Effect of resveratrol and melatonin on oxidative stress enzymes, regeneration, and hepatocyte ultrastructure in rats subjected to 70% partial hepatectomy

AIM: We sought to compare the antioxidant effects of resveratrol (R) and melatonin (M) after 70% partial hepatectomy (PH) as evidenced by ultrastructural alterations and effects on hepatocyte proliferation and apoptosis. METHODS: Twenty-six male Wistar albino rats were randomized into four groups: group A (n = 8) resveratrol (R); group B (n = 8) melatonin (M); group C (n = 5) control PH; group D (n = 5) sham operated animals. The rats that received either R or M were sacrificed a week after PH. The malondialdehyde, glutathione, glutathione S-transferase, and nitric oxide levels were estimated in liver homogenates. The morphological changes were investigated using light and electron microscopy (EM). Cell proliferation was detected by immunohistochemical staining with monoclonal antibodies to Ki-67. Apoptosis was detected by the transferase-mediated dUTP nick end-labeling method. RESULTS: PH induced hepatic LP, decreased GSH and NO, and inhibited GST activity (P < .05). R and M completely prevented PH-induced lipid peroxidation, decreased hepatic GSH and NO levels (P < .05). The inhibition of GST activity was prevented by R (P < .05), but not with M (P > .05). In the PH group EM showed severe morphological changes: mitochondrial degeneration, vacuoles, lipid droplets, and myelin-like figures. In both the R and M groups, morphological alterations repaired protective effects more prominently in the R group. Ki-67 indices (KI) were increased in the PH group and decreased in both R and M groups (P < .001). In the M group, KI was the lowest, but the difference compared with R was not significant (P > .05). Apoptosis was slightly increased in PH, but in either the R or M groups, apoptosis was intensively increased (P < .001). Increased apoptosis was greatest in the M group and the difference compared with the R group was statistically significant (P < .05). CONCLUSION: R and M suppressed PH-induced oxidative damage, attenuated proliferation, and stimulated apoptosis. When we compared R and M, R showed more potent antioxidative effects and was morphologically more protective to hepatocytes. Antiproliferative effects of M were more potent. Because of their potent antioxidative effects, R and M can be effective for oxidative damage like ischemia-reperfusion injury; however, because of the adverse effects on proliferation and apoptosis more studies are needed in states in which regeneration is critical.     

血晶素对大鼠70%肝脏切除后再生的影响

目的 探讨血晶素(Hemin)对大鼠70%肝脏切除术后肝脏再生的影响.方法 复制大鼠70%肝脏切除模型,随机分成血晶素治疗组和生理盐水对照组,在术后第1天、第2天、第3天和第7天测定比较肝重/体重、血清肿瘤坏死因子α(TNF-α)、肝脏组织中血晶素加氧酶1(HO-1)含量及肝细胞核增殖蛋白抗原(PCNA)表达指数.结果 术后第7天治疗组肝重/体重明显高于对照组(P<0.05),第3天开始肝细胞 PCNA表达指数较对照组升高,差异有统计学意义(P<0.01).治疗组术后肝脏组织中HO-1浓度比对照组高,血清TNF-α的含量比对照组低(P<0.05).结论 血晶素大鼠肝脏70%切除术后肝脏再生的速度明显提高,这可能跟HO-1表达升高有关.     

Effect of steroid on mitochondrial oxidative stress enzymes, intestinal microflora, and bacterial translocation in rats subjected to temporary liver inflow occlusion

Protective effects of steroids against ischemia-reperfusion (I/R) injury are well known, but there is little information about the influence of temporary inflow occlusion on intestinal barrier function or bacterial translocation. The aim of this experimental study was to investigate the effects on liver, kidney, spleen, ileal mitochondrial stress enzymes, and bacterial translocation of methylprednisolone (MP) in rats undergoing temporary liver inflow occlusion. Twenty-seven pathogen-free Wistar albino rats were randomized into three groups: group A: I/R (n = 10); group B: I/R + MP (n = 10); and group C: sham (n = 7). Rats in groups A and B were subjected to 20 minutes of portal vein and hepatic artery occlusion with 3 mg/kg MP injected into group B animals intraperitoneally during the occlusion. Twenty-two hours later, all rats were sacrificed to measure mitochondrial oxidative stress enzymes in liver, kidney, spleen, and ileum. We evaluated intestinal bacterial counts, intestinal mucosal histopathology, bacterial translocation to mesenteric lymph nodes (MLN), liver, spleen, and kidney. Decreased levels of malondialdehyde and increased levels of glutathione were observed in all examined tissues of group B compared to those of group A rats. Statistically significant increases in the intestinal counts of Klebsiella spp and Proteus spp and of bacterial translocation to liver, kidney, spleen, and MLN were measured in group B with respect to group A.     

