The role of endotoxin,TNF-α,and IL-6 in inducing the state of growth hormone insensitivity

AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1, which may play a very important role in inducing the growth hormone insensitivity. The objective of this current study was to investigate the role of endotoxin, TNF-alpha and IL-6 in inducing the growth hormone insensitivity at the receptor and post-receptor levels. METHODS: Spague-Dawley rats were injected with endotoxin, TNF-alpha, and IL-6, respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously. All rats were killed at different time points. The expression of IGF-I, GHR, SOCS-3 and beta-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay, the levels of TNF-alpha and IL-6 were detected by ELISA. RESULTS: There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection, however, liver IGF-I mRNA expression had been obviously down-regulated in endotoxemic rats. Liver GHR mRNA expression also had a predominant down-regulation after endotoxin injection. The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection, being decreased by 53% compared with control rats. For GHR mRNA expression, the lowest expression occurred at 8h and had a 81% decrease. Although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats. Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats. Endotoxin stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression. The liver GHR mRNA expression was obviously down-regulated after TNF-alpha iv injection and had a 40% decrease at 8h, but the liver SOCS-3 mRNA expression was the 4.94 times up-regulation occurred at 40 min after IL-6 injection. CONCLUSION: The growth hormone insensitivity could be induced by LPS injection, which was associated with down-regulated GHR mRNA expression at receptor level and with up-regulated SOCS-3 mRNA expression at post-receptor level. The in vivo biological activities of LPS were mediated by TNF-alpha and IL-6 indirectly, and TNF-alpha and IL-6 may exert their effects on the receptor and post-receptor levels respectively.

Operative stress response and energy metabolism after laparoscopic cholecystectomy compared to open surgery

AIM: To determine the least invasive surgical procedure by comparing the levels of operative stress hormones, responsereactive protein (CRP) and rest energy expenditure (REE)after laparoscopic (LC) and open cholecystectomy (OC).METHODS: Twenty-six consecutive patients with noncomplicated gallstones were randomized for LC (14) and OC (12). Plasma concentrations of somatotropin, insulin, cortisol and CRP were measured. The levels of REE were determined.RESULTS: In the third postoperative day, the insulin levels were lower compared to that before operation (P＜0.05).Tn the first postoperative day, the levels of somatotropin and cortisol were higher in OC than those in LC. After operation the parameters of somatotropin, CRP and cortisol increased, compared to those in the preoperative period in the all patients (P＜0.05). In the all-postoperative days,the CRP level was higher in OC than that in LC (7.46±0.02;7.38±0.01, P＜0.05). After operation the REE level all increased in OC and LC (P＜0.05). In the all-postoperative days, the REE level was higher in OC than that in LC (1438.5±A18.5;1222.3±L80.8, P＜0.05).CONCLUSION: LC results in less prominent stress response and smaller metabolic interference compared to open surgery. These advantages are beneficial to the restoration of stress hormones, the nitrogen balance, and the energy metabolism. However, LC can also induce acidemia and pulmonary hypoperfusion because of the penumoperitonium it uses during surgery.

Alterations of intestinal mucosa structure and barrier function following traumatic brain injury in rats

AIM: Gastrointestinal dysfunction is a common complication in patients with traumatic brain injury (TBI). However, the effect of traumatic brain injury on intestinal mucosa has not been studied previously. The aim of the current study was to explore the alterations of intestinal mucosa morphology and barrier function, and to determine how rapidly the impairment of gut barrier function occurs and how long it persists following traumatic brain injury.METHODS: Male Wistar rats were randomly divided into six groups (6 rats each group) including controls without brain injury and traumatic brain injury groups at hours 3,12, 24, and 72, and on day 7. The intestinal mucosa structure was detected by histopathological examination and electron microscopy. Gut barrier dysfunction was evaluated by detecting serum endotoxin and intestinal permeability. The level of serum endotoxin and intestinal permeability was measured by using chromogenic limulus amebocyte lysate and lactulose/mannitol (L/M) ratio, respectively.RESULTS: After traumatic brain injury, the histopathological alterations of gut mucosa occurred rapidly as early as 3 hours and progressed to a serious state, including shedding of epithelial cells, fracture of villi, focal ulcer, fusion of adjacent villi, dilation of central chyle duct, mucosal atrophy,and vascular dilation, congestion and edema in the villous interstitium and lamina propria. Apoptosis of epithelial cells,fracture and sparseness of microvilli, loss of tight junction between enterocytes, damage of mitochondria and endoplasm, were found by electron microscopy. The villous height, crypt depth and surface area in jejunum decreased progressively with the time of brain injury. As compared with that of control group (183.7±41.8 EU/L), serum endotoxin level was signnificantly increased at 3, 12, and 24 hours following TBI (434.8±54.9 EU/L, 324.2±61.7 EU/L and 303.3±60.2 EU/L, respectively), and peaked at 72 hours (560.5±76.2 EU/L), then declined on day 7 (306.7±62.4 EU/L,P＜0.0L). Two peaks of serum endotoxin level were found at hours 3 and 72 following TBI. L/M ratio was also significantly higher in TBI groups than that in control group (control,0.0172±0.0009; 12 h, 0.0303±0.0013; 24 h, 0.0354±0.0025;72 h, 0.0736±0.0105; 7 d, 0.0588±0.0083; P＜0.01).CONCLUSION: Traumatic brain injury can induce significant damages of gut structure and impairment of barrier function which occur rapidly as early as 3 hours following brain injury and lasts for more than 7 days with marked mucosal atrophy.

