Protective Effect Of Total Glucosides Of Paeonia On Mesangial Proliferative Glomerulonephritis Rat Model Via PI3K/AKT Pathway

ObjectiveMesangial proliferative glomerulonephritis (MsPGN), a common form of chronic glomerulonephritis in clinic, has been highly studied at present. Researches recently indicated that control of mesangial cells proliferation and reduction inflammatory response could effectively ameliorate kidney injury in MsPGN. Phosphatidylinositol 3-kinase(PI3K) /protein kinase B(AKT) pathway is involved in adjustment of proliferation, differentiation and apoptosis in renal inherent cells, which plays a crucial role in regulation of the pathogenesis of MsPGN. As a kind of glycoside extracted from Radix Paeoniae Alba, total glucosides of paeonia (TGP) has been widely used in secondary kidney disease for its immunomodulation and anti-inflammation effects. However, there are few studies about the effects of TGP on primary glomerulonephritis. Besides, it is unclear whether could TGP regulate the PI3K/AKT pathway in MsPGN.The classical MsPGN model, chronic serum sickness (CSS) model, was used in the present study to observe the protective effect of TGP. Further, the interventional mechanism of TGP in MsPGN was explored by regulation of inflammation and the PI3K/AKT signal pathway.MethodsTo observe the protective effects and interventional mechanism of TGP on MsPGN model rats, the modified CCS model established by immunological method was to imitate the pathologic process of MsPGN.1. To observe the effects of TGP on MsPGN model rats8 SD rats were randomly divided as control group, and the other rats were induced by bovine serum albumin (BSA) and lipopolysaccharide (LPS) in 4 weeks. The successful modeling rats were randomly divided into four groups, model group, TGP high dose group, TGP low dose group and prednisone acetate group. The treatment groups were orally administered the drugs in 5 week, meanwhile the general condition and 24h urinary protein were detected during the treatment. After 5 weeks drug treatments, the levels of serum creatinine (SCr), urea nitrogen (BUN), triglycerides (TG), total cholesterol (TC), albumin (propagated), total protein (TP), malondialdehyde (MDA) and total superoxide dismutase (SOD) in serum of all the rats were measured. Meanwhile, the morphological changes in the kidney were evaluated by HE staining, the deposition of IgG and C3 in glomeruli were observed by immunofluorescence, the expression of Desmin which indicates the level of injury in podocyte was detected by immunohistochemistry.2. To explore the anti-inflammation effect of TGP on MsPGN model ratsAfter been treated in 5 weeks, the contents of IL-α、IL-2、IL-10、IFN-γ in serum of the rats were tested by magnetic beads capture method. Besides, the expression of CD68, a macrophage marker protein, were detected by immunohistochemistry. All these methods above could show the improvement of TGP on inflammation caused by MsPGN.3. To discuss the regulation of PI3K/AKT pathway in MsPGN model rats after TGP treatmentAfter drug treatments and the total RNA and protein in rats were extracted, the mRNA levels of AKT、GSK3-β、Cyclin D1、IκB、NF-κB in kidneys were measured by qRT-PCR, the expression levels of PI3K, T-AKT, P-AKT, P-GSK3-α, P-GSK3-β,Cyclin D1 were determined by Western blot. Thus, the effect of TGP on PI3K/AKT pathway and the downstream signal molecules could be observed.Results1. The effects of TGP on MsPGN model ratsCompared with model group, better general condition, decreased 24h urinary protein, lower kidney weight, less deposition of IgG and C3 and less Desmin were observed in both TGP high dose and TGP low dose group. Relative to model group, TGP treatment groups could also reduce the contents of SCr, BUN, TG, TC and MDA, but increase the levels of ALB, TP and SOD in serum. Furthermore, TGP treatment groups could ameliorate the level of inflammatory infiltration, and decrease the mesangial cell hyperplasia, as well as heal glomerular morphology to some extent.2. The anti-inflammation effect of TGP on MsPGN model ratsRelative to model group, TGP treatment groups could decrease the levels of IL-α, IL-10, IFN-y, and increase the level of IL-2. It is showed that TGP treatment groups could reduce CD68.3. The regulation of PI3K/AKT pathway in MsPGN model rats after TGP treatmentCompared with model group, the qRT-PCR results showed that TGP treatment groups could decrease the mRNA levels of Cyclin D1、IκB、NF-κB, increase IκB. Western blot results showed that TGP treatment groups could reduce PI3K, P-AKT, P-GSK3-0, Cyclin D1, NF-κB expression, but there are no significant difference with T-AKT, and T-GSK3-α.Conclusions1. From the results of reducing 24h urinary protein, ameliorating renal function, attenuating pathologic changes, regulation of the serum lipid level, less deposition of IgG and C3 and alleviation podocyte injury, TGP shows a good therapeutic effect on curing MsPGN model rats.2. TGP could regulate the immune disorders in MsPGN model rats, adjust the levels of cytokines, restrain the activation of CD68.3. TGP could regulate PI3K/AKT signal pathway, ameliorate MsPGN model rat kidney damage.