Establishment of a cell model of insulin-resistant 3T3-L1 adipocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes.</p><p><b>METHOS</b>Dexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10(-8), 10(-7), and 10(-6) mol·L(-1)) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin- resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1µmol·L(-1) DEX, and the changes of glucose concen- tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay.</p><p><b>RESULTS</b>Treatment of 3T3-L1 cells with DEX, IBMX and 10(-6) mol·L(-1)) insulin for 9 days resulted in the differentiation of >90% of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 µmol·L(-1) DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001).</p><p><b>CONCLUSION</b>3T3-L1 cells can be induced into mature adipocytes by exposure to 1 µmol·L(-1) DEX, 0.5 mmol·L(-1) IBMX and 10(-6) mol·L(-1)) insulin. A 96 h exposure to 1 µmol·L(-1) DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.</p>

Advance in studies on regulatory mechanism of phenotype polarization of macrophages and intervention with traditional Chinese medicines / 中国中药杂志

Macrophages are heterogeneous and diversified, and can be polarized into different phenotypes in various microenvironments and physiological or pathological conditions. Major macrophage subpopulations including classically activated(M1) and alternatively activated(M2) macrophages, which represent different surface receptors, secret different cytokines and chemokines, are regulated by different signal paths of transcriptions and epigenetic levels, and play distinctive roles in tumor progress. TCMs may improve the microenvironment by regulating phenotype polarization of macrophages. So far, specific biomarkers and polarized molecules mechanisms generated through the macrophage polarization approach are still unclear. In this article, we merely summarize the advance in domestic and foreign studies on phenotype polarization of macrophages and regulatory mechanisms and look into the future of intervention with TCMs.

Total flavonoid from Glycyrrhizae Radix et Rhizoma and its ingredient isoliquiritigenin regulation M2 phenotype polarization of macrophages / 中国中药杂志

To study the mechanisms of total flavonoid from Glycyrrhizae Radix et Rhizoma (TFGR) and its ingredient isoliquiritigenin (ISL) on their regulation of M2 phenotype polarization of macrophages. IL-4 (60 μg x L(-1)) induced RAW264.7 cells for 6 h to establish the M2 macrophage model. TFGR and ISL restrained breast cancer cells migration with the aid of M2 macrophages in vitro. TFGR and ISL inhibited gene and protein expression of Arg-1, up-regulated gene of HO-1 and protein expression of iNOS, enhanced the expression of microRNA 155 and its target gene SHIP1, meanwhile down-regulated.the phosphorylation of STAT3 and STAT6. So TFGR and ISL were the bioactive fraction and ingredient in Glycyrrhizae Radix et Rhizoma to reverse M2 phenotype macrophages polarization. TFGR and ISL inhibited the promotion of M2 macrophages to breast cancer cells migration in vitro, STAT signal pathways and miR155 were partly involved.

Research on functions and mechanisms of MicroRNA-203 on migration of human hypopharyngeal carcinoma cells / 局解手术学杂志

Objective To determine the roles and underlying molecular mechanism of MicroRNA-203 on the migration of human hypo-pharyngeal carcinoma cells. Methods The potential MicroRNA-203 target genes were searched by bioinformatic miRNA target prediction tools and KEGG database,and a large number of candidates was identified. The MEKK1 was selected for further investigation. This gene is known to play a role in tumor metastasis. The MicroRNA-203’s binding sites in MEKK1’s mRNA 3’UTR were analyzed by luciferase report-er assays. Nextly,the protein expression of MEKK1 in Fadu-Lv-MicroRNA-203 cells was determined by Western blot assay. The regulation of MEKK1’s mRNA expression by MicroRNA-203 was analyzed by qRT-PCR. Transwell cell migration assays were performed to confirm the im-pact of MicroRNA-203 on hypopharyngeal carcinoma metastasis. Results The expression level of endogenous MicroRNA-203 was negatively correlated with the mRNA and protein expression levels of MEKK1 in hypopharyngeal carcinoma cells. Transwell migration assay results showed that MicroRNA-203 overexpression inhibited hypopharyngeal carcinoma cell migration ability. Furtherly,MEKK1 can promote hypo-pharyngeal carcinoma cell migration ability. Conclusion MEKK1 is a direct target of MicroRNA-203. MicroRNA-203 plays a role in hypo-pharyngeal carcinoma cell migration ability through MEKK1.

