Therapeutic Effect and Mechanism of Oxytropis falcata Gel on Deep Second-Degree Burn in Rats.

Oxytropis falcata has long been used to treat inflammation, sores, and bleeding in Tibet. However, the burn remedy and underlying molecular mechanisms are not well understood. This study is aimed at assessing the effect of Oxytropis falcate gel (OFG) on deep second-degree burn rats and exploring its mechanism. Wistar rats with second-degree burn were treated with OFG and silver sulfadiazine. Immunohistochemical detections for EGF and VEGF were performed, and ELISA detections for EGF, VEGF, p38, and IL-1ß in serum were determined. Rats treated with OFG (25, 50 g/kg) consisted of the major rhamnocitrin-3-O-ß-neohesperidoside significantly accelerated incrustation (P < 0.001) and decrustation (P < 0.001). According to HE staining, edema and infiltration of inflammatory cells decrease apparently with good hyperplasia and incrustation in administration groups (7 d). The expressions of EGF and CD34 in OFG (25, 50 g/kg) treatment increased obviously from immunohistochemical assessment (7 d). Serum EGF expression reached 321.27 ± 7.20 ng/mL by OFG treatment, while p38 (P < 0.05) and IL-1ß (P < 0.05) levels were significantly lower than the model and vehicle groups from day 1 to day 7. OFG possesses potential wound healing activities. The mechanism may be related to the increasing of biosynthesis and the releasing of EGF and CD34 and the decreasing p38 and IL-1ß levels.

Development of an indirect competitive ELISA for simultaneous detection of enrofloxacin and ciprofloxacin.

Modified 1-ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method was employed to synthesize the artificial antigen of enrofloxacin (ENR), and New Zealand rabbits were used to produce anti-ENR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (ELISA) standard curve was established. This assay was sensitive and had a linear range from 0.6 to 148.0 µg/kg (R(2) = 0.9567), with the half maximal inhibitory concentration (IC(50)) and limit of detection (LOD) values of 9.4 µg/kg and 0.2 µg/kg, respectively. Of all the competitive analogues, the produced pAb exhibited a high cross-reactivity to ciprofloxacin (CIP) (87%), the main metabolite of ENR in tissues. After optimization, the matrix effects can be ignored using a 10-fold dilution in beef and 20-fold dilution in pork. The overall recoveries and coefficients of variation (CVs) were in the ranges of 86%-109% and 6.8%-13.1%, respectively. It can be concluded that the established ELISA method is suitable for simultaneous detection of ENR and CIP in animal tissues.

Inhibitory effects of 16-hydroxycleroda-3,13(14)E-dien-15-oic acid on superoxide anion and elastase release in human neutrophils through multiple mechanisms.

Reactive oxygen species and granule proteases produced by neutrophils contribute to the pathogenesis of inflammatory diseases. In this study, a cellular model in isolated human neutrophils was established to elucidate the anti-inflammatory functions of 16-hydroxycleroda-3,13(14)E-dien-15-oic acid (PL3S), a clerodane diterpenoid from Formosan Polyalthia longifolia var. pendula. PL3S significantly inhibited the generation of superoxide anion and the release of elastase in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion with IC50 values of 3.06+/-0.20 and 3.30+/-0.48 microM, respectively. PL3S did not affect cAMP-dependent pathway, and the inhibitory effect of PL3S was not reversed by protein kinase A inhibitor. PL3S did not display antioxidant or superoxide anion-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. PL3S concentration-dependently inhibited calcium mobilization caused by FMLP but not thapsigargin. Furthermore, PL3S attenuated the FMLP-induced protein kinase B (AKT) and p38 mitogen-activated protein kinase phosphorylation. However, PL3S had no effect on FMLP-induced phosphorylation of extracellular regulated kinase and c-Jun N-terminal kinase. In summary, these results indicate that the suppressive effects of PL3S on human neutrophil respiratory burst and degranulation are at least partly mediated by inhibition of calcium, AKT, and p38 signaling pathways.

Nitric oxide-independent lipid metabolism in RAW 264.7 macrophages loaded with oleic acid.

The role of nitric oxide (NO) in the regulation of lipogenesis and lipolysis in RAW 264.7 macrophages loaded with oleic acid (OA) was investigated in this paper. Magnolol stimulated full lipolysis without affecting NO levels. Both inhibition and elevation of NO production in OA-loaded macrophages did not induce lipolysis. Besides, lipopolysaccharide (LPS)-induced increased accumulation of lipid droplets was not reduced by down-regulation of NO levels. Moreover, incubation of macrophages with sodium nitroprusside (SNP), an NO donor, stimulated significant NO production without altering the lipid droplet accumulation. All these results clearly demonstrate that NO is not involved in the modulation of lipid metabolism in macrophages loaded with OA.

Magnolol stimulates lipolysis in lipid-laden RAW 264.7 macrophages.

This study investigated the effect of magnolol, a compound isolated from Magnolia officinalis, on lipolysis in lipid-laden RAW 264.7 macrophages. Treatment of macrophages with magnolol led to dissolution of lipid droplets. This phenomenon was accompanied by a dose-dependent release of glycerol and cholesterol and a concomitant reduction in intracellular levels of glycerol and cholesterol. Furthermore, adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, was down-regulated by magnolol in a dose- and time-dependent manner by Western blot analysis. Immunofluorescence studies also showed that ADRP became detached from the surface of lipid droplets after magnolol treatment. The lipolytic effect of magnolol was not mediated through the cAMP-protein kinase A (PKA) system, an authentic lipolytic pathway for macrophages, since magnolol did not induce an increase of intracellular cAMP levels, and pretreatment with either of PKA inhibitors, PKI and KT5720, did not abrogate the lipolytic response to magnolol. We conclude that magnolol induce-lipolysis of lipid-laden macrophages by down-regulation of ADRP expression and detachment of ADRP from the lipid droplet surface by a cAMP-independent mechanism. Lipolysis of lipid-laden macrophages may occur when the amount of ADRP on the surface of lipid droplets is not enough to stabilize the lipid droplets.

Oleic acid-induced PKC isozyme translocation in RAW 264.7 macrophages.

Fatty acids are important second messengers that mediate various cellular functions, but their role in the formation of macrophage foam cells is not known. High plasma levels of oleic acid (OA) in obese patients are often associated with a high risk for atherosclerosis. In this study, we investigated the protein kinase C (PKC) isozymes involved in OA-induced lipid accumulation in RAW 264.7 macrophages. The results show that OA induces translocation of PKC alpha, beta1, and delta from the cytosol to the cell membrane 5 min after the treatment. After 16 h incubation with OA, PKC delta was found to be colocalized with adipose differentiation-related protein (ADRP) on the surface of lipid droplets, but immunoprecipitation experiments showed that PKC delta was not biochemically associated with ADRP. After 16 h incubation with OA plus phorbol 12-myristate 13-acetate (PMA), PKC delta staining on the lipid droplet surface was not seen, whereas the accumulation of lipid droplets was unaffected. Furthermore, downregulation of PKC delta was confirmed by immunoblotting. These results demonstrate possible involvement of specific PKC isozymes in the early phase of lipid accumulation, possibly during the uptake of OA.