Melatonin restores muscle regeneration and enhances muscle function after crush injury in rats

The goal of this study was to provide evidence that melatonin improves muscle healing following blunt skeletal muscle injury. For this purpose, we used 56 rats and induced an open muscle injury. After injury, all animals received either daily melatonin or vehicle solution intraperitoneally. Subsequent observations were performed at day 1, 4, 7, and 14 after injury. After assessment of fast twitch and tetanic muscle force, we analyzed leukocyte infiltration, satellite cell number, and cell apoptosis. We further quantified the expression of the melatonin receptor and the activation of extracellular-signal-regulated kinase (ERK). Chronic treatment with melatonin significantly increased the twitch and tetanic force of the injured muscle at day 4, 7, and 14. At day 1, melatonin significantly reduced the leukocyte infiltration and significantly increased the number of satellite cells when compared to the control group. Consistent with this observation, melatonin significantly reduced the number of apoptotic cells at day 4. Furthermore, phosphorylation of ERK reached maximal values in the melatonin group at day 1 after injury. Additionally, we detected the MT1a receptor in the injured muscle and showed a significant up-regulation of the MT1a mRNA in the melatonin group at day 4. These data support the hypothesis that melatonin supports muscle restoration after muscle injury, inhibits apoptosis via modulation of apoptosis-associated signaling pathways, increases the number of satellite cells, and reduces inflammation.     

Melatonin protects against thoracic aortic aneurysm and dissection through SIRT1-dependent regulation of oxidative stress and vascular smooth muscle cell loss

Melatonin functions as an endogenous protective molecule in multiple vascular diseases, whereas its effects on thoracic aortic aneurysm and dissection (TAAD) and underlying mechanisms have not been reported. In this study, TAAD mouse model was successfully induced by β-aminopropionitrile fumarate (BAPN). We found that melatonin treatment remarkably prevented the deterioration of TAAD, evidenced by decreased incidence, ameliorated aneurysmal dilation and vascular stiffness, improved aortic morphology, and inhibited elastin degradation, macrophage infiltration, and matrix metalloproteinase expression. Moreover, melatonin blunted oxidative stress damage and vascular smooth muscle cell (VSMC) loss. Notably, BAPN induced a decrease in SIRT1 expression and activity of mouse aorta, whereas melatonin treatment reversed it. Further mechanistic study demonstrated that blocking SIRT1 signaling partially inhibited these beneficial effects of melatonin on TAAD. Additionally, the melatonin receptor was involved in this phenomenon. Our study is the first to report that melatonin exerts therapeutic effects against TAAD by reducing oxidative stress and VSMC loss via activation of SIRT1 signaling in a receptor-dependent manner, thus suggesting a novel therapeutic strategy for TAAD.     

Effect of growth hormone and estrogen administration on hepatocyte alterations in old ovariectomized female wistar rats

Aging could be due to the accumulation of oxidative damage. On the other hand, growth hormone (GH) and estrogen deficiency induce deleterious effects on different tissues, and hormonal replacement could counteract these effects. We have investigated whether GH and estrogen administration modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female rats. Twenty-two month-old ovariectomized animals were divided into control rats, rats treated with GH, rats treated with estradiol, and rats treated with GH+estradiol. Two-month-old intact female rats were used as young reference group. Hepatocytes were isolated, cultured, and CO and NO release, ATP, cyclic-guanosyl monophosphate (cGMP), and lipid peroxide (LPO) content of cells, as well as phosphatidylcholine (PC) synthesis, were measured. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content and PC synthesis compared to young rats. Age also induced an increase in LPO, NO, CO, and cGMP. Treating old rats with GH significantly increased ATP and reduced CO and cGMP levels. Estradiol administration improved all the parameters that were altered. Co-administration of GH and estrogens induced a more marked effect than estrogens alone only in cGMP content. In conclusion, administration of estrogens to old ovariectomized females seemed to prevent oxidative changes in hepatocytes, whereas the effect of GH is not so evident.     