Effects of rutaecarpine on inflammatory cytokines in insulin resistant primary skeletal muscle cells / 中国中药杂志

It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 μmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P < 0.05 to P < 0.001) IL-1, IL-6 and TNF-α production by cultured IR-PSMC cells when incubating 24 hours with Rut, beginning from 20 to 180.0 μmol x L(-1). IL-1, IL-6 and TNF-α in the Rut treated groups were dose-dependently decreased compared with that in the IR-PSMC control group. Our results demonstrated that the Rut promoted glucose consumption and improved insulin resistance possibly through suppression of inflammatory cytokines in the IR-PSMC cells.

Advance in studies on molecular mechanisms of cisplatin resistance and intervention with traditional Chinese medicines / 中国中药杂志

Cisplatin is a first-line anticancer drug widely used in clinic. However, its resistance reduces its efficacy. With a non-specific cell cycle, cisplatin's main targets are nucleophilic protein, DNA and RNA in cells. Among cisplatin's multi-factorial resistance mechanisms, abnormal expression of transport protein, intracellular detoxification enhancement, DNA repair capacity increase and apoptosis blocking are the main mechanisms. Because traditional Chinese medicines (TCMs) have unique advantages in cancer treatment, their combination with cisplatin can improve the efficacy. In this paper, the authors summarized the advance in studies on cisplatin's resistance and the combination of TCMs and cisplatin in recent years.

Relationship of stroke and carotid intima-media thickness and traditional Chinese medicine intervention progress: a review / 中国中药杂志

Stroke is one of the major diseases that threaten human health, early diagnosis and treatment are very important for stroke. Carotid intima-media thickness (CIMT) is measured noninvasively to diagnosis stroke, and it is a independent predictor for stroke because its thickening can timely predict the incidence and development of stroke. As an important predictor of cardiovascular disease, more and more attention is played on CIMT. In this review, we will make a summary on the important role of CIMT in stroke and the mechanisms of carotid intima-media thickening in stroke as well as the potential use of traditional Chinese medicine in treating carotid intima-media thickening.

Effects of four kinds of Chinese medicine monomer on growth of PANC-1 xenograft tumor and studying of molecular mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>The antitumor effects of icarisid II, timosaponin A-III, neferine and salidroside were studied in PANC-1 xenograft tumor.</p><p><b>METHOD</b>To establish of the nude mice xenograft tumor model, PANC-1 cells were injected. When the tumor major diameter was reached 3-5 mm, the treatment was initiated. The mice were randomized into vehicle control and treatment groups of six animals per each. Chinese medicine monomer was injected intraperitoneally every day. In 23th day, mice were killed once a day, tumor tissue were isolated and weighed and divided into two parts. One part was fixed with formaldehyde for tissue section and immunohistochemistry, the another of tissue was frozen in liquid nitrogen then in - 80 degrees C refrigerator for gene and protein expression analysis.</p><p><b>RESULT</b>In PANC-1 tumor xenograft experiment, compared with model group, timosaponin A-III (1.0 mg x kg (-1)) exerted significant inhibitory effects on tumor growth. Timosaponin A-III suppressed mRNA expressions of VEGF (P < 0.05), reduced protein expressions of VEGF (P < 0.05), activated Caspase-3 protein. Icarisid II, neferine and salidroside had not an excelled antitumor effect.</p><p><b>CONCLUSION</b>Timosaponin A-III exerted an excelled antitumor effect. The antitumor mechanisms include anti-angiogenesis, apoptosis promotion.</p>

Study on in vitro anti-inflammatory activity of total flavonoids from Glycyrrhizae Radix et Rhizoma and its ingredients / 中国中药杂志

