Effect of Wuwei Xiaoduyin on NF-κB Signaling Pathway in Lipopolysaccharide-induced Rat Mesangial Cells / 中国实验方剂学杂志

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.

Anti-inflammatory Mechanism of Wuwei Xiaoduyin on IgA Nephropathy Rats Based on NF-κB Inflammatory Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of Wuwei Xiaoduyin on the nuclear factor-<italic>κ</italic>B （NF-<italic>κ</italic>B） signaling pathway in immunoglobulinA nephropathy（IgAN） rats， and to explore its mechanism of action in the treatment of IgA nephropathy. Method:The 40 SD rats were randomly divided into control group， model group， benazepril group （10 mg·kg·d^-1） and Wuwei Xiaoduyin group （2.75 g·kg·d^-1）， with 10 rats in each group. The IgA nephropathy rat model was established by intragastric administration of bovine serum albumin （BSA）， subcutaneous injection of carbon tetrachloride （CCl₄） and tail vein injection of lipopolysaccharide （LPS） for 9 weeks. The rats in each group were given corresponding doses of drugs by gavage， while the rats in the control group and model group were given the same amount of normal saline for successive 28 days. The levels of 24-hour urinary protein （UTP）， serum creatinine （SCr）， blood urea nitrogen （BUN） and serum albumin （ALB） were detected. The contents of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-1<italic>β</italic> （IL-1<italic>β</italic>） and interleukin-6 （IL-6） in serum were detected by enzyme-linked immunosorbent assay （ELISA）， the hematoxylineosin staining （HE）， immunofluorescence and transmission electron microscopy were used to observe the renal pathological changes， the expressions of IL-6， I<italic>κ</italic>B kinase <italic>β</italic> （IKK<italic>β</italic>）， phosphorylated I<italic>κ</italic>B kinase <italic>β</italic> （p-IKK<italic>β</italic>）， NF-<italic>κ</italic>B inhibitor protein <italic>α</italic> （I<italic>κ</italic>B<italic>α</italic>）， phosphorylated NF-<italic>κ</italic>B inhibitor protein <italic>α</italic> （p-I<italic>κ</italic>B<italic>α</italic>）， NF-<italic>κ</italic>B p65 and p-NF-<italic>κ</italic>B p65 were detected by immunohistochemistry （IHC）， real-time PCR （RT-PCR） and Western blot， respectively. Result:Compared with the control group， the level of UTP in the model group significantly increased （<italic>P</italic><0.01）， cultured glomerular mesangial cells proliferated， mesangial matrix and electronic dense deposit increased， mesentery thickened. A large amount of IgA was deposited in the glomerular mesangial area and showed irregular particles and mass distribution， the levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， IL-6 in serum significantly increased （<italic>P</italic><0.01）， the expression levels of IL-6， IKK<italic>β</italic>， p-IKK<italic>β， </italic>NF-<italic>κ</italic>B p65 and p-NF-<italic>κ</italic>B p65 in renal tissue significantly increased （<italic>P</italic><0.01）.Compared with the model group， the levels of UTP in each administration group significantly decreased （<italic>P</italic><0.01）， and the renal tissue injury alleviated， the levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， IL-6 in serum significantly decreased （<italic>P</italic><0.01）， the expressions of IL-6， IKK<italic>β</italic>， p-IKK<italic>β</italic>， I<italic>κ</italic>B<italic>α</italic>， p-I<italic>κ</italic>B<italic>α</italic>， NF-<italic>κ</italic>B p65 and p-NF-<italic>κ</italic>B p65 in the renal tissue significantly decreased （<italic>P</italic><0.01）. There were no significant differences in serum creatinine， blood urea nitrogen and serum albumin among the groups. Conclusion:Wuwei Xiaoduyin can reduce proteinuria in IgA nephropathy rats， and its mechanism may be related to the inhibition of NF-<italic>κ</italic>B signaling pathway and the over expression of related inflammatory factors.

Study on chemical fingerprint of Wuwei Xiaoduyin Oral Liquid and simultaneous determination of six characteristic components / 中草药

Objective: To establish the HPLC chemical fingerprints of Wuwei Xiaoduyin Oral Liquid (WXOL) and simultaneous determination method for six major characteristic components in this herbal preparation, i.e. chlorgenic acid, luteoloside, luteolin, linarin, caffeic acid, and esculetin. Methods: Both chemical fingerprint analysis and quantitative determination were implemented by Agilent 1260 high performance liquid chromatography system with an Agilent 5 TC-C18 column (250 mm × 4.6 mm, 5 μm). A gradient elution program, with the mobile phase of 0.5% glacial acetic acid aqueous solution (A) and methanol (B), was employed as following: 0—10 min, 10%—32% B; 10—20 min, 32% B; 20—25 min, 32%—46% B; 25—31 min, 46%—48% B; 31—41 min, 48%—80% B at the flow rate of 1.0 mL/min. The detection wavelength and column temperature were set at 320 nm and 30 ℃, respectively. Additionally, fingerprint similarity of 10 batches of WXOL was evaluated by software “Similarity Evaluation System for Chromatographic Fingerprint of TCM” published by GPC (Version 2012). The amounts of six characteristic components in WXOL were also simultaneously determined. Results: The common fingerprint pattern derived from 10 batches of samples was obtained with the similarity over 0.98 and 17 common peaks defined. Meanwhile, some common peaks were identified via peak pattern match with standard substances, showing that the peak No.5, No.7, No.8, No.12, No.16, and No.17 was chlorgenic acid, esculetin, caffeic acid, luteoloside, linarin, and lutelin, respectively. Chlorgenic acid content range of 54.038 3—105.551 1 μg/mL, esculetin content range of 4.122 1—31.359 9 μg/mL, caffeic acid content range of 2.413 0—4.420 7 μg/mL, luteoloside content range of 4.042 8—11.312 8 μg/mL, linarin content range of 3.866 3—46.271 9 μg/mL, and luteolin content range of 0.990 8—2.126 8 μg/mL. In combination with the non-significant difference of multiple characteristic components content among 10 batches of samples, the quality of home-made WXOL would be stable. Conclusion: A novel quality control method, which is HPLC fingerprint in combination with simultaneous quantitative analysis of multiple components, was established in this study, with high repeatability and reliability. Therefore, this method provides an applicable approach for the quality control of WXOL.