| The reabsorption function of proximal tubule is not only one of the most importantindex of kidney function, but also plays an important role in the excretion andreabsorption of drugs in vivo. The method of Supplementing Qi and Activating BloodCirculation of Traditional Chinese Medicine is a very important therapy for DiabeticNephropathy (DN) leads function injury of renal tubular reabsorption. In present study,the influence of couplet medicine of Huangqi and Danshen, which is a representative ofSupplementing Qi and Activating Blood Circulation, on reabsorption function of renaltubule in DN rats and high glucose harmed renal tubular epithelial cell (NRK-52E)has been investigated.PART IImprovements of Huangqi and Danshen combination on reabsorption function ofrenal tubule in DN ratsObjective： To investigate the effect of Huangqi and Danshen combination onproteinuria, kidney function, and tubular reabsorption in rat model of DN.Methods: The rat model of DN was established by intraperitoneal injection ofstreptozotocin (STZ,55mg/kg). The DN rats were randomly assigned to the modelgroup, combined administration group with Huangqi and Danshen by intraperitonealinjection, or positive control group with Gliquidone, which was a referencehypoglycemic drug, by intragastric administration, with the normal rats as the blankcontrol group. Physiological and biochemical indexes of24h urinary protein excretion,plasma glucose, serum creatinine (Cr), blood urea nitrogen (BUN), serum triglyceride(TG), and serum total cholesterol (T-CHO) were measured at regular time during thetherapeutic procedure of6weeks, as well as the Lipid Peroxidation (LPO), Superoxide Dismutase (SOD), Glutathion Peroxidase (GSH-Px), and Advanced GlycationEndoproducts (AGEs) in renal tissue homogenate. Hematoxylin-eosin (HE) andPeriodic Acid-schiff (PAS) stain were used to observe the structural changes in renal.After6weeks administration, fluorescein isothiocyanate labeled bovine serum albumin(FITC-BSA) was tail intravenous injected to investigate the reabsorption function,through the fluorescence microscope observation, and the megalin expression level inrenal tubular epithelial cell was detected by immunohistochemistry stain (IHC).Result: After DN model preparation, the plasma glucose,24h urinary protein excretion,Cr, BUN, TG and T-CHO levels in serum, and LPO and AGEs levels in renal tissuehomogenate of STZ-induced diabetic rats were significantly higher than those of normalrats (P<0.01), and the antioxidant enzyme activity of both GSH-Px and SOD weredecreased significantly (P<0.01). The administration of Huangqi and Danshencombination for6weeks in DN rats could significantly reduce the24h urinary proteinexcretion (P<0.01), serum levels of Cr, BUN, TG and T-CHO (P<0.05), and contentvariety of LPO and AGEs in renal tissue (P<0.05), and significantly increase theGSH-Px activity (P<0.05). Tissue dyeing assays also showed significantly improvementon tubular disorder, reabsorption function of FITC-BSA, and expression level ofmegalin in proximal tubule, in DN rats by administration of Huangqi and Danshencombination, with comparison of other groups.Conclusion: The administration of Huangqi and Danshen combination could decreaseproteinuria, and improve tubular lesions and tubular function of reabsorption. Theseprotective effects of Huangqi and Danshen combination was conjectured to beassociated with the enhancement of antioxidant enzyme activity in renal tissue andmegalin expression in renal tubular epithelial cells in diabetic rats. PART IIImprovement of Huangqi and Danshen combination on protein absorption by highglucose injured renal tubular epithelial cellObjective: To investigate the effect of Huangqi and Danshen combination on proteinabsorption by high glucose injured renal tubular epithelial cell of NRK-52E.Methods:(1) Preparation of high glucose injured cell model: Renal tubular epithelialcell of NRK-52E was respectively incubated in the medium with glucose at theconcentration of5.5,30,40,50,60and80mmol/L, and cultured for24,48and72hours. The inhibition of cell proliferation by glucose was evaluated by MTT assay, asthe basis for the glucose injured renal tubular epithelial cell model preparation.