Melatonin attenuates hypoxic pulmonary hypertension by inhibiting the inflammation and the proliferation of pulmonary arterial smooth muscle cells

Hypoxia‐induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Melatonin possesses anti‐inflammatory and antiproliferative properties. However, the effect of melatonin on HPH remains unclear. In this study, adult Sprague–Dawley rats were exposed to intermittent chronic hypoxia for 4 wk to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/LV+S) ratio, and median width of pulmonary arterioles. Melatonin attenuated the elevation of RVSP, RV/LV+S, and mitigated the pulmonary vascular structure remodeling. Melatonin also suppressed the hypoxia‐induced high expression of proliferating cell nuclear antigen (PCNA), hypoxia‐inducible factor‐1α (HIF‐1α), and nuclear factor‐κB (NF‐κB). In vitro, melatonin concentration‐dependently inhibited the proliferation of PASMCs and the levels of phosphorylation of Akt and extracellular signal‐regulated kinases1/2 (ERK1/2) caused by hypoxia. These results suggested that melatonin might potentially prevent HPH via anti‐inflammatory and antiproliferative mechanisms.     

Melatonin is able to prevent the liver of old castrated female rats from oxidative and pro-inflammatory damage

The aim of this study was to investigate the effect of aging and ovariectomy on various physiological parameters related to inflammation and oxidative stress in livers obtained from old female rats, and the influence of chronic administration of melatonin on these animals. Twenty-four female Wistar rats of 22 months of age were used. Animals were divided into four experimental groups: two intact groups that were untreated or given melatonin (1 mg/kg/day), and two ovariectomized groups that also untreated and treated with melatonin (1 mg/kg/day). After 10 wk of treatment, rats were sacrificed by decapitation, and livers were collected and homogenized. A group of 2-month-old female rats was used as young controls. Protein expression of inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), IL-6, TNF-alpha and IL-1beta were determined by Western blot analysis. The levels of nitric oxide metabolites (NO(x)), lipid hydroperoxide (LPO), TNF-alpha, IL-1beta, IL-6 and IL-10 were determined. Levels of LPO in the liver homogenates as well as iNOS protein expression and NO(x) levels were increased in old rats as compared with young animals; this effect was more evident in ovariectomized animals. Pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 were significantly increased and anti-inflammatory IL-10 decreased during aging and after ovariectomy. Aging also significantly increased the expression of HO-1 protein, and ovariectomized rats showed an additional increase. Administration of melatonin, both to intact and to the ovariectomized animals significantly reduced NO(x), LPO levels and pro-inflammatory cytokines in the liver as compared with untreated rats. Significant rice in IL-10 and reductions in the iNOS, HO-1, IL-6, TNF-alpha and IL-1beta protein expression were also found in rats treated with melatonin. Oxidative stress and inflammation induced during aging in the liver are more marked in castrated than in intact females. Administration of melatonin reduces both these situations.     

促甲状腺激素释放激素对结肠平滑肌钾电流的影响

目的探讨促甲状腺激素释放激素(TRH)对大鼠结肠平滑肌细胞膜瞬时外向钾电流(Ito)和延迟整流钾电流(Ik)的影响。方法酶解法急性分离单个大鼠结肠平滑肌细胞,运用膜片钳方法检测10、20、50、100μmol/L的TRH对结肠平滑肌细胞膜Ito和Ik的影响。结果 TRH浓度依赖性减少Ito(P<0.05),10、20、50、100μmol/L的TRH可使峰值Ito降低13.4%、28.0%、39.5%、53.4%,但Ik降低不明显(P>0.05)。结论 TRH阻滞大鼠结肠平滑肌细胞的瞬时外向钾电流,但不能降低延迟整流钾电流,TRH可能通过调节细胞的兴奋性对肠道动力产生影响。     