Effects of portal-systemic shunt following 90% partial hepatectomy in rats

We studied the effects of portal-systemic shunt after massive hepatectomy. Male Wistar rats were divided into two groups: one group underwent laparotomy alone (C group) and in the other group a portal-systemic shunt was placed through laparotomy (S group). After 90% hepatectomy was performed, 3-day and 1-week survival rates and histopathology were examined, and hepatic hemodynamics during the early stage after hepatectomy were measured using dye-containing microspheres. The 3-day survival rate in the S group was significantly higher, and the 1-week survival rate was slightly higher, than those in C group. Sinusoidal dilation 7 days after hepatectomy in the S group was significantly milder than that in the C group. Fatty degeneration of hepatocytes in the S group was significantly milder than that in the C group. With respect to hepatic hemodynamics during the early stage after hepatectomy, the rate of shunt (26.3%) in the S group was significantly higher than that (9.5%) in the C group. Portal pressure, total hepatic blood flow, and total hepatic blood flow per gram of liver in the S group were significantly lower than those in the C group. These results suggest that approximately 26% shunt after 90% hepatectomy in rats increases the early survival rate and improves histological changes in surviving rats 7 days after resection.     

Effects of TVE application during 70% hepatectomy on regeneration capacity of rats

BACKGROUND: For adequate control of excess bleeding during liver resection, total vascular exclusion (TVE) is preferred by surgeons, especially when the tumor is located in the posterior liver lobes or near the cava. To the authors' knowledge, the effects of TVE technique on the postoperative liver regeneration process have not thus far been evaluated yet in the literature. This study was planned to compare the effects of liver resections performed either with portal pedicle clamping or with TVE on the regeneration process. MATERIALS AND METHODS: Seventy percent hepatectomy was performed with portal pedicle clamping (n=10, Group A) or with TVE (n=10, Group B) in rats. At 48 h after resection, sampling was performed for the measurement of serum transaminase, alkaline phosphatase (ALP), tissue malondialdehyde (MDA), and glutathione (GSH) levels. Liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling, and mitotic indices were also evaluated. RESULTS: Liver injury determinants (serum transaminases, ALP, and tissue MDA levels) were found significantly higher in group B than in group A. Liver regeneration rate, liver GSH levels, PCNA labeling index, and mitotic index were significantly lower in group B than in group A. CONCLUSIONS: The injury during TVE seems to be greater than during resection with portal pedicle clamping. The negative effect of this oxidative damage may influence the regenerative capacity of the remnant liver tissue.     

Effect of photobiomodulation therapy on oxidative stress markers of gastrocnemius muscle of diabetic rats subjected to high-intensity exercise

This study aimed to determine whether photobiomodulation therapy (PBMT) in diabetic rats subjected to high-intensity exercise interferes with the expression of the oxidative stress marker in the gastrocnemius muscle. Twenty-four male Wistar rats were included in this study comprising 16 diabetic and eight control rats. The animals were allocated into three groups—control, diabetic fatigue, and diabetic PBMT fatigue groups. Diabetes was induced via the intraperitoneal administration of streptozotocin (50 mg/kg). We subsequently assessed blood lactate levels and PBMT. The animals of the diabetic fatigue group PBMT were irradiated before the beginning of the exercises, with dose of 4 J and 808 nm, were submitted to treadmill running with speed and gradual slope until exhaustion, as observed by the maximum volume of oxygen and lactate level. The animals were euthanized and muscle tissue was removed for analysis of SOD markers, including catalase (CAT), glutathione peroxidase (GPx), and 2-thiobarbituric acid (TBARS) reactive substances. CAT, SOD, and GPx activities were significantly higher in the diabetic PBMT fatigue group (p?<?0.05) than in the diabetic fatigue group. Outcomes for the diabetic PBMT fatigue group were similar to those of the control group (p?>?0.05), while their antioxidant enzymes were significantly higher than those of the diabetic fatigue group. PBMT mitigated the TBARS concentration (p?>?0.05). PBMT may reduce oxidative stress and be an alternative method of maintaining physical fitness when subjects are unable to perform exercise. However, this finding requires further testing in clinical studies.     