Levels of vasoactive intestinal peptide,cholecystokinin and calcitonin gene-related peptide in plasma and jejunum of rats following traumatic brain injury and underlying significance in gastrointestinal dysfunction

AIM:To study the alterations of brain-gut peptides following traumatic brain injury (TBI) and to explore the underlying significance of these peptides in the complicated gastrointestinal dysfunction.METHODS:Rat models of focal traumatic brain injury were established by impact insult method,and divided into 6 groups (6 rats each group) including control group with sham operation and TBI groups at postinjury 3,12,24,72h,and d 7.Blood and proximal jejunum samples were taken at time point of each group and gross observations of gastrointestinal pathology were recorded simultaneously.The levels of vasoactive intestinal peptide (VIP) in plasma,calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK) in both plasma and jejunum were measured by enzyme immunoassay (EIA). Radioimmunoassay (RIA) was used to determine the levels of VIP in jejunum.RESULTS:Gastric distension,delayed gastric emptying and intestinal dilatation with a large amount of yellowish effusion and thin edematous wall were found in TBI rats through 12h and 72h, which peaked at postinjury 72h. As compared with that of control group (247.8±29.5ng/L), plasma VIP levels were significantly decreased at postinjury 3,12 and 24h (106.7±34.1ng/L, 148.7±22.8ng/L,132.8±21.6ng/L,respectively),but significantly increased at 72h (405.0±29.8ng/L) and markedly declined on d 7 (130.7±19.3ng/L).However,Plasma levels CCK and CGRP were significantly increased through 3h and 7d following TBI (126-691% increases),with the peak at 72 h.Compared with control (VIP, 13.6±1.4ng/g;CGRP,70.6±17.7ng/g);VIP and CGRP levels in jejunum were significantly increased at 3h after TBI (VIP,35.4±5.0ng/g;CGRP,103.8±22.1ng/g),and declined gradually at 12 h and 2d h (VIP,16.5±1.8ng/g,5.5±1.4ng/g;CGRP,34.9±9.7ng/g, 18.5±7.7ng/g),but were significantly increased again at 72 h (VIP, 48.7±9.5ng/g; CGRP,142.1±24.3ng/g),then declined in various degrees on d 7 (VIP, 3.8±1.1ng/g; CGRP, 102.5±18.1ng/g).The CCK levels in jejunum were found to change in a similar trend as that in plasma with the concentrations of CCK significantly increased following TBI (99-517% increases) and peaked at 72h.CONCLUSION:Traumatic brain injury can lead to significant changes of brain-gut peptides in both plasma and small intestine, which may be involved in the pathogenesis of complicated gastrointestinal dysfunction.