Exaggerated prostaglandin production by colonic smooth muscle in rabbit colitis

Enhanced production of arachidonic acid metabolites by colonic mucosa has been reported in ulcerative colitis as well as in experimental models of colitis. However, production of these compounds by colonic smooth muscle from colitis subjects has not been described. To evaluate arachidonic acid metabolism in colonic tissue, we studied the production of prostaglandin E₂ (PGE₂) by mucosa and muscularis propria in two experimental models of acute colitis in which inflammation was virtually confined to the mucosa. Colitis was induced in New Zealand white rabbits by either of two methods, dinitrochlorobenzene (DNCB) sensitization or formalin followed by intravenous soluble immune complexes (F-IC). Arachidonic acid metabolites were identified from in vitro incubations of tissue with [¹⁴C]arachidonic acid by thin layer chromatography followed by autoradiography. The major eicosanoid metabolites of colitis mucosa and muscularis were¹⁴C-labeled prostaglandin E₂, prostaglandin F_2a and 6-keto prostaglandin f_1a. PGE₂ was quantitated from incubations without labeled arachidonic acid by radioimmunoassay. PGE₂, expressed as picograms per milligram protein per 20 min (mean±sem), was increased in F-IC mucosa (1093±141 vs 645±189, P < 0.05) and DNCB mucosa (1354±487 vs 527±222, P < 0.05) compared to normals. PGE₂ production by uninflamed colitis muscularis propria was also increased five-to eightfold compared to normals for F-IC muscularis (1594±329 vs 189±35, P < 0.005) and DNCB muscularis (1287±171 vs 225±72, P < 0.005). Thus, the adjacent inflammation in colonic mucosa may induce increased eicosanoid production by the uninflamed smooth muscle. Increased PGE₂, in turn, may contribute to the smooth muscle dysfunction seen in colitis.This work was supported by the National Institutes of Health through BRSG RR-05551, by the National Foundation for Ileitis & Colitis, and by the James L. Stamps Foundation, Inc.A preliminary report of this work was presented at the American Gastroenterological Association Meeting, New Orleans, Louisianna, in May 1984, and appeared in abstract form inGastroenterology 86:1129, 1984.     

Optimisation of isolation of richly pure and homogeneous primary human colonic smooth muscle cells

BACKGROUND: Inherent properties of gastrointestinal smooth muscle can be assessed using isolated cell suspensions. Currently available isolation techniques, based on short 2-h enzymatic digestion, however, present the disadvantage of low cellular yield with brief viability. These features are an important limiting factor especially in studies in humans in which tissue may not be available daily and mixing of samples is not recommended. AIMS: To optimise the isolation procedure of cells from human colon to obtain a richly pure primary smooth muscle cell preparation. METHODS: Slices of circular muscle layer, obtained from surgical specimens of human colon, were incubated overnight in Dulbecco's modified eagle's medium supplemented with antibiotics, foetal bovine serum, an ATP-regenerating system and collagenase. On the following day, digested muscle strips were suspended in HEPES buffer, and spontaneously dissociated smooth muscle cells were harvested and used either immediately or maintained in suspension for up to 72 h. Cell yield, purity, viability, contractile responses, associated intracellular calcium signals and RNA and protein extraction were evaluated and compared to cell suspensions obtained with the current short digestion protocol. RESULTS AND CONCLUSION: The overnight isolation protocol offers the advantage of obtaining a pure, homogeneous, long-life viable cell suspension that maintains a fully differentiated smooth muscle phenotype unchanged for at least 72 h and that allows multiple functional/biochemical studies and efficient RNA extraction from a single human specimen.     