<p><b>OBJECTIVE</b>To examine the anti-inflammatory mechanism of total flavonoids of Glycyrrhizae Radix et Rhizoma (TFGR) and its ingredient on IFN-gamma and LPS-induced macrophage RAW264.7.</p><p><b>METHOD</b>Solvent extraction and macroporous resin enrichment were adopted for preparing ethanol extracts of Glycyrrhizae Radix et Rhizoma, components and total flavonoids. Ultraviolet spectroscopy was used to determine the content of total flavonoids. An IFN-gamma and LPS-induced cell inflammatory model was established. Griess reaction was used for detecting the effect of extracts at all levels and flavonoid monomers on nitrite content in cell culture supernatant. FRAP was used for measuring anti-oxidation capacity. RT-PCR was used for determining the effect of TFGR and isoliquiritigenins on intracellular inducible nitric oxide synthase iNOS, COX-2, IL-6 and PPAR-gamma. Western blot was used for detecting the effect of TFGR and isoliquiritigenins on iNOS, COX-2 and MAPK signal transduction pathways.</p><p><b>RESULT</b>Compared with other extracts, ethyl acetate fractions from Glycyrrhizae Radix et Rhizoma showed the highest inhibition ratio on nitrite content at the same concentration. After being enriched with macroporous resin, TFGR (60. 08% of liquiritin) of ethyl acetate extracts from Glycyrrhizae Radix et Rhizoma showed dose-dependence, and inhibited the nitrite content in cell culture supernatant, which was superior to ethyl acetate extracts, and had the protective effect on post-stimulated cell activity, with a stronger total anti-oxidation than other extracts. TFGR inhibited iNOS, IL-6 mRNA, protein expressions of iNOS, COX-2 and IL-6. Isoliquiritigenin, a flavonoid monomer, could inhibited iNOS, COX-2 gene and protein expression and gene expressions of IL-1beta and IL-6, and upside-regulated gene expression of PPAR-gamma.</p><p><b>CONCLUSION</b>Activity-oriented extraction suggests that ethyl acetate fractions from Glycyrrhizae Radix et Rhizoma is one of components with anti-inflammatory activity. TFGR obtained by enriching the active component showed dose-dependence, and inhibited the nitrite content in cell culture supernatant. The anti-inflammatory effect is partially achieved by regulating ERK signal pathway and inhibiting iNOS and COX-2 gene and protein expressions through extracellular signals of mitogen activated protein kinases (MAPKs). Specifically, isoliquiritigenin may be a component with TFGR anti-inflammatory activity.</p>

Analysis on anti-vascular inflammatory mechanism in vitro of total flavones from Artemisia anomala / 中国中药杂志

<p><b>OBJECTIVE</b>To study the impact of total flavones from Artemisia anomala (TFAS) on activation of macrophages, cell oxidative stress, auto-nitration of CuZn-SOD, platelet aggregation and isolated vascular tension.</p><p><b>METHOD</b>LPS and IFN-gamma induced activation of macrophages and oxidative stress in rats; H2O2 and nitrite induced auto-nitration of CuZn-SOD; ADP, AA and collagen induced platelet aggregation in vitro in mice; PE stimulates isolated vascular tension; nitrite content of macrophages was measured by Griess assay; MTT assay and FRAP assay was applied for cell viability and total cell antioxidant capacity; auto-nitration of CuZn-SOD was measured by Western blot and colorimetric methods; platelet aggregation was detected by turbidimetry; and aorta ring relaxation was recorded by isolated vascular function experience devices for rats.</p><p><b>RESULT</b>TFAS demonstrated dose dependence (25, 50, 100, 200 mg x L(-1)) on inhibiting induced macrophages NO production from generating, while increasing cell viability and total anti-oxidant capacity. Auto-nitration of CuZn-SOD was suppressed by TFAS in dose dependence (0.5, 5, 50 mg x L(-1)). TFAS showed an inhibitory effect on collagen-induced platelet aggregation at 50 mg x L(-1) and an endothelium-dependent relaxation effect on PE-induced vasoconstriction at 1 g x L(-1).</p><p><b>CONCLUSION</b>TFAS shows effect on anti-inflammation, anti-oxidation, anti-nitration, anti-platelet aggregation and vasodilatation in experiment in vitro, which may inhibit vascular inflammatory by regulating multiple target points. It is among material bases for promoting blood circulation and removing blood stasis.</p>

