The Renoprotective Effects Of Berberine And Its Regulatory Effect On The Prostaglandin EP1 Receptor Mediated Signaling Pathway In Glomerular Mesangial Cell Of Diabetes In Rats

Objective To investigate the renoprotective effects of berberine and clarify its characteristics in ameliorating the renal function in diabetic rats, I studied the following contents. In my study, I try to elucidate the partial mechanism of berberine for its renoprotective effects through detecting the alterations of the main components of the extracellular matrix, the changes of corresponding regulating system i.e. the matrix metalloproteinases and matrix metallo-proteinase inhibitors (MMPs/TIMPs) and the expression levels of PGE2, EP1 and Gaq in the kidney tissue under diabetic state. Simultaneously, I trying to discuss the therapeutic features and the possible mechanisms by mean of detecting regulatory effects of berberine for the PGE2-EP1-Gaq signaling pathway in glomerular mesangial cells of DN rats. In order to provide fuller theoretical basis for the reasonable application for the clinical treatment of berberine and try to find new drug targets and lay the foundation for the development of new drugs that can treat DN, We further elucidate the molecular mechanism of DN and expanded understand themolecular mechanism of berberine in the treatment of DN.Methods Rats were purchased from animal laboratory center of Anhui Medical University. The model of diabetes was induced by feeding the high-sugar and high-lipid diet combined with appropriate doses of STZ. After 72h in the determination of fasting blood glucose (FBG), the FBG leve1≥11.1mol/L indicated that the model of diabetes was successfully established. Then the rats were divided into five groups including the DN control group, three different berberine treatment groups and enalapril (lmg/kg) as positive drug group. The three treatment groups received different dosage of berberine while sodium carboxymethylcellulose was giving to the normal and model group. Respectively weighting the body weight and measuring the FBG at 2,4,6,8 weeks. In the meantime, UTP and UCr were detected through collecting the urine sample and BUN and Scr were measured through femoral arterial blood sampling. Calculate the ratio of kidney weight and body weight and observe the pathologic changes in kidney. Immunohistochemical was used to detect the expression levels of Type IV collagen, Fibronectin, MMPs and TIMPs; Western blot was used to measure the expression levels of transforming growth factor-β1, MMP and the corresponding TIMP; Western blot was used to detect the protein levels of EP1 and Gaq in renal cortex; ELISA kit was used to explore the changes of protaglandin E2.In vitro study, glomerular mesangial cells (GMCs) were choosed as target cell with PGE2 as stimulating factor. In the following study, the changes of EP1, Gaq, GRK2, P-arrestin2 and Ca2+ expression in the activated GMCs were detected to explore the mechanism of EP1 receptor desensitization and EP1 related signaling pathway. Ⅰ try to elucidate the explicit mechanism that berberine inhibited the abnormal changes of GMCs according to the above study. The primary cultured GMCs were identified by immunofluorescent staining with characteristic including a-SMA, Desmin and Vimentin; The level of PGE2 in the cultured cell supernatant was detected by ELISA; MTT assay was used to measure the cell proliferation inhibiting effect of berberine; FCM and Western blot were both used to detect the desensitization of EP1 receptor at different time points; The levels of membrane and total protein of EP1 were measured by Western blot, also the expression levels of Gaq, GRK2 and β-arrestin2. Besides, the laser confocal method was used to observe the changes of Ca2+ in GMCs.ResultsSection one: Renoprotective effects of BBR in DN rats and partial mechanism1. Effects of BBR on ameliorating the biochemical and renal functional parameters of different stages of DN ratsCompared with the NC group, the levels of FBG, the ratio of KW/BW and the concentration of UCr, UTP/C, SCr and BUN were both significantly increased at different stages of DN model group rats, but with opposite effect from the body weight at the 2th week. When treated with different dosage of berberine, there were no obvious effects on body weight. The FBG of BBR (100,200mg/kg) group was significantly decreased compared with DN model group (P＜0.05), while BBR (50mg/kg) administration had no significant effect on FBG level of DN rats, and the positive drug enalapril treatment group can slightly decrease the FBG levels. From the 4th week, the results showed that BBR (200mg/kg) treatment shows visible improvements both in the abnormal renal function and biochemical indexes of DN rats, while BBR (100mg/kg) works at the 6th week. BBR (50mg/kg) have no obvious use for improving these biochemical and renal functional parameters. One other thing to note is that enalapril treatment could ameliorate these parameters from the 2nd week, and the changes were quite remarkable compared with those in the model group.2. Effects of BBR on ameliorating renal histopathological changes of DN ratsHistopathological results showed varying degrees of glomerular volume contraction, GMCs swelling, accumulation of ECM and basement membrane thickening. Always some tubular epithelial cells with a number of inflammatory cell infiltration appeared. BBR (200mg/kg) can significantly improve the abnormal pathological changes, while BBR (100mg/kg) works partly. Positive drug treatment group was similar ameliorating effects with BBR (200mg/kg) on renal histopathological.3. Effects of BBR on the expression of Type IV collagen and Fibronectin in kidney tissue of DN ratsImmunohistochemical results showed that the protein levels of type IV collagen and fibronectin, as significantly increased in DN model group compared with NC group. BBR (200, 100mg/kg) treatment could prevented the enhanced expression of these two proteins, while BBR (50mg/kg) could only partially reduce the expression level of fibronectin.4. Effects of BBR on the expression of MMP2/9 and TIMP 1/2 in kidney tissue of DN ratsWestern blotting showed the relatively lower expression level of MMP-2, while the elevated levels of MMP9, TIMP1 and TIMP2 compared with NC group. And then the ratio of MMP-2/TIMP-2 and MMP9/TIMP1 