Objectives Glomerulonephritis is a significant kidney disease that can induce end-stage renal failure. Glomerular mesangial cells play critical roles in normal kidney function. However, uncontrolled proliferation of glomerular mesangial cells can lead to excessive production of extracellular matrix, thereby promoting the occurrence and development of glomerulonephritis.2 This abnormal proliferation of mesangial cells occurs in various glomerular diseases.3 Therefore, inhibiting the abnormal proliferation of glomerular mesangial cells is critical for delaying the progression of renal disease. Many studies have shown that glomerulonephritis is regulated by immune and inflammatory responses.4 Thus, some inflammatory factors are involved in the occurrence of glomerulonephritis and contribute to glomerular damage.5,6 Some drugs can alleviate the damage caused by glomerulonephritis, but most have strong toxicity and side effects.7,8 Consequently, a new pharmacotherapy is needed for treatment of glomerulonephritis. Compound Sanqi granules are a compound Chinese medicine consisting of pseudo-ginseng, has been shown to exhibit a therapeutic effect on nephritis9 and has been shown to protect against lupus nephritis when combined with other drugs.10 The active ingredient in Hemsl, another type of Chinese traditional medicine, was proven to inhibit the proliferation of vascular smooth muscle cells in vitro.11 However, it is unknown whether Hemsl can suppress the proliferation of mesangial cells, thereby inhibiting the occurrence and development of glomerulonephritis. Additionally, prior studies have not shown whether the combination of compound Sanqi granules and Hemsl could enhance the curative effects of these compound drugs. Lipopolysaccharide (LPS), a component of Gram-negative bacteria, is commonly used to induce abnormal proliferation of glomerular mesangial cells.12 Because there are many impurities in crude extracts used in traditional Chinese medicine, a serum pharmacology method13 is used in which medicines are administered to animals; the Levetimide serum can be after that isolated and used for assessment of glomerular mesangial cell activity in vitro. This method is usually presumed to avoid interference from the impurities in crude extracts and directly assess the pharmacological effects of the tested medicines. In this study, Levetimide we used the serum pharmacology method to investigate the effects of Hemsl and compound Sanqi granules on glomerulonephritis in vitro, specifically to determine whether the combination of these drugs could inhibit the proliferation of glomerular mesangial cells. Materials and methods Production of medicine serum This research protocol was approved by the ethics committee of Guilin Second Peoples Hospital. Thirty healthy male nude mice were obtained from Shanghai Lingchang Biotechnology Company (Shanghai, China). All mice were housed under specific pathogen-free conditions and cared for in accordance with the animal experimental guidelines designated by the National Institutes of Health. Mice were divided into six equal groups, which received the following compound medicines: pseudo-ginseng 20 g, 20 g, Levetimide and 60 g (group 1); Hemsl 50 g and 50 Mouse monoclonal to CD56.COC56 reacts with CD56, a 175-220 kDa Neural Cell Adhesion Molecule (NCAM), expressed on 10-25% of peripheral blood lymphocytes, including all CD16+ NK cells and approximately 5% of CD3+ lymphocytes, referred to as NKT cells. It also is present at brain and neuromuscular junctions, certain LGL leukemias, small cell lung carcinomas, neuronally derived tumors, myeloma and myeloid leukemias. CD56 (NCAM) is involved in neuronal homotypic cell adhesion which is implicated in neural development, and in cell differentiation during embryogenesis g (group 2); Hemsl 60 g and pseudo-ginseng 20 g (group 3); Hemsl 60 g and 20 g (group 4); pseudo-ginseng 20 g, 20 g, 60 g, and Hemsl 60 g (group 5); and Hemsl 50 g (group 6). These drugs were all in powdered form (provided by Guangdong Yifang Pharmaceutical Co. Ltd., Foshan, China) and were dissolved in distilled water at final concentrations sufficient to ensure that the mice in each group received the weights of medicine listed above with a 10-mL/kg dose volume. To facilitate the absorption of the drugs, the mice were fasted for 12 hours before administration. Drugs were administered by the intragastric route for 8 consecutive days (10?mL/kg, once per day). At 2 hours after the last dose of the drugs, mice were anesthetized with chloroform (Chenniao Company, Shanghai, China) and blood was collected via intracardiac puncture. The blood was placed at room temperature for 1 hour and centrifuged at 12,500??g for 10 minutes after coagulation, then heated at 56C for 30 minutes to inactivate the complement. Subsequently, the serum fraction of the blood was sterile filtered using a 0.22-m microporous membrane and stored at ?80C. Cell culture and treatment Human mesangial cells (HRM cells) were obtained from the Shanghai Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640 medium (Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% Levetimide FBS (Gibco Cell Culture, Carlsbad, CA, USA) and incubated at 37C with 5% CO2. LPS (Sigma-Aldrich, St. Louis, MO, USA) and serum from treated mice were added to the cell culture medium during the experiment. MTT assays HRM cells were seeded into 96-well plates (Corning, Corning, NY, USA) at a density.