Research On The Mechanism Of Renal Injury Induced By Ox-LDL And The Effect Of Shen Kangling Intervention In Children With Nephrotic Syndrome

Purpose:In vitro, oxidized low density lipoprotein(ox-LDL) induced MCs proliferation, observe CXCL16, CD36, ADAM10, ADAM17levels and mitogen-activated protein kinase (MAPK) signaling pathway protein phosphorylation levels; In vivo, rats with adriamycin nephropathy (AN) as the research object, observe the CXCL16,CD36,disintegrin and metalloproteinase10, the ADAM17level and MAPK signaling pathway related proteins in renal tissue to clarify the mechanism of action of lipids on renal injury. Before and after intervention of Yishenhuoxue Chinese medicine Shenkangling,through observing the change of CXCL16,CD36,ADAM10, ADAM17and MAPK signal transduction pathway,and to clarify the mechanism of action of the intervention Yishenhuoxue, from the level of molecular biology and genetics, to improve the clinical application of the PNS clinical efficacy and Yishenhuoxue law, providing the basis for the clinical application of Yishenhuoxue.Methods:(1) basic research:in vitro, MCs were randomly divided into seven groups:①normal control group;②ox-LDL group;③ox-LDL+CXCR6group;④ox-LDL+low Shenkangling concentration group;⑤ox-LDL+high Shenkangling concentration group;⑥ox-LDL+low atorvastatin concentration group;⑦ox-LDL+high atorvastatin concentration group. MCs proliferation ratio was determined by MTT assay; CXCL16, CD36, IFN-γ, IL6, TNF-α content detected by ELISA, CXCL16, CD36, ADAM10level of ADAM17gene and its protein content assayed by Real Time-PCR, Western blot. MAPK pathway related protein (p38, JNK, ERK) and NF-κB phosphorylation levels detected by Western Blot.In Vivo, rats were divided into five groups according to body mass stratified random method:①normal group;②AN disease module;③low Shenkangling concentration group+prednisone;④high Shenkangling concentration group+prednisone;⑤prednisone group. The each group intragastrically continuous22days. After gavage, respectively, using Real Time-PCR, Western blot to assay CXCL16, CD36, ADAM10and ADAM17gene level and its protein content of rat kidney tissue, and detect urine protein and serum albumin, the level of cholesterol, analysis of renal Pathomorphology by HE staining and electron microscopy.(2) clinical research:PNS children randomly divided into the Shenkangling+western medicine group and the western medicine group, healthy children as normal control group selected in the examination center. Serum CXCL16, ADAM10and AD AM17Detected by ELISA in children with PNS before treatment and after-treatment, at the same time,serum ALB, CH,24-hour urinary protein excretion efficacy evaluation.Results:(1) basic research:in vitro, ox-LDL-induced proliferation of rat mesangial cells, CXCL16, CD36, IFN-γ, IL6, TNF-α expression was significantly increased (P<0.01), after adding CXCR6receptor, its expression level was further increased (P<0.01). The serum containing Shenkangling reducing the above indicators dose-dependently (P<0.01); ox-LDL-induced proliferation of rat mesangial cells, CXCL16, CD36, ADAM10and ADAM17gene level and protein content were significantly increased (P<0.01), after adding CXCR6receptor, gene level and protein content were further increased (P<0.01), Shenkangling containing serum reduced these indicators dose-dependently (P<0.01or P<0.05); ox-LDL-induced proliferation of rat mesangial cells, phosphorylation levels of NF-κBp65, p38, ERK1/2, SAPK/JNK were significantly increased (P <0.01), after adding CXCR6receptor phosphorylation levels further increased (P<0.01), The Shenkangling containing serum dose-dependently reduced ERK1/2, SAPK/JNK, NF-κB p65phosphorylation level (P<0.01).In vivo, urinary protein excretion increased in AN rats, serum ALB lower, CH increased, CXCL16, CD36, ADAM10and ADAM17gene level and protein content increased in renal tissue, p38, ERK1/2, SAPK/JNK, NF-κB p65phosphorylation levels increased (P<0.01), Shenkangling+prednisone group showed a concentration-dependent reduction in CXCL16, CD36, ADAM10, ADAM17expression, reduced p38, ERK1/2, SAPK/JNK NF-κBp65in phosphorylation level (P<0.01); CH level significantly reduced in Shenkangling (high, low concentration)+the prednisone group compared with the prednisone group (P<0.01or P<0.05).(2) clinical study:elevated PNS children with urinary protein excretion, serum ALB reduce, CH increased CXCL16, ADAM10, ADAM17content increased (P<0.01), Shenkangling+western medicine group and western medicine group compared to reduce urinary protein quantitation, CH and serum CXCL16, ADAM10, ADAM17’s content more significant (P<0.01or P<0.05).Conclusion:(1) ox-LDL may promote the release of inflammatory mediators such as CXCL16, CD36, ADAM10, ADAM17in mesangial cells, CXCR.6may mediated this path. ox-LDL activates the MAPK signal transduction pathway, the p38, ERK1/2, SAPK/JNK phosphorylation levels increased, activated NF-κBp65,CXCR6-CXCL16mediated MAPK signaling pathways.(2) Yi shen huo xue Shenkangling by reducing the release of inflammatory mediators such as CXCL16, CD36, ADAM10, ADAM17and inhibit the activation of the MAPK signal transduction pathway, thereby inhibiting the proliferation of mesangial cells, reducing proteinuria, CH, improve AN rats pathological damage, effectively improve the clinical symptoms of children with PNS.