Diabetes mellitus ulcers treatment with Bletilla striata polysaccharide / 中国中药杂志

The aim of this study was to evaluate the efficacy of Bletilla striata polysaccharide on diabetes mellitus ulcers. Diabetes mellitus animal model was established by single ip injection of streptozotocin (STZ, 50 mg x kg(-1)) with the criteria of blood glucose > or = 16.7 mmol x L(-1) after 72 h. 4 weeks after STZ injection, each animal received two full thickness incisional wounds (1.8 cm in diameter). The wounds then were divided into B. striata polysaccharide group and PBS group. Wound closure rate, fibroblast (FB) infiltration, hydroxyproline (OHP) content and myeloperoxidase (MPO) levels were examined on day 3, 7, 14, 21 post wound. The treatment of B. striata polysaccharide significantly facilitated diabetes mellitus ulcers healing compared to PBS group. Histological analysis showed that B. striata polysaccharide markedly increased inflammatory cell infiltration in wound area. The herb also strongly evaluation of FB, OHP demonstrated a significantly increased in B. striata polysaccharide group. B. striata polysaccharide group promoted wound closure by means of enhanced inflammatory cell infiltration and re-epithelialization, and the promotion of FB and OHP levels.

Mitochondrial dysfunction induced by excessive ROS/RNS-metabolic cardiovascular disease and traditional Chinese medicines intervention / 中国中药杂志

Metabolic cardiovascular disease is a type of disease which almost caused by body carbohydrate and lipid metabolism dysfunction. Type 2 diabetes mellitus is a typical metabolic disease. It not only lead to the insulin resistance but also related to atherosclerosis. Oxidative stress is produced by the reactive oxygen/nitrogen species (ROS/RNS). Oxidative stress and its consequence events play important roles in atherosclerosis (AS). Mitochondria are both sources and targets of reactive oxygen and/or nitrogen species (ROS/RNS), and there is growing evidence that mitochondrial dysfunction may be relevant intermediate mechanism by which cardiovascular risk factors lead to the formation of vascular lesions. Several cardiovascular risk factors are demonstrated causes of mitochondrial damage. This review starts with excessive ROS/RNS-induced mitochondrial dysfunction. The authors emphasize the relationship among axis of excessive ROS/RNS-mitochondrial dysfunction-apoptosis-atherosclerosis. They also introduce several traditional Chinese medicines such as Ophiopogon japonicus, butin, Panax ginseng, Pueraria lobata, Solanum lyratum and so on in the treatment of relevant diseases through anti-ROS/RNS mechanism. Moreover, the TCMs also can anti-cancer and anti-fatigue,which show the speciality of TCMs different from the single effect of classical western medicines.

Progress in co-culture and differentiation in adipose-derived stem cells: a review / 生物工程学报

Stem cells can be differentiated into many kinds of somatic cells under defined culture conditions. In addition, the homing possess can be partially imitated by co-culture of stem cells with mature somatic cells. Regarding the importance of clinical application of adipose-derived stem cells (ADSCs), our review first introduced the sources and signs of ADSCs, and then the current knowledge of ADSCs co-culture technology, including drug and chemical induced culture, two-dimensional (2D) and three-dimensional (3D) co-culture, mechanisms of ADSCs differentiation, and application development in recent years in details. Finally, we also addressed prospects of ADSCs.