Effect of tacrolimus and partial hepatectomy on transthyretin metabolism in rats

Liver transplantation, which serves as treatment of familial amyloidotic polyneuropathy (FAP), and domino liver transplantation, which utilizes resected livers from patients with FAP for treatment of liver diseases, may induce changes in transthyretin (TTR), a pathogenic FAP-related protein. To evaluate this possibility, we performed a 70% hepatectomy or administered tacrolimus to Dark Agouti (DA) rats for 7 days and then measured changes in liver TTR mRNA levels and changes in serum TTR concentrations. After hepatectomy, TTR mRNA levels decreased by 77%; at day 3, they returned to preoperative levels. Except for slightly elevated serum TTR concentrations 12 h after operation, serum TTR levels remained unchanged. Thus, partial hepatectomy did not influence serum TTR concentrations. After tacrolimus administration, TTR mRNA declined by 56% 12 h after the experiment started; however, after day 3, a rebound phenomenon occurred until day 7. Tacrolimus may facilitate serum TTR degradation, although production of TTR in the liver also increased. This finding -- that TTR, the source of FAP-inducing amyloid, did not increase after transplantation -- may help post-transplantation treatment of patients who have FAP and other liver diseases.     

帕瑞昔布对肝部分切除术老年大鼠海马炎症反应的影响

目的研究帕瑞昔布对肝部分切除术老年大鼠海马炎症反应的影响。方法选择SPF级雄性SD大鼠70只,20月龄,体重500~600g,随机分为三组:对照组(C组,n=10),手术组(S组,n=30)及帕瑞昔布组(P组,n=30)。采用肝部分切除术建立手术创伤导致海马炎症反应模型。P组大鼠在异氟醚麻醉下行肝部分切除术,并于术前1h腹腔注射帕瑞昔布10 mg/kg,术后每日腹腔注射帕瑞昔布2mg/kg直至处死。S组大鼠在异氟醚麻醉下行肝部分切除术,术前1h腹腔注射与P组等量的生理盐水。C组腹腔注射与以上各组等量的生理盐水。干预后第1、3、7天处死大鼠,取海马组织,采用实时定量PCR法检测环氧合酶-2(COX-2)mRNA的表达量,Western blot法检测COX-2、半胱氨酸蛋白酶-3(caspase-3)的蛋白表达量,ELISA法检测前列腺素E2(PGE2)的含量。结果干预后第1、3天S组和P组COX-2mRNA及蛋白表达、PGE2含量、caspase-3蛋白表达明显高于C组(P0.05或P0.01),但P组明显低于S组(P0.05或P0.01)。干预后第7天三组大鼠COX-2mRNA及蛋白表达、PGE2含量、caspase-3蛋白表达差异无统计学意义。结论肝部分切除术可导致海马炎症反应,帕瑞昔布可显著抑制此种炎症反应,其机制可能与下调海马COX-2mRNA和蛋白表达、PGE2含量和caspase-3蛋白表达有关。     

Protective effects of resveratrol on spleen and ileum in rats subjected to ischemia-reperfusion