Effect of melatonin administration on parameters related to oxidative damage in hepatocytes isolated from old Wistar rats

Aging induces changes in several organs and tissues, such as the liver, and this process might be due to oxidative damage caused by free radicals and inflammatory mediators. Melatonin is a secretory product with well-known antioxidant properties. The aim of this study was to investigate the effect of melatonin administration on age-induced alterations in hepatocytes. Twenty-two-month old male Wistar rats were treated with oral melatonin for 10 wk. At the end of the treatment, hepatocytes were isolated and cultured, and different parameters were measured in both cells and medium. Aging induced a significant increase in lipid peroxidation, nitric oxide, carbon monoxide and cyclic guanosyl-monophosphate, as well as a reduction in adenosine triphosphate content and phosphatidylcholine synthesis when compared to young animals. Melatonin administration significantly ameliorated all these age-related changes in males. Melatonin administration seems to exert beneficial effects against age-induced changes in hepatocytes.     

Melatonin reduces oxidative stress in erythrocytes and plasma of senescence-accelerated mice

It has been suggested that oxidative stress is a feature of aging. The goal of the present study was to assess the oxidant effects related to aging and the protective role of exogenous melatonin in senescence-accelerated mice (SAMP8). Two groups of SAMP8 mice (males and females) were compared with their respective control groups of SAMR1 mice (senescence-resistant inbred strain) to determine their oxidative status without melatonin treatment. Four other groups of the same characteristics were treated with melatonin (10 mg/kg/day) in their drinking water. The melatonin concentration in the feeding bottles was titrated according to water consumption and body weight (i.e. 0.06 mg/mL for 30 g of body weight and 5 mL/day of water consumption). The treatment began when animals were 1-month old and continued for 9 months. When mice were 10-month old, they were anesthetized and blood was obtained. Plasma and erythrocytes were processed to examine oxidative stress markers: reduced glutathione (GSH), oxidized glutathione (GSSG), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferase (GST), thiobarbituric acid reactive substances (TBARS), and hemolysis. The results showed greater oxidative stress in SAMP8 than in SAMR1, largely because of a decrease in GSH levels and to an increase in GSSG and TBARS with the subsequent induction of the antioxidant enzymes GPX and GR. Melatonin, as an antioxidant molecule, improved the glutathione-related parameters, prevented the induction of GPX in senescent groups, and promoted a decrease in SOD and TBARS in almost all the groups.     

Functional assessment of beta adrenoceptor subtypes in human colonic circular and longitudinal (taenia coli) smooth muscle

BACKGROUND AND AIMS: The subtype and species related heterogeneity of beta adrenoceptors prompted a functional reappraisal of these molecular targets of motility inhibition in the human colon. METHODS: Relaxation of muscle strips was measured in vitro. RESULTS: The following agonists had decreasing relaxing potency (effective concentration range 10(-8)-10(-4) mol/l): (-)isoprenaline (non-selective), terbutaline (beta(2) selective), CGP 12177 (beta(3) selective, also beta(1), beta(2) antagonist), and SR 58611A (beta(3) selective). Isoprenaline and terbutaline were more potent on circular than taenia strips; CGP 12177 and SR 58611A weakly and partially relaxed taenia but had little effect on circular strips. The potency of isoprenaline on circular strips was greatly reduced by the beta(1) selective antagonist CGP 20712 (10(-7) mol/l), and less so by ICI 118551 (10(-7) mol/l, beta(2) selective). CGP 20712 and ICI 118551 together (both 3 x 10(-6) mol/l) had no effect on taenia relaxation by SR 58611A and rendered isoprenaline and terbutaline virtually inactive on circular strips, although not on taenia, which was relaxed at higher than control concentrations and maximally by isoprenaline. Propranolol, a beta(1), beta(2) non-selective antagonist, at high concentrations (10(-5) mol/l) prevented taenia relaxation by CGP 12177 and SR 58611A; its quantitative antagonism of isoprenaline (in common with that of CGP 12177 used as an antagonist) was competitive in circular strips but not on taenia. CONCLUSIONS: beta(1), beta(2), and beta(3) adrenoceptors are functionally detectable in the human colon; agonist stimulation of any one type relaxed taenia but only isoprenaline was fully effective at the beta(3) subtype.     

