Renoprotective Effect Of Gallotannin Through Inhibition Of Poly (ADP-ribose) Polymerase Activation In Diabetic Rats

With the improvement of living standards. whether the developed countries in Europe and America, or the developing countries including China, diabetes mellitus (DM) has a trend of increased prevalence year by year. Currently, there are more than 150 millions DM patients in the world. Diabetic nephropathy (DN) is glomerular lesion induced by the microangiopathy of DM. known as diabetic glomerulosclerosis. DN is the most serious and common complication of DM. The glomerular hypertrophy, the thickening of the glomerular basement membrane and extracellular matrix (ECM) accumulation are the main pathological feature of DN. DN is a leading cause of end-stage renal disease, and threatens seriously the human healthy. At present, the pathogenesis of DN has not been fully clarified, and the effective measures to control its onset and progeression are deficient.Gallotannin (GLTN), also known as tannic acid or tannin, is widely spread in the plants. about 70% of Chinese herbal medicine contains this compound. GLTN has a variety of biological activity, such as antioxidation. anti-inflammation, blood lipid- and glucose-lowering action, anti-aging, etc. The previous studies showed that administration of tannin components extracted from Chinese herbal medicines decreased serum glucose, serum creatinine and blood urea nitrogen in rats with chronic renal failure. Also, GLTN treatment significantly reduced the degree of renal injury in cisplatin-treated rats. suggesting that GLTN had a preventive and therapeutic effect on the different reasons-induced renal injury. However, the effect of GLTN on onset and progeression of DN and its molecular mechanism have not yet been illuminated.Poly(ADP-ribose) polymerase (PARP) is a chief modified enzyme of post-translation, involved in many important processes of cellular activity. As an important stress protein. PARP transfers ADP-ribose from NAD+ to the modified protein and itself when it is activated. The excessive activation of PARP depletes NAD- and ATP, regulates some of cytokines and transcriptive regulatory factors, leading to the occurrence of cell lesions. PARP1 is the most main member of regulating cell function in the PARP superfamily and approximately occupies above 90% of cellular PARP activity. Numerous studies have demonstrated that activation of PARP1 plays an important role in the onset and progeression of DN. PARP1 overactivation depletes its substrate NAD- and ATP in poly(ADP-ribosyl)tion, slowing the rate of glycolysis, as well as up-regulation of cytokine expression. e.g. endothelin-1. TNF-a. nitric oxide synthase, eventually leading to functional impairment or death of cells and DN.Inhibition of PARP1 activity in the therapeutic value for certain diseases has attracted the concern of researchers. Currently there are two ways that can prevent the excessive activation of PARP1:the first way, use of PARP inhibitors, such as 3-AB and PJ34:another way is inhibition of PARG activity. Because PARG is an enzyme responsible for degrading poly(ADP-ribose) and prevents the cleavage of poly (ADP-ribose) chain, thereby indirectly inhibit PARP activation. At present, PARP inhibitors using in the experimental research, such as 3-AB. PJ34, and 1,5-isoquinoline diol. are the chemical reagents, their toxic and side-effects for human body have not been determined. Numerous studies have indicated that GLTN is a PARG inhibitor. GLTN has been used in clinic for many years as an active ingredient in some Chinese herbal medicines. GLTN as indirect inhibitors of PARP may have more extensive and practical prospects.In the present study, STZ-induced DM in rats and rat mesangial cells (GMC) as the research object, the main investigative issues are as follows:(1) the effects of GLTN treatment on blood glucose, renal function and morphology in DM rats and the levels of 8-OhdG and 3-NT in DM rat kidney; (2) the changes of PARP1 and PARG contents in DM rat kidney and GMC cultured in high glucose, and relationship between PARPl and PARG: (3) the effect of GLTN treatment on PARP1 and RPRG activities in DM rat kidney and GMC cultured in high glucose:(4) the effects of PARP1 and RPRG activities on ECM anabolism (ColⅣ, FN) and catabolism (PA1-1. TIMP-2). and ameliorating action of GLTN:(5) the relationship between PARP activity and growth factors(TGF-β1,CTGF); (6) the effect of GLTN on expression of TGF-β1 and CTGF in DM rat kidney and the GMC cultured in high glucose.The main results of this study are as follows:1. Administration of GLTN significantly reduced the levels of serum glucose, serum creatinine, blood urea nitrogen and 24 hours urinary protein excretion in DM rats, and ameliorated renal lesions. 