Study in the damage of endothelial function and administration recovery among different arteries during the developing progress of SHR / 中国药理学通报

Aim To evaluate the endothelial dysfunction level of different arteries at different stages of SHR,and the recovery after administration.Methods SHR model was used,captopril(3.375 g·kg~(-1)·d~(-1)) was administered from week 7 to week 24 and the effects were observed continuously until 8 weeks post treatment.Pathological changes of aorta,mesenteric and apex cordis arteries were examined at the time points of 6,18,24,32 wk,and the endothelial-dependent relaxation of the former two preparations were tested by acetylcholine(ACh)(n=6).Results There were pathological changes in the thoracic aorta,mesenteric artery and arteriole at 18 wk,and aggravated along the age.The thoracic aorta demonstrated the most severe pathological changes appearing endothelial cells ablated and tunica media thickening.The significant decline of endothelium-dependent relaxation in aorta,and mesenteric arteries of SHR reflected an aging dependent change of vascular function with the most severe situation in the aorta(P=0.10,18 wk,P<0.01 24,32 wk);captopril increased the aorta vasodilatation of SHR at 18 wk time point,without the effect in mesenteric artery(P<0.05 vs SHR).Conclusions During the progress of SHR,endothelial damages have been observed in all three kinds of vasculatures together with the reduced endothelial-dependent relaxation.The aorta presents earlier and deeper damage than middle and small size vessels,and is sensitive towards the antihypertensive therapy such as the angiotensin converting enzyme inhibitor.

Characterizing effects of solvent specific morus alba components on rat platelet aggregation, vascular tension and macrophage nitrite production / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of different solvent extractions of Mori Ramulus on platelet aggregation, vascular tension, and nitrite production from macrophages stimulated by lipopolysaccharides and interferon-gamma.</p><p><b>METHOD</b>The components of Mori Ramulus were extracted by EtoAc, n-BuOH and chloroform respectively. Platelet aggregation was induced by ADP, arachidonic acid and collagen in vitro; nitrite production of activated macrophages was measured by Griess assay, and the vasodilatory effects of three extractions were investigated by isometric tension changes of aortic rings.</p><p><b>RESULT</b>Chloroform extraction concentration-dependently inhibited platelet aggregation by arachidonic acid, and reduced vascular tension of PE preconstricted aorta rings with or without endothelium. On the other hand, extractions of EtoAc and n-BuOH demonstrated dose-dependent inhibition on macrophage NO production stimulated by LPS/IFN-gamma.</p><p><b>CONCLUSION</b>Pharmacological activities of Mori Ramulus depend on solvent specific components. Chloroform extraction of Mori Ramulus may benefit cardiovascular diseases through its properties of anti-platelet aggregation and vasodilatation. The inhibition of macrophage activity by EtoAc and n-BuOH extractions suggested an anti-inflammation effect of the compound.</p>

Study on effect of total saponins from Semen Nigellae on inflammatory mediators and ERK/MAPK pathway in stimulated macrophage / 中国中药杂志

<p><b>OBJECTIVE</b>To study the anti-inflammatory mechanism of total saponins from Semen Nigellae (TSSN).</p><p><b>METHOD</b>IFN-gamma plus LPS stimulated RAW 264. 7 macrophage has been used as inflammatory experimental model. Griess reaction for nitric oxide production, FRAP assay for total antioxidant capacity, RT-PCR for mRNA expression and Western blot for protein expression examination were performed.</p><p><b>RESULT</b>TSSN inhibited NO production in a dose-dependent manner. The gene and protein expression of iNOS were also suppressed by the herb extract. TSSN treatment significantly attenuated mRNA of inflammatory mediators such as COX-2, IL-1beta, IL-6 while increased PPAR-gamma gene and protein expression. Furthermore, phosphorylation of ERK (p-ERK) was markedly inhibited by TSSN.</p><p><b>CONCLUSION</b>TSSN suppressed pro-inflammatory mediators such as COX-2, IL-1beta, IL-6 and increased anti-inflammatory mediator PPAR-gamma expression. Meanwhile, TSSN inhibited over production of NO and iNOS expression through ERK/MAPK pathway.</p>

Inflammatory response and peroxisome proliferator-activated receptor isotypes expression in the brain of spontaneously hypertensive rats / 中华神经科杂志

