The Effect Of Compound Guben Huayu Tongluo Containing Serum On The Podocyte Injury Induced By High Glucose

Objective: Diabetic kidney diseases is a serious chronic complication of diabetes mellitus,which is the main systemic microvascular disease in diabetes mellitus.With a high incidence and insidious onset,it be the main causes of premature death in patients with diabetes,has been a serious threat to human health.The pathogenesis of diabetic kidney diseases?DKD?is very complex.It involves many factors,such as the metabolism disorder of glucose and lipid,abnormal renal hemodynamics,cytokines activation and so on.Genetic predisposition has also been shown to be involved.Proteinuria is the most common early clinical symptoms of diabetic kidney diseases,which is also an important reason of kidney damage cause by diabetes mellitus.Pathophysiologic changes of the thickening of the glomerular basement membrane and expansion of the mesangial area have long been considered at the heart of presence of proteinuria in diabetic kidney diseases,but many studies in recent years have confirmed that diabetic kidney diseases is essentially a podocytopathy,podocyte injury occurred throughout the whole process of the occurrence and development of diabetic kidney diseases,stabilizing the morphology and function of podocyte become a key to control proteinuria in diabetic kidney diseases.Combining the basic theory of Chinese medicine of yin-Yang and the clinical practice experiences,Professor Ye proposed the primary pathogenesis of diabetic kidney diseases is deficiency of spleen and kidney,intermingled phlem and blood stasis,channels stasis.On the basis of this,our research group has established a traditional Chinese medicine compound that named Compound Guben Huayu Tongluo,which effect is invigorating spleen and tonifying kidney,resolving phlegm and removing stasis,promoting blood circulation to remove meridian obstruction,harmonizing yin and yang.In the preliminary clinical observation,we used the method of Guben Huayu Tongluo as the treatment of diabetic kidney diseases,it has been proved that the compound can significantly reduce the urinary protein excretion rate in patients with diabetic kidney diseases,and the effect is better than that of Angiotensin II receptor antagonist?ARB?Irbesartan.In this study,we intend to establish a cell model of podocyte with hyperglycemia intervention,prepared serum containing Compound Guben Huayu Tongluo by serum pharmacological methods and apply it to the cell model,The abnormal changes of podocyte morphology and cell apoptosis were regarded as markers of podocyte injury,to observe the effects of Compound Guben Huayu Tongluo on podocyte injury under high glucose environment,to further verify its clinical efficacy,so as to provide a theoretical basis for Compound Guben Huayu Tongluo applying to the treatment of proteinuria in diabetic kidney disease.Methods: 1.Preparation of Compound Guben Huayu Tongluo containing serum and normal rat serum.Referring to the principle of serum pharmacology,20 healthy male SD rats of 6⁸ weeks old,SPF grade with body weight of 200±20g were selected.After a week of adaptive feeding,they were randomly divided into Compound Guben Huayu Tongluo group?Chinese medicine group?and blank control group?10 per group?.The rats in the Chinese medicine group was intragastric injection administration with Compound Guben Huayu Tongluo,which dose equivalent to 1 times human clinical equivalent dose.While the blank control group was given the same volume of normal saline.The dosage of gastric lavage was converted to 1ml/100 g weight,once a day,for 7 days in arow.1hour after the last intragastric administration?before gavage fasting for 12 hours but do not restricting water?,blood samples of each group were collected from abdominal aorta at the same time.Isolating serum separat after centrifugation,then filtration sterilization by micro filtration membrane with pore size of 0.22?m,56 ? water bath inactivated 30 min,packaged into small copies,stored at-80 ? standby.2.Culture and identification of podocyte Cultivating conditional immortalized mouse podocytes,The undifferentiated podocytes were cultured in RMPI-1640 medium containing 10% fetal bovine serum?FBS?,10 U / ml mouse recombinant IFN-?,1%?penicillin and streptomycin?double solution?which consist of 100 U / ml penicillin,100 ?g / ml streptomycin?at 33 ?,5% CO₂ incubator.After subculture,the cells were induced to differentiate in RMPI-1640 medium containing 10% FBS,1% double