Effect of glutamine on change in early postoperative intestinal permeability and its relation to systemic inflammatory response

AIM: To study the effects of glutamine (Gin) on the change of intestinal permeability and its relationship to systemic inflammatory response in early abdominal postoperative patients.METHODS: A prospective, randomized, double-blind and controlled trial was taken. Twenty patients undergoing abdominal surgery were randomized into Gin group (oral administration of glutamine, 30 g/d, for 7 d, n=10) and placebo group (oral administration of placebo, 30 g/d, for 7 d, n=-10). Temperatures and heart rates of all patients were daily recorded. White blood cell counts(WBC) and biochemical variables were measured before operation and 4 and 7 d alter drug administration. Serum concentrations of glutamine, endotoxin, diamine oxidase and malondialdehyde and urine lactulose/mannito (L/M) ratio were measured before and 7 d alter drug administration.RESULTS: The patients in the 2 groups were comparable prior to drug administration. Serum Gin concentration was significantly decreased in the placebo group and increased in the Gin group 7 d alter drug administration. Urine L/M ratio was significantly increased in the placebo group and decreased in the Gin group. The serum concentration of endotoxin, diamine oxidase and malondialdehyde was significantly decreased in the Gin group compared with those in the placebo group. Temperatures, heart rates and WBC counts were significantly lower in the Gin group than those in the placebo group.CONCLUSION: Gut is one of the sources of systemic inflammatory response in abdominal postoperative patients and glutamine can decrease intestinal permeability, maintain intestinal barrier and attenuate systemic inflammatory response in early postoperative patients.

Functional hepatic flow in patients with liver cirrhosis

AIM:To evaluate hepatic reserve function by investigating the change of functional hepatic flow and total hepatic flow in cirrhotic patients with portal hypertension.METHODS:HPLC method was employed for bhe determination of concentration of D-sorbitol in human plasma and urine.The functional hepatic flow (FHF) and total hepatic flow (THF) were determined by means of modified hepatic clearance of D-sorbitol combined with duplex doppler color sonography in 20 patients with cirrhosis and 10 healthy volunteers.RESULTS:FHF,evaluated by means of the D-sorbitol dearance,was significantly reduced in patients with drrhosis in comparison to controls (764.74±167.91 vs 1195.04±242.97mL/min,P<0.01). While THF was significantly increased in patients with cirrhosis in comparison to controls (1605.23±279.99 vs 1 256.12±198.34mL/min, P<0.01).Portal blood flow and hepatic artery flow all were increased in cirrhosis compared to controls (P<0.05 and P<0.01). D-sorbitol total clearance was significantly reduced in cirrhosis compared to control (P<0.01), while D-sorbitol renal clearance was significantly increased in cirrhosis (P<0.05). In controls FHF was similar to THF (1195.05±242.97 vs 1256.12±198.34mL/min, P=0.636),while FHF was significantly reduced compared with THF in drrhosis (764.74±167.91 vs 1605.23±279.99mL/min,P<0.01).CONCLUSION:Our method that combined modified hepatic clearance of D-sorbitol with duplex doppler color sonography is effective in the measurement of FHF and THE FHF can be used to estimate hepatic reserve function.

Up-regulation of intestinal nuclear factor kappa B and intercellular adhesion molecule-1 following traumatic brain injury in rats

AIM: Nuclear factor kappa B (NF-κB) regulates a large number of genes involved in the inflammatory response to critical illnesses, but it is not known if and how NF-KB is activated and intercellular adhesion molecule-1 (ICAM-1) expressed in the gut following traumatic brain injury (TBI). The aim of current study was to investigate the temporal pattern of intestinal NF-κB activation and ICAM-1 expression following TBI. METHODS: Male Wistar rats were randomly divided into six groups (6 rats in each group) including controls with sham operation and TBI groups at hours 3, 12, 24, and 72, and on d 7. Parietal brain contusion was adopted using weight-dropping method. All rats were decapitated at corresponding time point and mid-jejunum samples were taken. NF-KB binding activity in jejunal tissue was measured using EMSA. Immunohistochemistry was used for detection of ICAM-1 expression in jejunal samples. RESULTS: There was a very low NF-κB binding activity and little ICAM-1 expression in the gut of control rats after sham surgery. NF-KB binding activity in jejunum significantly increased by 160% at 3 h following TBI (P<0.05 vs control), peaked at 72 h (500% increase) and remained elevated on d 7 post-injury by 390% increase. Compared to controls, ICAM-1 was significantly up-regulated on the endothelia of microvessels in villous interstitium and lamina propria by 24 h following TBI and maximally expressed at 72 h post-injury (P<0.001). The endothelial ICAM-1 immunoreactivity in jejunal mucosa still remained strong on d 7 post-injury. The peak of NF-κB activation and endothelial ICAM-1 expression coincided in time with the period during which secondary mucosal injury of the gut was also at their culmination following TBI. CONCLUSION: TBI could induce an immediate and persistent up-regulation of NF-κB activity and subsequent up-regulation of ICAM-1 expression in the intestine. Inflammatory response mediated by increased NF-κB activation and ICAM-1 expression may play an important role in the pathogenesis of acute gut mucosal injury following TBI.

