ABSTRACT
Objective:To explore the protective effect of methyl eugenol (Me) on islet ischemia/reperfusion (I/R) injury and elucidate its underlying mechanism.Methods:The islets were isolated and purified from 6-8 week male BALB/c mice and divided into four groups of normal control (normal culture without any treatment), hypoxia/reoxygenation (H/R treatment), H/R+ dimethyl sulfoxide (DMSO dosing plus H/R treatment) and H/R+ Me (Me dosing plus H/R). Viability of islet cells in each group was detected by acridine orange (AO)/propidium iodide (PI) double stain.Function of islet cells (insulin secretion) was measured by enzyme-linked immunosorbent assay (ELISA). Murine islet β Min6 cells were selected for detecting the effect of Me on the proliferative activity of normal cultured and H/R treated islet cells under different concentration gradients by CCK8.Then Min6 cells were divided into four groups of normal, H/R, H/R+ DMSO and H/R+ Me.The definition of group was the same as that of primary murine islets.Flow cytometry and Hoechst 33342 nuclear stain were utilized for detecting cell apoptotic rate in each group.The protein expressions of p-JNK, p-p38, JNK, p38, Bcl-2 and Bax were detected by Western blot.And the data were processed by one-way ANOVA or t test.Results:The proportion of dead islet cells in H/R group was (29.47±2.65)% and it was significantly lower than that in normal group (7.63±1.53)%.And the inter-group differences were statistically significant ( P<0.001). The proportion of dead islet cells was (20.63±3.07)% in H/R+ Me group.It was higher than that in H/R group (29.47±2.65)% and in H/R+ DMSO group (30.13±1.50)% and inter-group difference was statistically significant ( P<0.05 & P<0.01). Under the stimulation of high glucose, the insulin secretion level of islet in H/R+ Me group was (1.76+ 0.08) mg/L, which was higher than that in H/R group and H/R+ DMSD group(1.24±0.14)mg/L and(1.27±0.05)mg/L, and the difference was statistically significant[(1.76±0.08) vs. (1.24±0.14) mg/L; (1.76±0.08) vs.(1.27±0.05) mg/L, P<0.01]. There was no significant effect on cell viability after Me dosing within a certain concentration range (0-40 μmol/L). After Me dosing (5 μmol/L), cell viability of H/R-treated Min6 cells was significantly higher than that without Me.And the difference was statistically significant[(1.19±0.03) vs.(1.00±0), P<0.01]. As compared with H/R and H/R+ DMSO groups, overall apoptotic rate declined in H/R+ Me group (Hoechst 33342 stain: 14.50%±1.05% vs. 23.30%±1.18%, 14.50%±1.05% vs. 22.77%±1.75%, P<0.001; Flow cytometry: 4.36%±0.54% vs. 21.44%±1.02%, 4.36%±0.54% vs. 21.68%±3.06%, P<0.01). The expressions of p-JNK and p-p38 were down-regulated (p-JNK: 0.77±0.06 vs. 1.03±0.05, 0.77±0.06 vs.0.93±0.04, P<0.001; p-p38: 0.80±0.05 vs. 1.01±0.08; 0.80±0.05 vs. 1.00±0.05, P<0.05) while Bcl-2/Bax ratio rose (1.62±0.13 vs. 0.72±0.10, 1.62±0.13 vs. 0.74±0.13, P<0.01). Conclusions:Me can improve the viability and function of islets and suppress the apoptosis of Min6 cells after H/R.The mechanism is correlated with JNK and p38 MAPK signaling pathways.
ABSTRACT
This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 μmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.
