ABSTRACT
Objective: To investigate the effect of Yiqi Yangyin Zhuyu recipe on expressions of inflammatory factor and transformation of classically activated macrophages(M1)/alternatively activated macrophages (M2) inflammatory phenotype in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Method: Methyl-thiazdyl-tetrazolium(MTT) reduction assay was used to detect the effect of different concentrations of Yiqi Yangyin Zhuyu recipe on the proliferation of the cells. The release of nitric oxide was detected by the Griess method. Enzyme linked immunosorbent assay(ELISA) was used to detect the release of M1/M2 inflammatory cytokines in cell supernatant. The expressions of the pro-inflammatory factor genes of M1-macrophages and the anti-inflammatory factor genes of M2-macrophages were detected by Real-time PCR. The protein expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1,nitric oxide synthase(iNOS) were detected by Western blot. Result: Results of MTT showed that Yiqi Yangyin Zhuyu recipe with the concentration of 2.0 g·L-1 and below had no effect on the cell proliferation. Results of Griess indicated that compared with blank group, the release of nitric oxide of LPS-induced group was increased (PPPPPPPα,IL-6,IL-1β,iNOS were up-regulated (Pα,IL-6,IL-1β,iNOS were down-regulated in Yiqi Yangyin Zhuyu recipe group, especially at the concentration at 2.0 g·L-1 (PConclusion: Yiqi Yangyin Zhuyu recipe could effectively inhibit the inflammatory reaction induced by LPS. The anti-inflammatory mechanism of Yiqi Yangyin Zhuyu recipe may be related to inhibition of macrophages to M1 phenotype polarization, so as to play the role of regulating immune and reducing the release of inflammatory cytokines, like NO,TNF-α,IL-6,IL-1β.
ABSTRACT
<p><b>OBJECTIVE</b>To study the effects of Guanxinping Tablet (GT) containing serum on H2O2-induced apoptosis and the nuclear factor kappa B (NF-kappaB) expression in vascular endothelial cells (VECs).</p><p><b>METHODS</b>Rabbits were randomly divided into the normal control group (treated with normal saline, 10 mL/kg), the verapamil group (0. 02 g/kg, 10 mL/kg), the small dose GT group (2; 8 g/kg, 10 mL/kg), the middle dose GT group (5.6 g/kg, 10 mL/kg), and the large dose GT group (11.2 g/kg, 10 mL/kg), 3 in each group. The medication was given to rabbits by gastrogavage for 3 successive days. The gastrogavage was performed twice on the last day with an interval of 2 h. One h after the last medication the peripheral blood was sampled from the vein of the ear edge. The blood was put for 1 h and centrifuged at 2 500 r/min for 30 min. The serum was extracted and deactivated at 56 degrees C for 30 min to prepare the drug containing serum. The apoptosis injury model was established using 100 micromol/L H2O2 induced VECs in the log phase growth. After modeling they were divided into 6 groups, 5 samples in each group, i. e., the normal group (10% vehicle serum culture solution), the model group (10% vehicle serum culture solution +100 micromol/L H2O2), the verapamil group (10% verapamil serum culture solution +100 micromol/L H2O2), the low dose GT group (10% low dose GT culture solution +100 micromol/L H2O2), the middle dose GT group (10% middle dose GT culture solution + 100 micromol/L H2O2), and the high dose GT group (10% high dose GT culture solution + 100 micromol/L H2O2). THE VEC apoptotic rate was detected using flow cytometry. The protein expression of NF-kappaB was detected using Western blot.</p><p><b>RESULTS</b>The VEC apoptosis rate (9.00% +/- 1.18%) and the protein expression of NF-kappaB (0.39% +/- 0.06%) increased more in the model group than in the normal control group (P<0.01). Compared with the model group, the VEC apoptosis rate of the verapamil group (6.00% +/- 0.18%), the large dose GT group (5.30% +/- 0.08%), and the middle dose GT group (6.83% +/- 0.51%) were obviously lower. The expression of NF-kappaB of each treatment group significantly decreased (the verapamil group: 0.28% +/- 0.03%; the small dose GT group: 0.33% +/- 0.03%; the middle dose GT group: 0.30% +/- 0.03%; the large dose GT group: 0.28% +/- 0.04%, P<0.01, P<0.05).</p><p><b>CONCLUSIONS</b>GT could fight against H2O2-induced VEC cell apoptosis. Its mechanism might be correlated with regulating the expression of NF-kappaB protein.</p>