to take drugs or stop taking medications for the side effect.Objective:This research verify the curative effect of acupuncture therapy for T2DM combined with renal hypertension through the continued electroacupuncture to the Zusanli point of the rats model. We establish the rats model firstly and evaluate it by the result of physiology, pathology and iconography,and test its stability and sustainability.Next we exert high frequency electroacupuncture on the rats of compound model and detect the change of blood glucose, blood pressure and the value of GHbAlc,PRA,AngⅢ to verify the curative effect and provide a new basis for the clinical treatment and prevention of the disease of DM2 combined with renal hypertension.Methods:Part 1 The establishment and assessment of the model of type 2 diabetes mellitus(T2DM) complicated renal hypertensionFirst,we provide a high fat and sugar diet for the rats of model and provide an ordinary diet for the rats in blank control group as a comparison then the OGTT is taken on the rats of model 4 weeks later.When the insulin resistance is come into being,we take an intraperitoneal injection of STZ on rats at the dose of 28mg/kg,on the same way the rats in blank control group is treated by Citric acid and sodium citrate buffer at the same dose.Two weeks later the model is established successfully with the measure of FBG and RBG(FBG>7.8mmol/L,RBG>16.7mmol/L).Second,The rats above are randomly divided into diabetes mellitus group (diabetic rats), sham operation group(diabetic rats which perform laparotomy but without operation), compound group (diabetic rats with renal artery ligation).Among the compound group after anesthesia with right lateral were sterilized on their skin. A right subcostal incision about 2cm is cut off along the left side of ribs paralleling to spine about lcm until enterocoelia layer by layer.Wrapped the bare kidney in gauze wet out by NS(normal saline) in case the kidney loss water and make sure there isn’t a renal pedicle torsion.Adequately expose the kidney and aorta abdominalis and use the cotton swab to wipe fascia which wrap the outboard of renal artery and vein and separate them by glass needle in the bifurcation of abdominal aorta and renal artery until 0.5-1.0cm to thread the needle for preparation.Put the acupuncture needle with 0.25 mm diameter on one side of renal artery and parallel to renal artery,then draw out the acupuncture needle after ligature it by 3-0stylolite. Sprinkle the penicillin sodium powder after the operation for anti-inflammatory therapy for a week.The sham operation group is same with the compound group except the the ligation of left renal artery.We got the measured value of SBP>140mmHg and FBG>7.8mmol/L or RBG≥16.7mmol/L for consecutive 3 days two weeks later which indicate the formation of the model.Final,we evaluate the model by the multiple index in the subject of physiology, biochemistry, anatomy, pathology and iconography.Part 2 A study on the effect of electroacupuncture on the rat models of DM 2 combined with renal hypertension.We divided the rats into five groups:simple diabetes group,simple renal hypertension group,compound group without electric acupuncture,the compound group with electric acupuncture(All the rats are prepared again except the rats in compound group)Detect and record the level of Blood glucose.Blood pressure,GHbA1c,PRA,AngⅡ, Pathology.Through the comparison of the indicator of physiology,biochemistry,pathology to evaluate the treatment effect of electric acupuncture in the model of DM2 combined with renal hypertension.Results:Part 1 The establishment and assessment of the model of type 2 diabetes mellitus(T2DM) complicated renal hypertension.1. General behavior observationThe rats in blank control group has a glossy and clean fur and a good mental state and its weight has an obvious increase with the raising cycle and its state is fundamentally the same with other groups except the increase of ingestion,drink or fecal and urinary,the slow gain of weight,and part of them loose its weight.Compare to the rats in diabetes group and sham-operation group,the compound group have a mental strain and their fur are lack of gloss and part of them turn yellow and easy to fall off.Furthermore,the urinary incontinence defecation is very common especially the phenomenon of sloppy soft stool with putrefactive odor and an increase in amount of urine and dung.2. The change of body weightThere are no differences in weight between each groups before but there is a statistically significant increase (p<0.05)in weight after the high fat and high sugar diet compare to the blank control group. There is also an obvious statistics difference between the STZ group which have a continuously increase and the model group with a decreasing weight.3. OGTT, FBG and GHbAlc valueThe change of OGTT in rats.Results:In the blank control group there is an increase and a decrease of blood glucose during the time of 0.5h,1h and 2h and it comes to a normal level at last during the 2 hours.It has a significant difference in blood glucose at time points(P<0.05).There in an unconspicuous change of fasting blood glucose in both groups after the high fat and sugar diet for 4 weeks (P>0.05)while there is a significant statistical difference between the rats model with an obviously increase in blood glucose and the rats in blank control group whose change of blood glucose is not obvious (P<0.01).The change level of GHbAlc in rats.The rats model have a significant statistical increase in glycated hemoglobin level compare to the blank control after the measurement of serum seperated from the Orbital venous blood (P<0.01)4. Blood pressure valueThere is a significant statistical difference (P<0.01)in the increase of systolic blood pressure and diastolic blood pressure in compound groups compared with the former three groups.There is no significant statistical difference (P>0.05)in systolic blood pressure and diastolic blood pressure among the blank control group,diabetes group and sham group.5. Color ultrasonographyThe result show the number 1,2,3,4respeatively indicate the size of kidney which is 2.19×1.01cm,2.32×1.08cm,2.15×1.06cm and 1.39×0.70cm.Apparently the kidney of the former three groups is larger than the compound group and it indicates the shrink of the kidney after ligating the renal artery in compound group.The PSV and EDV in No.5,6,7,8 represent