Objective To evaluate the inflammatory response and the expressions of peroxisome proliferator-activated receptor(PPAR)isoforms(PPARα,PPARβ/δ,and PPARγ)in the brain(cortex,striatum,cerebellum)of spontaneously hypertensive rats(SHR).Methods Brain tissues(cortex,striatum,and cerebellum)were dissected from SHR and age-matched control Wistar-Kyoto rats.Myeloperoxidase(MPO)activity was measured in brain tissues as an index of neutrophil accumulation and the carbonyl protein content was analyzed by spectrophotometry to evaluate the protein oxidation.RT-PCR and Western blotting were performed to examine the expressions of inflammatory mediators(IL-1β,TNFα,ICAM-1,and iNOS)and nuclear factor PPARs(PPARα,PPARβ/δ,and PPARγ),respectively.Results(1)Systolic blood pressure of SHR was significantly higher than that of Wistar-Kyoto rats,(205.4±9.4)mm Hg versus(130.4±7.9)mm Hg(t=14.96,P＜0.01).(2)MPO activity of cortex,striatum,and cerebellum were markedly higher in SHR than in Wistar-Kyoto rats.Carbonyl protein levels of cortex,striatum,and cerebellum in Wistar-Kyoto rats and SHR were(3.27±0.43)nmol/mg versus(11.87±1.11)nmol/mg,(4.02±1.04)nmol/mg versus(14.06±1.36)nmol/mg,(5.94±0.71)nmol/mg versus(14.95±1.82)nmol/mg,indicating significantly higher levels of protein oxidation in SHR than Wistar-Kyoto rats(t=17.70,14.36,11.30,P＜0.05).Consistently,the expression of pro-inflammatory mediators(IL-1β,TNFα,ICAM-1,and iNOS)was upregulated when compared with Wistar-Kyoto rats.The difference between SHR and control Wistar-Kyoto rats was statistically significant except the mRNA expression of IL-1β in striatum,cerebellum and TNFα in cerebellum of SHR.All the above experimental data indicated the occurrence of inflammatory status in the brain tissue of hypertension.(3)mRNA and protein levels of brain PPAR isoforms(PPARα,PPARβ/δ,and PPARγ)of SHR increased significantly when compared with Wistar-Kyoto rats.Specifically.protein levels of PPARα in cortex.striatum,and cerebellum of SHR increased by 644.78%,791.95%,and 42.85%;PPARβ/δ increased by 106.72%,94.12%,and 161.44%;PPARγ was up-regulated by 2700.16%,790.81%,and 875.00%compared with that of Wistar-Kyoto rats,respectively.Conclusions The brain(cortex,striatum,and cerebellum)from SHR shows marked inflammatory status and increased expression of all PPAR isoforms.Increases in PPARs expression may play a compensatory role in the inflammatory response of the brain in SHR.

Inflammatory reaction versus endogenous peroxisome proliferator-activated receptors expression, re-exploring secondary organ complications of spontaneously hypertensive rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The chronic pathological changes in vascular walls of hypertension may exert destructive effects on multiple organ systems. Accumulating evidence indicates that inflammatory reactions are involved in the pathological changes of hypertension. Three peroxisome proliferator-activated receptors (PPARs) have been identified: PPARalpha, PPARbeta/delta, and PPARgamma, all of which have multiple biological effects, especially the inhibition of inflammation. The aim of this study was to evaluate PPAR isoforms expression profile in important organs of spontaneously hypertensive rats (SHR) and to understand the modulation of endogenous PPAR isoforms under inflammatory condition.</p><p><b>METHODS</b>Tissues (kidney, liver, heart, and brain) were dissected from SHR and age-matched control Wistar-Kyoto rats (WKY) to investigate the abundance of PPAR isoforms and PPAR-responsive genes (acyl-CoA oxidase and CD36). The expression of CCAAT/enhancer-binding protein delta (C/EBPdelta), which can trans-activate PPARgamma expression, was also observed. The inflammatory response was analyzed by the expression of inflammatory mediators inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, interleukin-1 beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha), and formation of carbonyl and nitrated proteins.</p><p><b>RESULTS</b>The expressions of 3 PPAR isoforms and PPAR-responsive genes were markedly upregulated in SHR compared with those of WKY. Specifically, the expression of PPARalpha protein in the kidney, liver, heart and brain increased by 130.76%, 91.48%, 306.24%, and 90.70%; PPARbeta/delta upregulated by 109.34%, 161.98%, 137.04%, and 131.66%; PPARgamma increased by 393.76%, 193.17%, 559.29%, and 591.18%. In consistent with the changes in PPARgamma, the expression of C/EBPdelta was also dramatically elevated in SHR. Inflammatory mediators expressions were significantly increased in the most organs of SHR than WKY. As a consequence, increased formation of carbonyl and nitrated proteins were also observed in the most organs of SHR.</p><p><b>CONCLUSIONS</b>These findings suggest an enhanced inflammatory response in the organs of SHR, which might play a key role in pathogenesis of hypertension and secondary organ complications. Changes (increases) in PPARs expression may reflect a compensatory mechanism to the inflammatory status of hypertensive rats.</p>