solution,but without added mouse recombinant IFN-? for 2 weeks at 37 ?,5% CO₂ incubator.The cells were identified by immunofluorescence technique,and the podocyte specific proteins nephrin,podocin and podocalyxin were selected,the identification results showed that the cells obtained were in accordance with the experimental requirements of podocyte.When podocytes become differentiation and maturation,the cells were inoculated in 6 holes with a density of about 1×10⁵¹×10⁶ cells /ml.Adherent for 24 hours,the serum-free medium of RMPI-1640 was used for synchronization for a period of 24 hours,cells can be used for subsequent experiments.3.the effects of Compound Guben Huayu Tongluo containing serum on podocyte injury under high glucose environment After synchronization treatment,the mature podocytes were randomly divided into the following four groups for drug intervention:?1?high glucose control group?Control?: high glucose medium?RMPI-1640 medium containing 30mmol/L Dglucose?+ 10% normal rat serum;?2?low concentration of Compound Guben Huayu Tongluo containing serum?low concentration?: high glucose medium?RMPI-1640 medium containing 30mmol/L D-glucose?+ 2.5% Compound Guben Huayu Tongluo containing serum;?3?moderate concentration of Compound Guben Huayu Tongluo containing serum group?moderate concentration?: high-sugar medium?RMPI-1640 medium containing 30mmol/L D-glucose?+ 5% Compound Guben Huayu Tongluo containing serum;?4?high concentration of Guben Huayu Tongluo ricipe containing serum group?high concentration?: high glucose medium?RMPI-1640 medium containing 30mmol/L D-glucose?+ 10% Compound Guben Huayu Tongluo containing serum.After 48 hours of intervention at 37 ?,the morphological changes of podocytes were observed under the inverted phase contrast microscope,CFSE/PI double staining was used to measure the viability of podocytes.Annexin V-FITC/PI double staining combined with flow cytometry were used to detect the apoptosis of podocytes.to observe the effect of Compound Guben Huayu Tongluo containing serum on podocytes injury under high glucose environment.Results: 1.Morphological observation of podocytes in inverted phase contrast microscope:After 48 h of high glucose stimulated,podocytes in high glucose group showed more obvious cell damage,sunch as cells shrunken and deformed,the volume became smaller,the vacuoles formed in the cytoplasm,the FPs were short,fused or disappeared,and the number of cells decreased.The cell morphology of the three groups treated with Compound Guben Huayu Tongluo containing serum was higher than that of the sugar group,The number of podocytes increased,the cell body did not shrink,the vacuoles in the cytoplasm decreased,and the arrangement of the podocytes was more compact.Among them,the high concentration of traditional Chinese medicine containing serum cells of the best form.2.CFSE/PI double staining cytotoxicity test:After 48 h of high glucose stimulated,The number of dead cells in the high glucose control group was significantly higher than that in the traditional Chinese medicine treatment group.Among them,there is only a few dead cells in the high concentration of Compound Guben Huayu Tongluo containing serum group.3.Flow cytometry?Annexin V-FITC/PI double staining?apoptosis detection: After 48 h of high glucose stimulation,The apoptosis of podocytes in the low concentration ?moderate concentration and high concentration of Compound Guben Huayu Tongluo containing serum group was significantly lower than that in high glucose group,the difference was statistically significant?P < 0.05?.In addition,the percentage of normal podocytes in the moderate concentration and high concentration of Compound Guben Huayu Tongluo containing serum group was significantly higher than that in the high glucose group,the difference was statistically significant?P < 0.05?,there was no significant difference in the percentage of normal podocytes between the low concentration of Compound Guben Huayu Tongluo containing serum group and the high glucose group?P>0.05?.Conclusion: 1?The serum contained Compound Guben Huayu Tongluo can significantly improve the morphological changes of podocyte injury induced by high glucose stimulation,and has a certain concentration gradient dependence.2? High glucose intervention can induce podocyte apoptosis,the serum contained Compound Guben Huayu Tongluo can reduce the apoptosis of podocytes induced by high glucose and improve the ratio of normal cells,the specific mechanism remains to be further explored.