ABSTRACT
ObjectiveTo observe the modulatory effect of modified Zhenwutang on the interleukin-6 （IL-6）， matrix metallopeptidase-9（MMP-9）， type Ⅳ collagen（COL-Ⅳ） in rats with chronic renal failure （CRF） and to investigate the potential mechanism of its treatment of CRF. MethodFifty male SD rats were randomly divided into a modeling group of 40 rats and a normal group of 10 rats， and the modeling group was prepared by continuous adenine gavage for 12 weeks. After successful modelling， the modelling group was divided into the model group， the low dose （7.2 g·kg-1·d-1） group， the medium dose （14.4 g·kg-1·d-1） group， the high dose （28.8 g·kg-1·d-1） group and the Benadryl hydrochloride （10 mg·kg-1·d-1） group for gavage according to the random number table method， In the normal group and the model group， equal volume of distilled water was administered by gavage for 4 weeks. After the administration， the levels of blood creatinine （SCr）， blood urea nitrogen （BUN） and 24 h urine protein （24 h-UTP） were measured， the levels of serum IL-6 were measured by enzyme linked immunosorbent assay（ELISA）. Immunohistochemistry （IHC） was used to detect intercellular cell adhesion molecule-1 （ICAM-1）， IL-6， MMP-9， and other molecules in the rat kidney. The expression of ICAM-1 mRNA， IL-6 mRNA， MMP-9 mRNA and COL-Ⅳ mRNA in rat kidney tissues was measured by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expression levels of ICAM-1， IL-6， MMP-9 and COL-Ⅳ in rat kidney tissues were measured by Western blot. ResultCompared with the normal group， the levels of SCr， BUN and 24 h-UTP were significantly increased in the model group （P<0.01）； the serum IL-6 level was significantly increased （P<0.01）， the tubular lumen was dilated with atrophy， the tubular epithelial cells were necrotic， swollen and vacuolated， the interstitium was infiltrated by a large number of inflammatory cells and collagen fibers were deposited， the levels of IL-6， ICAM-1 and COL-Ⅳ were strongly positive in the tubular interstitium of the model group （P<0.01）， The levels of ICAM-1 mRNA， IL-6 mRNA and COL-Ⅳ mRNA were significantly increased （P<0.01） and MMP-9 mRNA was significantly decreased （P<0.05） in the model rats. ICAM-1， IL-6 and COL-Ⅳ protein expression was significantly increased （P<0.01） and MMP-9 protein expression was significantly increased （P<0.01） in the renal tissue， and MMP-9 protein expression was significantly decreased （P<0.01）. Compared with the model group， the 24 h-UTP， SCr and BUN levels of rats were significantly reduced after treatment with modified Zhenwutang （P<0.01）， the serum IL-6 level was significantly decreased （P<0.01）， the renal lesions of rats were significantly improved and collagen fiber deposition was reduced； the expression of IL-6， ICAM-1 and COL-Ⅳ in renal tubules and interstitium was weakened， and MMP-9 in ICAM-1 mRNA， IL-6 mRNA and COL-Ⅳ mRNA levels were significantly reduced （P<0.01） and MMP-9 mRNA levels were significantly increased （P<0.05）， ICAM-1， IL-6 and COL-Ⅳ protein expression was significantly reduced （P<0.01） and MMP-9 protein expression was significantly The expression of ICAM-1， IL-6 and COL-Ⅳ proteins was significantly decreased （P<0.01） and MMP-9 protein expression was significantly increased （P<0.01）. ConclusionModified Zhenwutang may regulate the IL-6/MMP-9/COL-Ⅳ signaling pathway， thereby reducing proteinuria， improving renal function， reducing renal pathological damage and delaying the progression of CRF interstitial fibrosis.
ABSTRACT
ObjectiveTo study the effect and mechanism of Wuwei Xiaoduyin in treating rat renal mesangial cells （HBZY-1） induced by lipopolysaccharide （LPS） through the nuclear factor-κB （NF-κB） signaling pathway. MethodRat HBZY-1 cells were randomly assigned into the normal group， model group， benazepril （50 μmol·L-1） group， and high- and low-dose （2.75 and 0.69 g·kg-1） Wuwei Xiaoduyin groups. The normal group， model group， and benazepril group were treated with 10% normal rat serum， and the Wuwei Xiaoduyin groups with 10% medicated serum. Except the normal group， the other four groups were treated with LPS （100 ng·mL-1） for modeling in vitro. The changes of cell morphology were observed under optical microscope. The expression of NF-κB p65 was detected by immunofluorescence （IF） method. Methyl thiazolyl tetrazolium （MTT） colorimetry was employed to detect cytotoxicity and cell proliferation. The levels of interleukin-1β （IL-1β）， intercellular adhesion molecule-1 （ICAM-1）， laminin （LN）， and fibronectin （FN） in cell supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA levels of IL-1β， FN， and NF-κB p65 were measured by real-time fluorescence quantitative PCR. The protein levels of phosphorylated inhibitor of NF-κB kinase β （p-IKKβ）， phosphorylated NF-κB inhibitor （p-IκBα）， and NF-κB p65 were determined by Western blot. ResultCompared with the normal group， the modeling increased cell proliferation （P<0.01）， elevated the levels of IL-1β， ICAM-1， LN， and FN in cell supernatant （P<0.01）， and up-regulated the mRNA levels of IL-1β， FN， and NF-κB p65 （P<0.01） and the protein levels of p-IKKβ， p-IκBα， and NF-κB p65 （P<0.01）. Such changes were recovered by benazepril and Wuwei Xiaoduyin （P<0.05， P<0.01）. ConclusionWuwei Xiaoduyin can mitigate the inflammatory injury of renal mesangial cells induced by LPS by inhibiting the NF-κB signaling pathway.