| BackgroundPropofol (2,6-diisopropylphenol) is probably the most widely used intravenous hypnotic agent in daily practice. However, its anti-inflammatory properties have seldom been addressed. In this study, we evaluated the anti-inflammatory activity and mechanisms of propofol on lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro and found that propofol markedly inhibited LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and expression of inducible nitric oxide synthase (iNOS). At the same time, the expression of hepatocyte nuclear factor-la (HNF-la) and apolipoprotein M (apoM) was inhibited by treatment with LPS and LPS-induced down-regulation of HNF-la expression and apoM expression could be compensated by propofol treatment. However, propofol could not compensate LPS-induced down-regulation of apoM expression by treatment with HNF-la siRNA and the suppressive effect on LPS-induced pro-inflammatory cytokines production by propofol was significantly compensated by treatment with apoM siRNA. These results provide evidence that propofol may first up-regulate apoM expression by enhancing HNF-la expression and then inhibit pro-inflammatory cytokine production in LPS-stimulated cells. Therefore, our study may be useful in understanding the critical effect of propofol in patients with systemic inflammatory response syndrome.Aims1. To study the effects of propofol on expressions of apoM and HNF-la in LPS treated C57BL/6mice.2. To study the effects of propofol on expressions of apoM and HNF-la in LPS treated HepG2cells.3. To study wether HNF-la is involved in the propfol induced apoM expression in LPS treated HepG2cells.4. To study the anti-inflamation effects of propofol on LPS treated THP-1cells.5. To study propofol excert anti-inflammation effects whether through the apoM pathway in LPS treated THP-1cells.Materials and MethodsPropofol (2,’6-di-isopropylphenol) and lipopolysaccharides from Escherichia coli055:B5were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PrimeScript RT Reagent kit (Perfect Real Time; catalog no. DRR037A) and the SYBR(?) Premix Ex TaqTM II kit (Tli RNaseH Plus; catalog no. DRR820A) were obtained from TaKaRa Bio, Inc.(Shiga, Japan). All other chemicals were of pharmaceutical grade and purchased from commercial suppliers.AnimalsEight-week-old, female C57BL/6mice (Laboratory Animal Center of Peking University, Beijing, China) with a mean body mass of20g were randomized into four groups:(1) PBS-received intraperitoneal (i.p.) injections of phosphate-buffered saline (PBS, pH7.4);(2) LPS-received i.p.5mg/kg of LPS;(3) Propofol-received i.p.10mg/kg of propofol; and (4) LPS+Propofol-received i.p.5mg/kg of LPS plus10mg/kg of propofol. All animals were housed five per cage at25oC on a12-h light/dark cycle. The animal care and experimental procedures were approved by the Animal Experimental Committee at Nanfang Hospital (Guangdong, China).Cell CultureHuman hepatocytes (HepG2) and acute monocytic leukemia (THP-1) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The HepG2cells were cultured in25-cm2vented flasks containing Dulbecco’s modified Eagle’s medium (DMEM) with10%fetal calf serum (FCS) under standard culture conditions (5%CO2,37℃). THP-1cells were maintained in Roswell Park Memorial Institute (RPMI)1640medium containing10%FCS and differentiated for72h with100nM phorbol12-myristate13-acetate (PMA) under standard culture conditions (5%CO2,37℃). Prior to the experiment, cells were washed twice with PBS and once with serum-free medium without antibiotics. Experimental media contained DMEM or RPMI1640with0.2%human serum albumin and one or more additives (i.e., LPS, propofol, etc.) at the concentrations described in the figure legends.RNA isolation and quantitative real time PCR (qPCR)Total RNA from mouse tissues or cultured cells was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacture’s instructions. qPCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City. CA, USA). Melt curve analyses of all qPCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered. Quantitative measurements were determined using the△Ct method and expression of glyceraldehyde3-phosphate dehydrogenase was used as an internal control.Western blot analyses Proteins were extracted from mouse tissues or cultured cells using RIPA buffer (Biocolor Ltd., Belfast, Northern Ireland, UK), quantified using the BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China), and then subjected to western blot analyses (10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis;30μg protein per lane) using rabbit anti-APOM antibodies (BD Biosciences, San Jose, CA, USA), rabbit polyclonal anti-iNOS antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and rabbit polyclonal anti-HNF-la-and β-actin-specific antibodies (Abcam, Cambridge, MA, USA). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blot Detection System; Amerisham Biosciences, Foster City, CA, USA).Measurement of cytokine productionThe THP-1cells were differentiated into macrophages with100nM PMA for72h and incubated in the absence or presence of LPS with or without propofol. After stimulation and sample treatment, the cell-free supernatants of the THP-1macrophages were collected and used for cytokine analysis. The concentrations of TNF-a, IL-1β, and IL-6were measured in duplicate using a BiotrakTM enzyme-linked immunosorbent assay (ELISA) kit (Amersham Biosciences).Transfection with small interfering RNA (siRNA)The siRNAs against HNF-la and APOM and irrelevant21-nucleotide control siRNAs were purchased from RiboBio Co., Ltd.