were decreased subsequently. Compared with DN control group, DN rats treated with berberine (50mg/kg) had no effect on ameliorating these symptoms. However, the berberine (100,200mg/kg) treatment could significantly up-regulate the ratio of MMP-2/TIMP-2 and help to repair the damaged balance of MMP-9/TIMP-1. The results are are consistent with the result of immunohistochemical.5. Effects of BBR on the expression of TGF-β1 in kidney tissue of DN ratsWhen compared with NC group, increased TGF-β1 expression in diabetic rats was mainly observed. Berberine (50,100,200mg/kg) treatment could significantly decrease the expression of TGF-β16. Effects of BBR on the expression level of PGE2 in kidney tissue of DN ratsELISA analysis showed the significant increased level of PGE2 expression in kidney tissue of diabetic rats. Berberine (100,200mg/kg) treatment could significantly decrease its level, while significant changes did not appear in berberine (50mg/kg) treatment group.7. Effects of BBR on the expression level of EP1 receptor subtype in kidney tissue Western blot analysis showed the increased level of EP1 in kidney tissue of diabetic rats. Berberine (100,200mg/kg) treatment could significantly decrease its level, while no obvious changes emerged in berberine (50mg/kg) treatment group.8. Effects of BBR on the expression of Gaq protein in renal tissue of DN ratsWestern blot analysis showed the increased level of Gaq in kidney tissue of diabetic rats compared with those in NC group. Berberine (100,200mg/kg) treatment could significantly decrease its level and the differences are statistically significant, while no significant changes emerged in berberine (50mg/kg) treatment group.Section two:Regulatory effects of BBR on the EP1 mediated signaling pathway in primary cultured GMC of DN rats1. Effect of BBR on primary cultured GMCs proliferation induced by PGE2 under DN stateThe results of MTT experiment showed that PGE2 could induced the GMCs proliferation and BBR (15、30、60、90、120、240μM) groups could significantly inhibit the above cell proliferation reaction. But, in the experiment, BBR (120、240μM) groups exhibited a severe cytotoxicity, while BBR(7.5μM) treatment group could not exert the above function.2. Receptor desensitization of EP1 in primary cultured GMCs induced by PGE2 under DN stateResults from the flow cytometry detection showed that the fluorescence intensity gradually increased as time goes by from time 0 to 10 min under the stimulation of PGE2 125μg·L-1. When that fluorescence intensity is at its peak, the time is 10min and the fluorescence intensity has obviously statistical difference compared with Omin (p< 0.01). Then, the fluorescence intensity wore off as time goes on between 10min and 120min. Western blot tests found that the increasing expression level of EP1 receptor on the membrane of glomerular mesangial cell and the peak time is 10min, then followed by the gradually decreased EP1 expression on the membrane. The results observed by Western blot were in accord with data got from flow cytometry.3. The expression of GRK2 and β-arrestin2 at different time points in primary cultured GMCs induced by PGE2 under DN stateResults from Western blot approved that the expression levels of β-arrestin2 and GRK2 on the membrane were began to increase at 10 min significant difference compared with NC group (p<0.05). Then the level of β-arrestin2 has been slowly and the tendency of GRK2 is more significant.4. Effect of BBR on PGE2 secretion in primary cultured GMCs under DN stateELISA analysis showed the significant increased level of PGE2 expression in the supernatant of primary cultured glomerular mesangial cell compared with those in NC group. Both berberine (30,60,90μM) treatments could significantly decrease the level of PGE2 at different levels and the differences are statistically significant.5. Effects of BBR on the expression level of EP1 receptor and Gaq protein in primary cultured GMCs under DN stateWestern blot analysis showed the increased level of EP1 and Gaq protein in GMCs of DN group. BBR (60,90μM) treatments could significantly decrease their expression levels, while no obvious changes emerged in berberine (50mg/kg) treatment group, while no significant changes emerged in berberine (30μM) treatment group.6. Effects of BBR on the level of Ca2+ in primary cultured GMCs under DN stateThe laser confocal detection results showed the increased concentration of Ca2+ in GMCs of DN group compared with NC group. BBR (30,60,90μM) treatments could significantly decrease levels of Ca2+ in GMCs and the differences are statistically significant.Conclusion1. BBR (100,200mg/kg) treatment could improve the biomechanics of blood and urine and correlated renal function indicators, which indicated that BBR had a protective effect on diabetic kidney disease. And meantime, BBR treatment down-regulate the abnormal expression level of PGE2 and TGF-β1 in diabetic rats suggested that there may be some relationships between renoprotective effects of BBR and its regulatory effect of inflammatory cytokines including PGE2 and TGF-β1;2. MMPs/TIMPs system is the most important degrading enzyme system of ECM in the kidney. The expression levels and activities of the system directly impacted the ECM balance. The focus of ongoing study reminds us that there may be some relationships between renoprotective effects of BBR and its’ regulatory effect on the regulatory system of ECM accumulation;3. The regulatory effects of BBR on the expression of EP1 and Gaq indicated that there may be some relationships between renoprotective effects of BBR and it’s regulate effect on the signaling pathway of PGE2-EP1-Gaq in renal tissue of DN rats.4. Membrane protein of EP1 markedly decreased in number may be associated with the process of receptor desensitization and the abnormal transient rise of EP1 receptor may be correlated with the glomerular mesangial cells proliferation induced by PGE2.5. The changes of GRK2 and β-arrestin2 may play important roles in the progression of EP1 receptor desensitization. Therefore, GRK2 and β-arrestin2 may play pivotal roles in glomerular mesangial cell proliferation induced by PGE2.6. BBR may play an important role in inhibiting the abnormal proliferation of glomerulus mesangial cell under DN state via the PGE2-EP1-Gaq pathway regulation by EP1 receptor.