the blood flow velocity.RI stands for the vascular resistance.The blood flow velocity in the former three groups has increased but vascular resistance has decreased compare with the compound group and it indicates the decreased diameter of renal artery and blood flow velocity after the ligation.6. Comparison between groups, serum creatinine (Cr) and urea nitrogen (BUN) values.We collect the blood of all rats and measure the BUN and Cr,the results are show in the table 5 and there isn’t significant differences between each groups (P>0.05).7. Pathological observationsThe Pancreas HE staining results show that there is a great number of oval cells with plenty of plasma in blank control group and we have also observed an increase of islet cell volume, vacuolar degeneration to varying degrees and part of nuclear pyknosis necrosis in the rest three groups.HE staining results of left kidney show that there is atrophy to varying degrees in both renal tubules and glomeruli in compound group,especially he trenal tubule atrophy which is obviously and accompanied by renal interstitial fibrosis.Moreover,there isn’t an obvious atrophy about the renal tubules and glomeruli in the rest three groups.Part 2 A study on the effect of electroacupuncture on the rat models of DM 2 combined with renal hypertension.1. observation of generally behaviorThe rats of blank group and the group with simple renal hypertension have the good mental state and the smooth and clean hair,and their weight has increased with the feeding time pass by. Compared with the two groups above, ingestion,drinking and excretion of the rats in the other three groups have increased, the gain of weight is slow and even part of the rats had weight loss.Compared with other rats,the rats in composite group became more upset and the hair is less shiny and easy to fall off.The phenomenon of enuresis and excretion is very common when the rats are grasped and the stool with obvious fusty-smelling become soft and even loose.The amount of urine and dung has increased.After the electric acupuncture intervention,part of the rats in the composite group with electric acupuncture intervention have died and their ambilateral knee-joint have extravasated blood. The rest of the group had no obvious abnormal phenomenon.2. The change of FBG in rats.From the analysis of variance(ANOVA)in repeated measurements we can reach such conclusion:FBG in different group has significant difference(P<0.05)Through the pairwise comparison,FBG of rats in simple DM group and compound group with electroacupuncture have dramatic decline(P<0.01)compared with electroacupuncture intervention before.After two weeks’electroacupuncture intervention, the FBG in simple DM group and compound group with electroacupuncture have dramatic decline (P<0.01)compared with electroacupuncture intervention before.There are no statistically significant differences (P>0.05) of FBG in the blank control group.the simple renal hypertension group and the compound group without acupuncture intervention.3. measurement of blood pressure.From the analysis of variance(ANOVA)in repeated measurements we can reach such conclusion:Blood pressure in different group has significant difference(P<0.05)Through the pairwise comparison,systolic blood pressure and diastolic blood pressure in simple renal hypertension group and compound group with electroacupuncture have significant difference(P<0.01)compared with electroacupuncture intervention before.The systolic blood pressure and diastolic blood pressure in simple DM group,blank control group and compound group without electroacupuncture have no significant difference(P>0.05).4. The change of GHbA1c、PRA、AngH in ratsAll the rats take venous blood collection in orbit and separate the blood serum to measure the concentration of GHbAlc,PRA,AngII.From the analysis of variance(ANOVA)in repeated measurements we can reach the conclusion that there are significant differences in the value of GHbAlc、PRA、AngⅡ among different groups(P<0.05).Through the pairwise comparison.comparing with the value before the electric acupuncture intervention,it has an obvious decline in the value of GHbAlc in the blank control group and compound group a week later(P<0.01).There is also a significant decline in the level of GHbAlc two weeks after the electric acupuncture intervention in the simple DM group and compound group with acupuncture intervention.Otherwise,there is a significant decline in the level of PRA, Ang Ⅱ in the simple renal hypertension group and compound group with acupuncture intervention (P<0.01) a week later.There is also a significant decline in the level of PRA and Ang Ⅱ two weeks after the electric acupuncture intervention in the simple DM group and compound group with acupuncture intervention.5. Serum creatinine (Cr), urea nitrogen (BUN) value.After the blood collection and the separation of blood serum in each group.we measure to get the Cr and BUN.There are no statistically significant differences of renal function in each groups (P>0.05).6. Pathology observation.6.1 HE-staining of pancreasResults:Islet cells of rats in the blank control group and simple renal hypertension group mainly to be the oval cells with the great number and rich plasm.The The islet cell volume in the other three groups have increased and we also find the vacuolar degeneration in varying degrees and karyopyknosis in part of cells.6.2 HE-staining of renalResults:the left side of renal tubules and glomerulus have atrophy in varying degrees in the simple renal hypertension group.compound group with electroacupuncture and the compound group without electroacupuncture.The atrophy and disappear of renal tubules in the left side companied with renal interstitial fibrosis is very obvious.The right side of renal in the other three groups has compensatory increase with the performance of hyperplasia and hypertrophy in renal tubular cell.There are no obvious renal lesions in the rest two groups.Conclusion:To sum up, the model of type 2 diabetes mellitus(T2DM) complicated renal hypertension we established successfully can simulate the clinical signs and symptoms of the disease.In addition,this study also verified acupuncture can treat the model of type 2 diabetes mellitus complicated renal hypertension and inhibit the progress of type 2 diabetes mellitus(T2DM) complicated renal hypertension and its possible mechanism has already discussed.