2. The levels of 8-OhdG and 3-NT in DM rat kidney were obviously elevated compared with control rats, Diabetic rats treated with GLTN showed a significantly decrease in 8-OhdG and 3-NT levels in kidney.3. Expression of PARP1 and PARG protein in DM renal tissues was markedly higher than in control rats (p><0.01). the expression levels of PARP1 and PARG were positively correlated (r=0.927, P<0.01). GLTN treatment inhibited expression of PARG at the same time. then expression of PARP1 was suppressed also.4. Expression levels of PARP1 and TGF-β1 proteins were positively correlated (r= 0.899. P<0.01) in DM renal tissue. GLTN treatment decreased mRNA and protein expression of PARP1, PARG, TGF-β1 and CTGF.5. GLTN treatment, effectively decreased expression levels of ColⅣ, FN, PAI-1 and TIMP-2 proteins (P<0.01) in DM rat kidney, thereby inhibiting ECM synthesis and promoting ECM degradation. reducing ECM accumulation.6. GMC cultured in high glucose was treated with GLTN showed inhibition for percentage of S phase and GMC proliferation. and this inhibitorY effect was a time- and concentration-dependent manner.7. After GMC cultured in high glucose was treated with GLTN. the levels of ColⅣ, FN. PAI-1 and TIMP-2 in GMC cultured supernatant were reduced, and revealed dependence between concentration and effect.8. Expression of PARP1 and PARG in GMC cultured in high glucose gradually increased with action time, and showed a time-dependent fashion. GMC cultured in high glucose was treated with GLTN revealed inhibition of expression PARP1 and PARG with time- and concentration-dependent manner. the best effect is GLTN at a concentration of 40μmol/L and action for 24 hour.9. GLTN treatment caused a significant decrease in expression of PARP1. PARG. TGF-β1 and CTGF at mRNA and protein in GMC cultured in high glucose(P<0.01). A positive correlation was observed between expression levels of PARP1 and TGF-β1.The conclusions of this study are as follows:1. GLTN treatment significantly reduces the blood glucose levels, ameliorates renal function and pathological lesions in DM rats, and obviously decreases 8-OhdG and 3-NT levels in DM rat kidney.2. Inhibition of PARG activation via GLTN in DM rat kidney exerts an inhibitory effect on PARP1 activity, and the inhibition of PARP1 activity down-regulates TGF-(31 expression in DM rat kidney, delaying the progeression of DN.3. The results in vivo and in vitro demonstrates that GLTN treatment inhibits ECM synthesis and promotes ECM degradation, reducing ECM accumulation.4. GLTN treatment inhibits proliferation of GMC cultured in high glucose. Expression of PARP1 and PARG (including gene and protein) in GMC cultured in high glucose is upregulated, and this up-regulation of expression is inhibited by GLTN.5. Expression levels of PARP1 and PARG in GMC cultured in high glucose are positively correlated, and expression level of PARP1 is positively correlated with expression level of TGF-β1.The creative points of the present study are:it is demonstrated in STZ-induced diabetic rat kidney and in GMC cultured in high glucose that the expression levels of PARP1 and PARG are up-regulated and the expression levels of these two are positively correlated. Treatment with GLTN prevents PARG activation and consequent indirectly inhibition of PARP1 activation, exerting renoprotective effect:PARP1 expression is positively correlated with TGF-β1 expression; GLTN treatment shows a decrease in expression of PARP1, PARG, TGF-β1 and CTGF; Treatment of GLTN down-regulates expression of ColⅣ, FN, PAI-1 and TIMP-2, thereby decreasing ECM synthesis and promoting ECM degradation, reducing ECM accumulation from two directions.