Inflammation of different tissues in spontaneously hypertensive rats / 生理学报

The hypertension is one of chronic vascular diseases, which often implicates multiple tissues causing stroke, cardiac hypertrophy, and renal failure. A growing body of evidence suggests that inflammatory mechanisms are important participants in the pathophysiology of hypertension. In this study, the inflammatory status of these tissues (kidney, liver, heart, and brain) in spontaneously hypertensive rats (SHR) was analyzed and its molecular mechanism was explored. The tissues were dissected from SHR and age-matched control Wistar-Kyoto (WKY) rats to investigate the abundance of inflammation-related mediators (IL-1beta, TNFalpha, ICAM-1, iNOS, C/EBPdelta and PPARgamma). mRNA levels were determined by reverse transcription-polymerase chain reaction and protein expression was evaluated by Western blot. To evaluate the oxidative stress of tissues, carbonyl protein content and total antioxidant capacity of tissues were detected by spectrophotometry and ferric reduction ability power (FRAP) method. The results suggest that: (1) Expressions of inflammation-related mediators (IL-1beta, TNFalpha, ICAM-1, iNOS, C/EBPdelta and PPARgamma) in SHR were higher compared with those in WKY rats except no evident increase of IL-1beta mRNA in liver and brain in SHR. (2) Tissues in SHR contained obviously increased carbonyl protein (nmol/mg protein) compared to that in WKY rats (8.93+/-1.08 vs 2.27+/-0.43 for kidney, 2.23+/-0.23 vs 0.17+/-0.02 for heart, 13.42+/-1.10 vs 5.72+/-1.01 for brain, respectively, P<0.05). However, no evident difference in the amount of carbonyl protein in liver was detected between SHR and WKY rats. (3) Total antioxidant capacities of kidney, liver, heart and brain were markedly lower in SHR than that in WKY rats (P<0.05). Thus, the present data reveal a higher inflammatory status in the important tissues in SHR and indicate that inflammation might play a potential role in pathogenesis of hypertension and secondary organ complications.

Molecular cloning of the survivin gene promoter and its specific expression in the human laryngeal cancer Hep-2 cell line / 医学研究生学报

Objective: To construct the eukaryotic expression vector pSurp-EGFP regulated by the survivin gene promoter and to detect the specific expression of the promoter in human laryngeal cancer Hep-2 cells by green fluorescent protein assay.Methods: Thesurvivin gene promoter was generated by polymerase chain reaction(PCR) and the CMV promoter of the pShuttle vector replaced by the survivin gene promoter to generate the plasmid pSurp.The three plasmids pShuttle,pSurp and pEGFP-C1 were respectively double-enzyme digested so as to produce the plasmids pCMV-EGFP and pSurp-EGFP carrying the CMV or survivin promoter.The purified pCMV-EGFP and pSurp-EGFP were transfected into Hep-2 cell and vascular endothelial cell ECV304 using liposome transfection reagent and the expressions of EGFP detected by the fluorescent microscope.Results: Thesurvivin gene promoter was successfully cloned by PCR,and thesurvivin gene promoter-regulated pSurp-EGFP was constructed.Green fluorescence was observed in Hep-2 cells but not in ECV304. Conclusion: The high specific activity of the survivin gene promoter in Hep-2 cells that we successfully constructed attributes to the studies of tumor specific gene therapy.