Resveratrol is as an antioxidant with free radical-scavenging activity and finds its clinical application in the prevention of postischemic tissue injury following solid organ transplantation. This study investigates the effect of Resveratrol on spleen and ileum tissues subjected to hepatic ischemia-reperfusion (I/R) in rats. Twenty-four rats were recruited in the study as follows: group A: I/R (n = 8), group B: I/R + Resveratrol (n = 8), and group C: sham operation (n = 8). After intraperitonealy pretreatment of eight rats with resveratrol (15 mg/kg/d) for 5 days, 16 rats were subjected to 45 minutes of hepatic ischemia followed by 30 minutes reperfusion period. Resveratrol was given 15 minutes prior to ischemia and just before the reperfusion in rats. After reperfusion period all rats were sacrificed. Spleen and ileum tissues were examined spectrophotometrically to measure malondialdehyde (MDA), glutathione (GSH), and total nitrite, nitrate as an end product of nitric oxide (NO) levels. Concerning the spleen, statistically significant decrease of GSH and increase of MDA and NO levels were found group A when compared to groups B and C (P = .040, P = .004, and P = .001 group A vs group B; P = .05, P = .003, and P = .001 group A vs group C, respectively). Parallel results were obtained in ileum. A statistically significant decrease in GSH and an increase in MDA and NO levels in group A in respect to group B and group C was obtained (P = .048, P = .034, and P = .001 group A vs group B; P = .004, P = .001, and P = .003 group A vs group C, respectively). The result of this study shows that resveratrol has a protective effect on spleen and ileal mitochondrial oxidative stress in rats subjected to I/R.     

大鼠部分肝缺血再灌注损伤后切除对肝再生的影响

目的 观察大鼠部分肝缺血再灌注损伤后切除对残肝再生的影响.方法 将75只健康雄性SD大鼠随机分为5组:肝脏左叶和中叶(约占全肝70%)切除组(Control组)、肝脏左叶和中叶缺血10min再灌注30min后切除组(I10R30组)、类推得到I60R30组、I90R30组、I90R60组.术后6、12、24h等时间点,测定再生肝重量(RLW);自动生化分析仪检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)含量;酶联免疫吸附试验(ELISA)检测血清肿瘤坏死因子(TNF)-α含量;通过免疫组织化学法检测残肝增殖细胞核抗原(Ki-67)表达.结果 术后12h,I60R30、I90R30和I90R60组RLW值分别为(1.80±0.03)%、(1.82±0.10)%、(1.87±0.05)%;Ki-67值分别为(58.35±2.18)%、(59.73±3.06)%、(62.65±2.24)%,均明显高于对照组(P<0.05).缺血再灌注干预各组ALT和AST明显高于对照组(P<0.05).术后6h和12h,I60R30、I90R30和I90R60组TNF-α明显高于对照组(P<0.05).结论 大鼠即将被切除的肝脏先缺血再灌注后切除,对残肝再生具有促进作用;诱导产生的TNF-α表达量增多是促进肝再生的原因之一.

Abstract：

Objective To investigate the effects of ischemia reperfusion injury before partial hepatectomy on liver regeneration in rats. Methods Seventy-five male healthy SD rats were randomly classified into 5 groups: group control, in which rats were only subjected to 70% hepatectomy; group I10R30, 70% liver hepatectomy after 10 min of ischemia and 30 min of reperfusion in the resected liver; By analogy, group I60R30, group I90R30 and group I90R60 were constructed. At 6th, 12th and 24th h after operation, RLW was determined; serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activities were measured by using autoanalyzer; the levels of serum tumor necrosis factor (TNF)-α were determined by ELISA and the expression level of Ki-67 was detected by using immunohistochemical methods in the residual liver tissues. Results At 12th h after partial hepatectomy, the rate of RLW in group I60R30, group I90R30 and group I90R60 was (1.80±0.03)%, (1.82±0.10)%, (1.87±0.05)% respectively; the rate of Ki-67 was (58.35±2.18)%, (59.73±3.06)%, (62.65±2.24)% respectively, which was significantly higher than that in the group control (P<0.05). The levels of ALT and AST in rats with ischemia reperfusion injury were higher than in the group control (P<0.05). At 6th h and 12th h after operation, the expression levels of TNF-α in groups I60R30, I90R30 and I90R60 were significantly higher than those in the group control (P<0.05). Conclusion Ischemia reperfusion injury in the resected liver before partial hepatectomy could improve liver regeneration of the remnant liver in rats. The high expression of induced TNF-α may be one of the reasons.     