Subject(s)
Humans , Apoptosis , Epithelial Cells/metabolism , Eugenol/pharmacology , Heme Oxygenase-1/metabolism , Hypoxia , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Reactive Oxygen Species , Reperfusion Injury/drug therapyABSTRACT
Abstract The essential oils from the fresh leaves of three Melalecua spp. viz; Melaleuca leucadendron (L.) Melaleuca linariifolia Sm. and Melaleuca bracteata F. Muell. growing in Tarai region of North India were analyzed by a combination of gas chromatography/mass spectrometry. The analysis revealed the presence of several constituents of industrial and pharmacological importance. M. leucadendron essential oil was found to be dominated by E-nerolidol (85.7%) rich chemotype. 1,8-cineole (61.1%) along with significant presence of α-terpineol (12.3%), α-pinene (4.0%), β-myrcene (3.8%), and E-caryophyllene (1.7%) were identified in the essential oil from M. linariifolia Similarly M. bracteata was dominated by the presence of phenylpropanoids viz; methyl eugenol (74.8%) and methyl cinnamate (8.0%). The essential oils were studied for their in-vitro antioxidant, anti-inflammatory and antimicrobial potential. All the oils revealed potential antioxidant activity with maximum in M. bracteata essential oil. All the oils exhibited significant antibacterial activity against Bacillus megaterium, Staphylococcus aureus, Escherichia coli,Salmonella typhimurium and anti-fungal activity against phytopathogenic fungi Fusarium oxysporum, Sclerotinia sclerotiorum, Exserohilum turcicum and Curvularia lunata. The observations from present study suggest further cultivation of Melaleucas and its commercialization as industrial crops.
ABSTRACT
Objective: To verify the feasibility of vapor-permeable membrane technology for the separation of water bodies containing essential oil of Asari Radix et Rhizoma (ARR) essential oil, and then to apply vapor permeate technology to the separation of more essential oils of traditional Chinese medicine. Methods: The polydimethylsiloxane/polyvinylidene fluoride (PDMS/PVDF) composite flat membrane and polyvinylidene fluoride (PVDF) flat membrane were collected as the membrane material. The oil-bearing water body of ARR volatile oil was separated by vapor permeate technology, and the oil penetration rate of two kinds of membranes was calculated. At the same time, the changes of the composition and content of the essential oil before and after the membrane were analyzed by gas chromatography-mass spectrometry (GC-MS). Results: The results showed that the essential oil penetration rate was significantly higher than that of PDMS/PVDF membrane when PVDF membrane was used as membrane material. GC-MS qualitative analysis results showed that the composition of the essential oil in the penetrants of the two membranes was basically the same as that of the essential oil obtained by the traditional steam distillation method. The content of α-pinene, β-pinene, 3,5-dimethoxytoluene, and methyl eugenol were determined by double internal standard method. The results showed that the content of each component in the PVDF membrane permeation was significantly higher than that of the PDMS/PVDF membrane permeation solution. Conclusion: It is feasible to separate the oil containing water from the essential oil of ARR by vapor permeation membrane technology. Compared with the PDMS/PVDF membrane, the PVDF membrane is more suitable for separating the oil containing water of the essential oil of ARR.
ABSTRACT
Objective: To clone the enzyme genes related with methyl eugenol synthesis and characterize the corresponding sequence information based on the transcriptome sequencing of Asarum heterotropoides. Methods: RT-PCR was performed to obtain the full length cDNA of the phenylalanine lyase (PAL) gene, cinnamic acid-4-hydroxylase (C4H) gene, 4-hydroxycinnamoyl-CoA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD) genes by using young leaves as materials. The acquired enzyme genes were analyzed by bioinformatics. Results: The ORF lengths of AhPAL, AhC4H, Ah4CL, and AhCAD were 2 157, 1 278, 1 623 and 1 071 bp, which respectively encoded 718, 425, 540, and 356 amino acids. Four proteins had respective conserved domains. The amino acid sequences of AhPAL, Ah4CL, and AhCAD were similar to those of other reported species except for AhC4H. Conclusion: Four enzyme genes related with methyl-eugenol synthesis in A. heterotropoides were separated and analyzed using bioinformatics method. These results would lay the important foundation for functional analysis of corresponding genes and for elucidating the regulation mechanism of methyl-eugenol biosynthesis.