(2) Screening of efficient conditions for Huangqi and Danshen to protect the highglucose injured renal tubular epithelial cell: The protective effect of Huangqi on cellproliferation of glucose injured NRK-52E was detected by MTT assay, after24hincubation at the concentration of1,5,10,20and40μg/mL, respectively. The protectiveeffect of Danshen was detected in the same way at the concentration of10,20,40,80,and160μg/mL respectively.(3) Protective effect of Huangqi and Danshen combination on high glucose injuredNRK-52E: The Huangqi and Danshen were combined into three dosages, based on theeffective concentration. With24h incubation, the protective effect of Huangqi andDanshen combination on cell proliferation of glucose injured NRK-52E was detected byMTT assay.(4) Influence of Huangqi and Danshen combination on protein absorption of highglucose injured NRK-52E: With synchronization, the NRK-52E cell was randomlydivided into four groups of normal, NRK-52E injure model, combined administration of Huangqi and Danshen, and positive control of gliquidone, and incubated in the mediumcontained FITC-BSA at the concentration of30mg/L for24h. The megalin expressionlevel in each group was detected by IHC. The fluorescence intensity and content ofabsorbed FITC-BSA in NRK-52E was observed by fluorescence microscope anddetected after lysis.Result:(l) Preparation of high glucose injured cell model: There was no significanteffect of30or40mmol/L glucose on cell proliferation, while the50,60and80mmol/Lglucose could inhibit cell proliferation obviously. With comparison of normal group(with5.5mmol/L glucose contained), the cellular proliferation inhibition rate of50,60and80mmol/L glucose on NRK-52E was17%,24%and22%respectively, after48hincubation, while the inhibition rate of80mmol/L glucose on NRK-52E with24hincubation was54%. Therefore, incubation with medium contained80mmol/L glucosefor24h was chosen as the best condition to establish high glucose injured renal tubularepithelial cell model.(2) Screening of efficient conditions for Huangqi and Danshen to protect the highglucose injured renal tubular epithelial cell: Either Huangqi or Danshen exhibitedprotective effect on high glucose injured renal tubular epithelial cell of NRK-52E, witha dose-dependent manner. In present study, the medium with Huangqi at10,20and40μg/mL and Danshen at40,80and160μg/mL was chosen as the efficient condition inthe next experiments.(3) Protective effect of Huangqi and Danshen combination on high glucose injuredNRK-52E: Each dosage of Huangqi and Danshen combination exhibited obviousrestoration on high glucose-induced cell proliferation inhibition, with a dose-dependentmanner. The high dosage of Huangqi and Danshen combination with40μg/mL Huangqiand160μg/mL Danshen exhibited much higher protective effect than separateadministration, which was equivalent to98%of gliquidone (20μmol/L).(4) Influence of Huangqi and Danshen combination on protein absorption of high glucose injured NRK-52E: Results of IHC exhibited that high glucose couldsignificantly decrease the megalin expression in cell membrane of NRK-52E, bothHuangqi and Danshen combination and gliquidone could enhance the megalinexpression in high glucose injured NRK-52E, and there was no significant differencebetween combined administration group and normal group (P>0.05). The fluorescentmicroscope pictures and fluorescence intensity analysis both exhibited that theabsorption of FITC-BSA in NRK-52E injure model group was significantly lower thanthat of normal group, and mostly distributed in cell membrane (P<0.05), while thepositive drug of gliquidone and combined administration of Huangqi and Danshencould enhance the FITC-BSA absorption, with the similar content and location asnormal group.Conclusion: Twenty-four hour incubation with80mmol/L high glueose could inhibitthe proliferation and protein absorption of NRK-52E, and reduce the expression ofprotein absorption-related receptor of megalin. The administration of Huangqi andDanshen combination could efficiently enhance the proliferation of high glucose injuredcells and improve the protein absorption, associated with the upregulation of megalinexpression. |
PDF Full Text Request