Retrovirus-mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin.The inhibitory effect of endostatin expressed by transfected 5MMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted.RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P＜0.01). In vivo experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P＜0.001).The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6±1.1, much fewer than that of 22.6±4.5 from SMMC-pLncx cells (P＜0.01).CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.

A randomized controlled trial of laparoscopic versus open cholecystectomy in patients with cirrhotic portal hypertension

AIM: To evaluate the characters, risks and benefits of laparoscopic cholecystectomy (LC) in cirrhotic portal hypertension (CPH) patients.METHODS: Altogether 80 patients with symptomatic gallbladder disease and CPH, including 41 Child class A,32 Child class B and 7 Child class C, were randomly divided into open cholecystectomy (OC) group (38 patients) and LC group (42 patients). The cohorts were well-matched for number, age, sex, Child classification and types of disease.Data of the two groups were collected and analyzed.RESULTS: In LC group, LC was successfully performed in 36 cases, and 2 patients were converted to OC for difficulty in managing bleeding under laparoscope and dense adhesion of Calot's triangle. The rate of conversion was 5.3%. The surgical duration was 62.6±15.2 min. The operative blood loss was 75.5±15.5 mL. The time to resume diet was 18.3±6.5 h. Seven postoperative complications occurred in five patients (13.2%). All patients were dismissed after an average of 4.6±2.4 d. In OC group, the operation time was 60.5±17.5 min. The operative blood loss was 112.5±23.5 mL. The time to resume diet was 44.2±10.5 h.Fifteen postoperative complications occurred in 12patients (30.0%). All patients were dismissed after an average of 7.5±3.5 d. There was no significant difference in operation time between OC and LC group. But LC offered several advantages over OC, including fewer blood loss and lower postoperative complication rate, shorter time to resume diet and shorter length of hospitalization in patients with CPH.CONCLUSION: Though LC for patients with CPH is difficult, it is feasible, relatively safe, and superior to OC.It is important to know the technical characters of the operation, and pay more attention to the meticulous perioperative managements.

Intestinal failure:Pathophysiological elements and clinical diseases

There are two main functions of gastrointestinal tract, digestion and absorption, and barrier function. The latter has an important defensive effect, which keeps the body away from the invading and damaging of bacteria and endotoxin. It maintains the systemic homeostasis. Intestinal dysfunction would happen when body suffers from diseases or harmful stimulations. The lesser dysfunction of GI tract manifests only disorder of digestion and absorption, whereas the more serious intestinal disorders would harm the intestinal protective mechanism, or intestinal barrier function, and bacterial/endotoxin translocation, of intestinal failure (IF) would ensue. This review disscussed the theory of the intestinal failure, aiming at attracting recognition and valuable comments by clinicians.

Modified technique for combined liver-small bowel transplantation in pigs

AIM: As the conventional combined liver-small bowel transplantation is complicated with many postoperative complications, the aim of this study was to describe a modified technique for the combined liver-small bowel transplantation with preservation of the duodenum, partial head of pancreas and hepatic biliary system in pigs.METHODS: Composite liver/small bowel allotransplantations were undertaken in 30 long-white pigs. The graft included liver, about 3 to 4 m proximal jejunum, duodenum and partial pancreatic head. Vessels reconstructions included subhepatic vena cava-vena cava anastomosis, aorta-aorta anastomosis and portal-splenic vein anastomosis.RESULTS: Without immunosuppressive treatment, the median survival time of the animals was 6 days (2 to 12days), and about 76.9 % (20/26) of the animals survived for more than 4 days after operation.CONCLUSION: The modified technique is feasible and safe for the composite liver/small bowel transplantation with duodenum and pancreas preserved in pigs. And also this technique can simplify the operation and decrease possible postoperative complications.