促肝细胞生长素对慢性肝损伤鼠肝部分切除术后肝细胞的影响

目的　 研究促肝细胞生长素 (主要成分为肝细胞生长因子 )对慢性肝损伤时肝部分切除术后肝再生和肝功能的影响。 方法　 以四氯化碳和乙醇联合诱导大鼠的慢性肝损伤模型 ,对其行 30 %肝部分切除术 ,术后 10d经腹腔注射促肝细胞生长素 (分对照组、小剂量组、大剂量组 ) ,同时建立正常对照组。观察不同剂量促肝细胞生长素对肝再生、肝功能和肝储备功能的影响。 结果　 小剂量组与对照组的总蛋白、白蛋白、谷丙转氨酶 (AST )、谷草转氨酶 (ALT)、总胆固醇、动脉血酮体比比较 ,差异无显著性 ( P >0 .0 5 )。大剂量组与对照组的AST ,ALT ,总胆固醇、动脉血酮体比的比较差异显著 ( P <0 .0 1)。电镜和光镜观察显示 ,大剂量的促肝细胞生长素能促进肝再生。 结论　 应用大剂量促肝细胞生长素可明显改善肝功能和肝储备功能 ,促进肝细胞再生     

Biological significance of enhanced mitochondrial ketogenesis during the early stages after 70% hepatectomy in rats

Ketogenic capacity of mitochondria from the remnant liver of 70% hepatectomized rats was studied in relation to mitochondrial phosphorylative activity. Ketogenic capacity increased to a maximum of 6.04 +/- 0.39 from 3.84 +/- 0.13 of control, with an enhancement of mitochondrial phosphorylative activity 6 hr after hepatectomy, and then decreased to normal levels within 24 hr. Adenylate energy charge, (ATP + 1/2ADP)/(ATP + ADP + AMP), of the remnant liver decreased to 0.825 +/- 0.006 as compared to 0.849 +/- 0.002 of control 6 hr after operation. At 12 hr, total ketone body concentrations of the arterial blood increased concomitant with a fall in ketone body ratio (acetoacetate/3-hydroxybutyrate) which reflects the decreased liver mitochondrial redox (NAD+/NADH) state. These findings suggest that an enhancement of mitochondrial fatty acid oxidation and ketogenesis occurs concomitant with an enhancement of mitochondrial phosphorylative activity in the remnant liver in response to a decreased energy charge after 70% hepatectomy.     

Augmentation of cell-mediated cytotoxicity following 50% partial hepatectomy

The cytolytic responses of C3H/HeJ mice after 50% hepatectomy (PH) were assessed in a 4-hr 51Cr-release assay. Spleen cells (SC) (50 x 10(6] from normal or PH C3H/HeJ (H-2k) mice were sensitized with equal numbers of irradiated allogeneic DBA/2 (H-2d) spleen cells in a five-day mixed lymphocyte culture. Generated cytolytic activity was measured against 51Cr-labeled P815 mastocytoma (H-2d) and EL4 lymphoma (H-2b) target cells. The wet weight and cell numbers per spleen following 50% partial hepatectomy were 70% and 75% higher than the control values for the first 20 days, and then returned to normal levels by 21 days. The cytolysis by spleen cells from 2-, 14-, and 31-day PH mice were 89.3 +/- 0.7, 86.9 +/- 5.3, and 90.1 +/- 1.3%, respectively, compared with control (sham-operated) values of 56.0 +/- 1.0, 57.0 +/- 2.0, and 49.9 +/- 7.0% (P less than 0.03 at E/T 100:1). This enhanced cytolysis by PH spleen cells remained high for at least 118 days after the liver resection before returning to control levels by 268 days. Cytolytic effector cells in PH SC were generated at least 24 hr earlier than in control SC. When normal and PH cytolysis were compared following primary and secondary in vitro sensitization, the cytolytic levels of primarily-sensitized PH spleen cells were comparable to secondarily sensitized normal spleen cells. Furthermore, the primarily sensitized normal spleen cells did not show crossreactive cytolysis with EL4 target cells (H-2b), while both the primarily sensitized PH spleen cells and the secondarily sensitized normal spleen cells were significantly cross-reactive against the third party EL4 target cells. Adherent PH spleen cells appear to be responsible for this augmented cytolytic capacity since their coculture with normal nonadherent responder spleen cells increased control cytolysis by approximately 30%. These studied demonstrate that, following 50% partial hepatectomy, there is an immediate and sustained increase in the allospecific cytolytic response.