Type 2 deiodinase expression is stimulated by growth factors in human vascular smooth muscle cells

Type 2 deiodinase (D2) catalyzes the conversion of the prohormone T4 to the biologically active T3. D2 is expressed in human aortic smooth muscle cells (hASMCs). In this study, we demonstrated that the D2 mRNA and activity in hASMCs were up-regulated by platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF). The induction of D2 mRNA by PDGF-BB and bFGF was dependent on de novo RNA and protein synthesis. PD98059, a specific inhibitor of the upstream kinase that activates extracellular signal-regulated kinase (ERK), significantly suppressed the induction by both PDGF-BB and bFGF. SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, and SP600125, a specific inhibitor of c-Jun N-terminal kinase (JNK), also reduced the induction by both PDGF-BB and bFGF. These results suggest that both PDGF-BB and bFGF induce D2 expression at least partly via ERK pathway. The p38 MAP kinase and JNK pathways may also be involved in the induction.     

Melatonin ameliorates low‐grade inflammation and oxidative stress in young Zucker diabetic fatty rats

The aim of this study was to investigate the effects of melatonin on low‐grade inflammation and oxidative stress in young male Zucker diabetic fatty (ZDF) rats, an experimental model of metabolic syndrome and type 2 diabetes mellitus (T2DM). ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups, each composed of 10 rats: naive (N), vehicle treated (V), and melatonin treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Pro‐inflammatory state was evaluated by plasma levels of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and C‐reactive protein (CRP). Also, oxidative stress was assessed by plasma lipid peroxidation (LPO), both basal and after Fe²⁺/H₂O₂ inducement. ZDF rats exhibited higher levels of IL‐6 (112.4 ± 1.5 pg/mL), TNF‐α (11.0 ± 0.1 pg/mL) and CRP (828 ± 16.0 µg/mL) compared with lean rats (IL‐6, 89.9 ± 1.0, P < 0.01; TNF‐α, 9.7 ± 0.4, P < 0.01; CRP, 508 ± 21.5, P < 0.001). Melatonin lowered IL‐6 (10%, P < 0.05), TNF‐α (10%, P < 0.05), and CRP (21%, P < 0.01). Basal and Fe²⁺/H₂O₂‐induced LPO, expressed as malondialdehyde equivalents (µmol/L), were higher in ZDF rats (basal, 3.2 ± 0.1 versus 2.5 ± 0.1 in ZL, P < 0.01; Fe²⁺/H₂O₂‐induced, 8.7 ± 0.2 versus 5.5 ± 0.3 in ZL; P < 0.001). Melatonin improved basal LPO (15%, P < 0.05) in ZDF rats, and Fe²⁺/H₂O₂‐ induced LPO in both ZL (15.2%, P < 0.01) and ZDF rats (39%, P < 0.001). These results demonstrated that oral melatonin administration ameliorates the pro‐inflammatory state and oxidative stress, which underlie the development of insulin resistance and their consequences, metabolic syndrome, diabetes, and cardiovascular disease.     

Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats

In an attempt to define the role of the pineal secretory melatonin and an analogue, 6-hydroxymelatonin (6-OHM), in limiting oxidative stress, the present study investigated the cisplatin (CP)-induced alteration in the renal antioxidant system and nephroprotection with the two indolamines. Melatonin (5 mg/kg), 6-OHM (5 mg/kg), or an equal volume of saline were administered intraperitoneally (i.p.) to male Sprague Dawley rats 30 min prior to an i.p. injection of CP (7 mg/kg). After CP treatment, the animals each received indolamine or saline every day and were sacrificed 3 or 5 days later and plasma as well as kidney were collected. Both plasma creatinine and blood urea nitrogen increased significantly following CP administration alone; these values decreased significantly with melatonin co-treatment of CP-treated rats. In the kidney, CP decreased the levels of GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, an index directly related to oxidative stress. When animals were treated with melatonin, the reduction in the GSH/GSSG ratio was prevented. Treatment of CP-enhanced lipid peroxidation in the kidney was again prevented in animals treated with melatonin. The activity of the antioxidant enzyme, glutathione peroxidase (GSH-Px), decreased as a result of CP administration, which was restored to control levels with melatonin co-treatment. Upon histological analysis, damage to the proximal tubular cells was seen in the kidneys of CP-treated rats; these changes were prevented by melatonin treatment. 6-OHM has been shown to have some antioxidative capacity, however, the protective effects of 6-OHM against CP-induced nephrotoxicity were less than those of melatonin. The residual platinum concentration in the kidney of melatonin co-treated rats was significantly lower than that of rats treated with CP alone. It is concluded that administration of CP imposes a severe oxidative stress to renal tissue and melatonin confers protection against the oxidative damage associated with CP. This mechanism may be reasonably attributed to its radical scavenging activity, to its GSH-Px activating property, and/or to its regulatory activity for renal function.     

Acceleration of wound healing by growth hormone-releasing hormone and its agonists

Despite the well-documented action of growth hormone-releasing hormone (GHRH) on the stimulation of production and release of growth hormone (GH), the effects of GHRH in peripheral tissues are incompletely explored. In this study, we show that GHRH plays a role in wound healing and tissue repair by acting primarily on wound-associated fibroblasts. Mouse embryonic fibroblasts (MEFs) in culture and wound-associated fibroblasts in mice expressed a splice variant of the receptors for GHRH (SV1). Exposure of MEFs to 100 nM and 500 nM GHRH or the GHRH agonist JI-38 stimulated the expression of α-smooth muscle actin (αSMA) based on immunoblot analyses as well as the expression of an αSMA-β-galactosidase reporter transgene in primary cultures of fibroblasts isolated from transgenic mice. Consistent with this induction of αSMA expression, results of transwell-based migration assays and in vitro wound healing (scratch) assays showed that both GHRH and GHRH agonist JI-38 stimulated the migration of MEFs in vitro. In vivo, local application of GHRH or JI-38 accelerated healing in skin wounds of mice. Histological evaluation of skin biopsies showed that wounds treated with GHRH and JI-38 were both characterized by increased abundance of fibroblasts during the early stages of wound healing and accelerated reformation of the covering epithelium at later stages. These results identify another function of GHRH in promoting skin tissue wound healing and repair. Our findings suggest that GHRH may have clinical utility for augmenting healing of skin wounds resulting from trauma, surgery, or disease.     

Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy

Abstract: Duchenne muscular dystrophy (DMD) is a severe X‐linked muscle‐wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx^5Cv mouse, an animal model for DMD. Male mdx^5Cv mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine^® implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx^5Cv mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force–frequency relationship of melatonin‐treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.     

Melatonin protects against lipopolysaccharide-induced intra-uterine fetal death and growth retardation in mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes, including intra-uterine fetal death (IUFD) and intra-uterine growth retardation (IUGR). However, the exact mechanism for LPS-induced IUFD and IURD remains unclear. LPS stimulates macrophages to generate reactive oxygen species (ROS). Therefore, we hypothesize that ROS may be involved in LPS-induced IUFD and IURD. Melatonin is a powerful endogenous antioxidant. In this study, we investigated the protective effects of melatonin on LPS-induced IUFD and IURD in ICR mice. All pregnant mice except controls received an intraperitoneal (75 microg/kg, i.p.) injection of LPS on gestational day (gd) 15-17. The experiment was carried out in two different modes. In mode A, the pregnant mice received two doses of melatonin within 24 hr, one (5 or 10 mg/kg) injected immediately after LPS and the other (5 or 10 mg/kg) injected at 3 hr after LPS. In mode B, the pregnant mice were pretreated with 10 mg/kg of melatonin 18 hr before LPS and then received two doses of melatonin in 24 hr, one (10 mg/kg) injected immediately after LPS and the other (10 mg/kg) injected 3 hr after LPS. The number of live fetuses, dead fetuses and resorption sites were counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that post-treatments with melatonin significantly attenuated LPS-induced IUFD in a dose-dependent manner. Surprisingly, pre- plus post-treatments with melatonin almost blocked LPS-induced IUFD. In addition, both post-treatments and pre- plus post-treatments with melatonin significantly alleviated LPS-induced decreases in crown-rump and tail lengths and reversed LPS-induced skeletal developmental retardation. However, melatonin had little effect on LPS-induced decrease in fetal weight. Furthermore, pre- plus post-treatments with melatonin significantly attenuated LPS-induced lipid peroxidation in maternal liver. These results indicate that melatonin protects against LPS-induced IURD and IUGR via counteracting LPS-induced oxidative stress.     