(Guangzhou, China). Cells (2×106/well) were transfected with siRNA targeting HNF-la, APOM, or the control in the absence or presence of appropriate plasmids using Lipofectamine2000(Invitrogen). qPCR and western blotting were performed48h post-transfection.Construction of recombinant plasmidsThe PIRES2-EGFP and PCR-XL-TOPO vectors (containing HNF-la which was assembled by the chemically synthesized oligos through PCR) were purchased from Invitrogen. Segments of EcoRI-HNF-la and IRES-EGFP XhoI were amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. EcoRI-HNF-la-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested by EcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent E. coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-HNF-la. The plasmid transfection process was performed using Lipofectamine2000transfection reagent according to the manu-facturer’s instructions.Results1. Effects of propofol on APOM and HNF-la expression in LPS-treated miceWe first investigated the role of propofol on APOM and HNF-la expression in hepatocytes of LPS-treated mice by qPCR and western blotting. As shown in Figures1A and C, APOM RNA and protein levels were markedly down-regulated by LPS treatment. In contrast, APOM expression was significantly up-regulated by propofol treatment. Moreover, propofol obviously compensated LPS-induced down-regulation of APOM expression in mouse hepatocytes. As shown in Figures IB and D, the transcript and protein levels of HNF-la were down-regulated by LPS treatment while up-regulated by propofol treatment, as propofol obviously compensated LPS-induced down-regulation of HNF-la expression in mouse hepatocytes.2. Effects of LPS and propofol on expression of apoM and HNF-la in HepG2cellsSince the expression of apoM and HNF-la was negatively regulated by LPS in mouse hepatocytes and apoM expression was directly regulated by transcription factor HNF-la, we next examined LPS effects on apoM and HNF-la expression in HepG2cells by qPCR (Figures2A and2B) and western blotting (Figures2C and2D). As shown, expression levels of apoM and HNF-la were decreased by LPS treatment at the transcriptional and translational levels. The inhibitory effects of LPS on apoM and HNF-la expression were increased with cell culture time and the strongest effects were seen after24h. We also observed the effects of propofol on APOM and HNF-la expression in HepG2cells by qPCR (Figures2E and2F) and western blotting (Figures2G and2H). We showed that expression of APOM and HNF-la was enhanced by propofol at the transcriptional and translational levels in a time-dependent manner in HepG2cells.3. HNF-la is involved in apoM expression regulation by propofol in LPS-stimulated HepG2cellsSince both apoM and HNF-la expression were inhibited by LPS while enhanced by propofol, we further investigated the involvement of HNF-la in apoM expression regulation following propofol treatment in LPS-stimulated HepG2cells. First, we examined the effects of HNF-la siRNA on apoM expression treated with propofol in LPS-stimulated HepG2cells. As shown in Figure3, treatment with siRNA targeting HNF-la downregulated HNF-la protein expression by89%in HepG2cells. The up-regulation of apoM expression via propofol treatment was nearly restored by HNF-la siRNA treatment, whereas propofol could not compensate LPS-induced down-regulation of apoM expression in HepG2cells. Next, we observed the effects of recombinant plasmids over-expressing HNF-la (pcDNA-HNF-1α) on apoM expression following propofol treatment in LPS-stimulated HepG2cells. As shown in Figure3, treatment with pcDNA-HNF-la upregulated HNF-la protein expression by613%in HepG2cells. Suppression of apoM expression by LPS was markedly compensated by treatment with pcDNA-HNF-la and propofol significantly enhanced apoM expression in LPS-stimulated HepG2cells.4. Anti-inflammatory effects of propofol on LPS-stimulated THP-1macrophagesWe explored the effects of propofol on apoM and HNF-la expression in LPS-stimulated THP-1macrophages by western blotting. As shown in Figure4A, apoM levels were markedly down-regulated by LPS treatment. In contrast, apoM expression was significantly up-regulated by propofol treatment, which obviously compensated LPS-induced down-regulation of apoM expression in THP-1 macrophages. Likewise, as shown in Figure4B, HNF-la levels were down-regulated by LPS treatment while up-regulated by propofol treatment, which obviously compensated LPS-induced down-regulation of HNF-la expression in THP-1macrophages. In order to study the anti-inflammatory effects of propofol on LPS-stimulated THP-1macrophages, we analyzed iNOS expression via western blotting. As shown in Figure4C, iNOS levels were markedly up-regulated by LPS treatment, whereas propofol notably inhibited the enhancement effect induced by LPS in THP-1macrophages. To evaluate the effects of propofol on pro-inflammatory cytokine production, including TNF-a, IL-1β and IL-6in the culture medium, THP-1macrophages were treated with LPS only or LPS with propofol. As shown in Table1, LPS treatment dramatically increased pro-inflammatory cytokine secretion into the culture medium as compared to the control group. However, pro-inflammatory cytokine production was decreased following propofol treatment in LPS-stimulated THP-1macrophages.5. apoM is involved in the anti-inflammatory effects of propofol in LPS-stimulated THP-1macrophages.Next, we investigated the role of apoM in propofol-induced anti-inflammatory effects on LPS-stimulated THP-1macrophages using apoM siRNA. As shown in Figure5, treatment with siRNA targeting apoM for48h downregulated apoM protein expression by86%in THP-1macrophages. The iNOS-induced up-regulation of LPS expression could not be compensated by propofol after treatment with apoM siRNA in THP-1macrophages. We also observed the effects of APOM siRNA on expression of pro-inflammatory cytokines by propofol in LPS-stimulated THP-1macrophages. As shown in Table2, the suppression of pro-inflammatory cytokines expression by propofol was markedly compensated by treatment with APOM siRNA in LPS-stimulated THP-1macrophages.Conclusion1. apoM and HNF-la mRNA and proteins expressions were up-regaulated by propofol in LPS-treated C57BL/6mice.2. Expression of apoM and HNF-la mRNA and proteins expressions were up-regaulated by propofol in HepG2cells.3. HNF-la is involved in apoM expression by propofol in LPS-stimulated HepG2cells.4. Propofol has anti-inflammatory effects in LPS-stimulated THP-1macrophages.5. apoM is involved in the anti-inflammatory effects of propofol in LPS treated THP-1macrophages cells. |
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