Association of two polymorphisms of tumor necrosis factor gene with acute biliary pancreatitis

AIM: To investigate TNF-α-308 and TNFB polymorphisms in acute biliary pancreatitis (ABP) and to related them to the plasma TNF-α levels.METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Genotypes and allele frequencies were determined in patients (n=127) and healthy controls (n=-102)using restriction fragment length polymorphism analysis of polymerase chain reaction (PCR) products. Reading the size of digested bands from polyacrylamide gel demonstrated the two alleles TNF1 and TNF2, or the two alleles TNFB1and TNFB2.RESULTS: The frequencies of TNF2 polymorphism and TNFB2 polymorphism were both similar in patients with mild or severe pancreatitis, so were in pancreatitis patients and in controls. Patients with septic shock showed a significantly higher prevalence of the TNF2 than those without. No significant differences were found in the genotype distribution of TNF-α-308 and TNFB among different groups. Plasma TNF-α levels did not differ significantly in ASBP patients displaying different alleles of the TNF gene studied.CONCLUSION: Results indicate that TNF gene polymorphisms studied play no part in determination of disease severity or susceptibility to acute biliary pancreatitis; however, TNF2polymorphism is associated with septic shock from ASBP.Genetic factors are not important in determining plasma TNF-α levels in ASBP.

Auxiliary en-bloc liver-small bowel transplantation with partial pancreas preservation in pigs

AIM: The aim of this study was to describe an auxiliary combined liver-small bowel transplantation model with the preservation of duodenum, head of pancreas and hepatic biliary system in pigs. The technique, feasibility, security and immunosuppression were commented.METHODS- Forty outbred long-white pigs were randomized into two groups, and the auxiliary composite liver/small bowel allotransplantations were undertaken in 10 longwhite pigs in each group with the recipient liver preserved.Group A was not treated with immunosuppressive drugs while group B was treated with cyclosporine A and methylprednisolone after operation. The hemodynamic changes and amylase of body fluid (including blood, urine and abdominal drain) were analyzed.RESULTS: The average survival time of the animals was 10±1.929 d (6 to 25 d) in group A while more than 30 d in group B. The pigs could tolerate the hemodynamic fluctuation during operation and the hemodynamic parameters recovered to normal 2 h after blood reperfusion. The transient high amylase level was decreased to normal one week after operation and autopsy showed no pancreatitis.CONCLUSION: Auxiliary en-b/oc liver-small bowel transplantation with partial pancreas preservation is a feasible and safe model with simplified surgical techniques for composite liver/small bowel transplantation. This model may be used as a preclinical training model for clinical transplantation method, clinical liver-small bowel transplantation related complication research, basic research including immunosuppressive treatment, organ preservation, acute rejection, chronic rejection, immuno-tolerance and xenotransplantation.

Effects of n-3 fatty acid,fructose-l,6-diphosphate and glutamine on mucosal cell proliferation and apoptosis of small bowel graft after transplantation in rats

AIM:To evaluate the effects of n-3 fatty acids (n-3FA), fructose-1,6-diphosphate (FDP) and glutamine (GLN) on mucosal cell proliferation and apoptosis of small bowel graft. METHODS:One hundred and ninety-six inbred strain Wistar rats were grouped as donors and recipients,and underwent heterotopic small bowel transplantation (SBT).n-3FA,FDP and GLN were administered via gastric tube as well as venous infusion for 10 days before and after surgery,respectively. The proliferation and apoptosis of mucosal cells were analyzed with flow cytometry and in situ cell death detection kits. RESULTS:Apparent apoptosis and minor proliferation of mucosal cells of small bowel graft after transplantation were observed.A higher mucosal cell proliferative index and lower apoptotic index were found in all small bowel grafts after supplying with n-3FA,FDP and GLN. CONCLUSION:Nutritional support with n-3FA,FDP and GLN promotes mucosal cell proliferation significantly,and prevents mucosal cell from undergoing apoptosis with different degrees.These regulatory effects on the apoptosis alter the structure and absorption function of transplanted small bowel favorably.

Modifications in combined liver-small bowel transplantation in pigs

AIM: To introduce combined liver-small bowel transplantation in pigs.METHODS: Eighteen transplantations in 36 large white pigs were performed. Three modifications in combined liver-small bowel transplantation model were applied: Veno-venous bypass was not used. Preservation of the donor duodenum and head of pancreas in continuity with the combined graft to avoid biliary reconstruction. The splenic vein of donor was anastomosed end-to-end with the portal vein of recipients by the formation of a 'cuff'.RESULTS: Without immunosuppressive therapy, 72-hour survival rate of the transplanted animals was 72% (13/18).Five of 18 pigs operated died of respiratory failure (3 cases)and bleeding during hepatectomy (2 cases). The longest survival time of animals was 6 days.CONCLUSION: Our surgical modifications are feasible and reliable, which have made the transplantation in pigs simpler and less aggressive, and thus these can be used for preclinical study.