ABSTRACT
ObjectiveTo observe the intervention of modified Sanrentang on the lipopolysaccharide-induced proliferation of rat glomerular mesangial cells， the phosphatidylinositol-3 kinase（PI3K）/protein kinase B（PKB/Akt）/nuclear factor kappa B（NF-κB） signaling pathway， and to investigate its mechanism in improving kidney inflammation in rats with immunoglobulin A nephropathy（IgAN）. MethodThe 18 rats were divided into 3 groups by serum pharmacology method： normal group， high-dose and low-dose （20.70，10.35 g·kg-1·d-1） groups with 6 rats in each group. Modified Sanrentang high- and low-dose groups were intragastric with the corresponding solution of modified Sanrentang， and normal group was intragastric with equal volume of distilled water. After 5 days of intragastric administration， blood samples were collected to prepare drug-containing serum. Rat mesangial （HBZY-1） were divided into five groups of normal group， LPS 10 mg·L-1 in the model group， benazepril（50 μmol·L-1）， modified Sanrentang high- and low-dose group. Preclude the use of methyl thiazolyl tetrazolium（MTT） method detect the proliferation activity of HBZY-1 cells， enzyme-linked immunosorbent assay（ELISA） was used to determine the content of each group type Ⅳ collagen（ColⅣ），Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） were used to detect protein and mRNA expression levels of PI3K/Akt/NF-κB signaling pathway. ResultAs compared with the normal group， MTT assay showed that exposure to LPS significantly enhanced the proliferative activity， the ColⅣ was increased significantly of HBZY-1 cells（P<0.01）， p-Akt， p-p65 was increased significantly （P<0.01）. Compared with the model group， the proliferation and ColⅣ of rat chronic glomerulonephritis cells induced by LPS by inhibiting PI3K/Akt/NF-κB signaling pathway（P<0.01）， and the phosphorylation of Akt was significantly inhibited（P<0.01）， the expression levels of NF-κB p65 was reduced in modified Sanrentang high-dose group（P<0.01）. ConclusionModified Sanrentang could inhibit cell proliferation and the content of ColⅣ in rat mesangial cells induced by LPS， and its mechanism might be related to suppression of PI3K/Akt signaling pathway.
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.