胰岛素样生长因子-1对大鼠结肠平滑肌细胞增殖、凋亡及MAPK通路的影响

目的 探讨胰岛素样生长因子-1(IGF-1)对大鼠结肠平滑肌细胞(SMCs)增殖、凋亡及丝裂原活化蛋白激酶(MAPK)信号转导通路的影响.方法 利用酶解法分离培养Sprague-Dawley大鼠结肠SMCs,进行免疫组化染色鉴定后,将其分为对照组、IGF-1组和IGF-1+ PD98059[细胞外信号调节激酶(ERK)抑制剂]组,分别采用噻唑蓝(MTT)法检测SMCs增殖,流式细胞术AnnexinV-FITC/PI检测SMCs凋亡,Western印迹检测磷酸化ERK、ERK、磷酸化p38MAPK、p38MAPK和磷酸化c-Jun氨基末端激酶(JNK)、JNK的表达.结果 分离培养的细胞经免疫组化鉴定为结肠SMCs,IGF-1组较对照组细胞增殖增强[(1.786 ±0.271)比(0.998±0.057),P＜0.01],凋亡率降低[(2.59±0.28)％比(20.68±2.48)％,P＜0.01],磷酸化ERK表达增强,磷酸化ERK/ERK比值升高[(42.71±3.74)％比(23.88±2.52％),P均＜0.01];磷酸化p38MAPK、p38MAPK、磷酸化JNK、JNK表达无差异(P均＞0.05).IGF-1+ PD98059组较对照组细胞增殖下降[(0.154±0.021)比(0.998±0.057),P＜0.01],凋亡率升高[(84.31±7.54)％比(20.68±2.48)％,P＜0.01],磷酸化ERK表达减弱,磷酸化ERK/ERK比值降低[(10.47±1.22)％比(23.88±2.52)％,P均＜0.01].结论 IGF-1可能通过激活结肠SMCs MAPK通路中的ERK途径,促进细胞增殖,抑制凋亡,可能与p38MAPK途径和JNK途径无关.     

老年大鼠血管平滑肌细胞对氧化应激反应的研究

目的观察老年大鼠血管平滑肌细胞（VSMC）对氧化应激损伤的反应。方法贴块法培养雄性Wistar大鼠胸主动脉VSMC，采用50umol／L H2O2处理细胞6h形成氧化应激，比较老年组和成年组VSMC对氧化应激反应的差异。RT—PCR法检测单核细胞趋化因子-1（MCP-1）mRNA的表达，比色法测定细胞丙二醛（MDA）含量。结果给予H2O2氧化应激前，老年组VSMC的MCP-1mRNA表达及MDA含量无显著差异（P〉0．05）；给予H2O2氧化应激后，老年组VSMC的MCP-1mRNA表达及细胞MDA含量的显著高于成年组VSMC。结论老年大鼠VSMC对氧化应激有着更强烈的反应，这可能是老年期易患动脉粥样硬化性血管疾病的重要机制之一。