We believe that our present experimental observations further support a possible benefit of MZR in the treatment of lupus nephritis. Poly IC was from Sigma (St. Louis, MO, USA). Primer oligo(dT)12–18, dNTP mix, and Moloney murine leukemia virus (MMLV) reverse transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). SsoFast EvaGreen
Supermix was from Bio-Rad (Hercules, CA, USA). Oligonucleotide primers for polymerase chain reaction (PCR) were custom synthesized by Greiner Japan (Atsugi, Japan). Enzyme-linked immunosorbent assay (ELISA) kits for MCP-1, CCL5, fractalkine and IL-8 were from R&D Systems (Minneapolis, MN, USA). Dexamethasone (DEX) was from Roche Diagnostics Depsipeptide chemical structure (Basel, Switzerland). MZR was from Asahi Kasei Pharma Corporation (Tokyo, Japan). Tacrolimus (Tac) was from Astellas Pharma Corporation (Tokyo, Japan).
Normal human mesangial cells (MCs) were purchased from Lonza (Walkersville, MD, USA), and the cells were cultured according to the manufacturer’s protocol.[13-17] Poly IC was dissolved in phosphate-buffered saline (PBS) and the cells were treated with 2–50 μg/mL poly IC for up to 48 h.[13-17] In the experiments using immunosuppressive reagents, the LEE011 cells were pretreated, with 1–100 μg/mL MZR, 10 μM DEX, or 5 μg/mL Tac, 1 h before the treatment with 30 μg/mL poly IC. We have already confirmed that viability of cells was not affected by the treatment of these reagents (not shown). To examine the effect of MZR in
more detail in this setting, the cells at the time of 16 h after the stimulation with 30 μg/mL poly IC were post-treated with 100 μg/mL of MZR for 24 h. Total RNA was extracted from cells using RNeasy RNA extraction kit. Single-strand cDNA was synthesized from 1 μg of total RNA using oligo(dT)12–18 primer and MMLV reverse transcriptase. The cDNA for MCP-1, CCL5, fractalkine, IL-8, or glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was amplified using SsoFast EvaGreen Supermix, as reported previously.[13-17] The primers were custom-synthesized by Greiner Japan (Atsugi, Japan), and the sequences of the primers were as follows: MCP-1: -forward, 5′-AAACTGAAGCTCGCACTCTCGC−3′, reverse, CHIR 99021 5′-ATTCTTGGGTTGTTGAGTGAGT−3′; CCL5: -forward, 5′-CTACTCGGGAGGCTAAGGCAGGAA−3′, reverse, 5′-GAGGGGTTGAGACGGCGGAAGC−3′; fractalkine: -forward, 5′-GACCCCTAAGGCTGAGGAAC-3′, reverse, 5′-CTCTCCTGCCATCTTTCGAG-3′; IL-8: -forward, 5′-AGGAGTGCTAAAGAACTTCGA−3′, reverse, 5′-TGAATTCTCAGCCCTCTTCAA-3′, and GAPDH: -forward, 5′-GCACCGTCAAGGCTGAGAAC−3′, reverse, 5′-ATGGTGGTGAAGACGCCAGT−3′. Each sample was run in triplicate. The concentration of MCP-1, CCL5, fractalkine and IL-8 in cell-conditioned medium was measured in triplicate in each, using an ELISA kit according to the manufacturer’s protocol. Statistical significance was evaluated using the paired t-test.
No related posts.