| BackgroundObstructive jaundice(0J), a common symptom in hepatobiliary surgery, is caused by an occlusion of the bile duct, and which is most commonly due to choledocholithiasis, pancreaticobiliary malignancies, biliary stricture, metastatic disease and iatrogenic injury of biliary tract, etc. when the bile duct is obstructed, the toxic substances (e.g. bile salts etc.) that regularly pass into the intestinal lumen will accumulate in the bile duct and reflex into the bloodstream of the host and result in systemic toxic effects. On the other hand, the reduced or absent bile in the intestinal lumen often lead to bacteria overgrowth, increased endotoxin release, damaged intestinal mucosal tight junction and eventually give rise to the gut barrier dysfunction and subsequent portal and systemic endotoxemia or bacterial translocation. In addition, the impaired endotoxin clearance of Kupffer cells and inhibited systemic immunity resulting from jaundice further aggravated the patients’ disturbance. Furthermore, most patients should be removed the obstruction via surgery, despite developments in surgical techniques and perioperative management, postoperative morbidity and mortality are still considered to be higher in patients with obstructive jaundice.There is an emerging consensus that obstructive jaundice impairs gut barrier function, resulting in the endotoxin and gut florae translocation, portal or systemic endotoxemia and bacteremia, contributing to the pathophysiology of the SIRS, sepsis or multiple organ dysfunctions. And this theory has been supported by many studies from animal models and clinical findings.However, in recent years, many scholars suggested that besides the endotoxin and bacteria translocated by the portal vein to the systemic circulation, the intestinal epithelial cells itself could be activated by the noxious stimuli (such as the hemorrhage shock, ischemic/reperfusion, acute pancreatitis, sepsis or some drugs, etc.), and then secret one or more kinds of "gut-derived factors" into circulation through the "gut-lymph" pathway. The high-mobility group protein box 1 (HMGB1), which was firstly proved as a late-acting proinflammatory mediator in 1999 by wang et al, is now been regarded as one key cytokines in many pathophysiology. On the one hand, it can be secreted by the activated monocytes、macrophages, dendritic cells, NK cells or epithelial cells etc, to act as a promoter of systemic inflammatory responses; on the other hand, it can also be passively released from the necrotic or damaged cells, serve as an originator to activate immune cells and start an inflammatory response.When HMGB1 was secreted into the extracellular matrix, it can bind to receptor for advanced glycation end-products (RAGEs), and toll-like receptors (TLRs), such as TLR-2, TLR-4 and TLR-9, and then activating the nuclear factor-κB (NF-κB) to produce tumor necrotic factor-α (TNF-a), interleukin-1β(IL-1β) and IL-6 etc, participating many acute inflammatory conditions. Many experimental animal models of trauma or hemorrhage shock, a variety of organ ischemia/reperfusion (I/R) including intestinal I/R, hepatic I/R, lung I/R, renal I/R etc. and acute panceatitis, sepsis as well as a number of clinical investigations of organ injury such as trauma, stroke, hepatic transplantation and I/R syndromes, are all demonstrated that HMGB1 takes part in these sterile inflammation and/or the infectious disease. Among the receptors that HMGB1 bind to and transduct signals across the cell membranes, the HMGB1-TLR4 axis is thought to be play an important role in the progression of the above mentioned pathophysiologic status, and which may be a therapeutic target in this population. The most recent report confirmed that the gut is the source where HMGB1 derived from in the trauma/hemorrhagic shock, when it is released and arrived at the remote organ, e.g. the lung, the tissue injury occurs.As we all know, patients suffered from obstructive jaundice usually complicated with multiple organ injury either, especially, the impairment of gut mucosa play a leading role in the development of disturbance of the obstructive jaundice subjects. However, detailed information about the role of HMGB1 in intestinal injury in patients with obstructive jaundice was still limited in current literature. What’s more, there’s little information concerning the change of "gut-lymph" pathway in obstructive jaundice.Omega-3 polyunsaturated fatty acid (PUFA) is one of the important elements of immunonutrients, and is mainly got from the fish oil, including the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and so forth. Contrary to the ω-6 PUFA derived primarily from vegetable oils (e.g. soybeans, rapeseed, etc.), ω-3 PUFA can exert regulatory and suppressive effects on innate immunity through numerous ways. It can directly substitute the phospholipid arachidonic acid in cell membranes to the nucleus, and then affect the membrane fluidity, the formation of functional microdomains, the expression of the receptors, altering intracellular signaling cascades, reducing the inflammatory factors derived from arachidonic acid, enhancing the production of anti-inflammatory mediators, and so on, eventually, controlling the inflammatory response. Furthermore, the immune suppressive influence of ω-3 PUFA have been implemented in the clinic and got varying degrees of success. A number of animal model experiments and clinical investigation demonstrated that ω-3 PUFA could play beneficial effects on a variaty of disease, such as, amelioration of acute pneumonia induced by Klebsiella and Streptococcus, improvement of liver regeneration after 90% hepatectomy, decrease of hepatic steatosis and consequently alleviation of ischemia-reperfusion injury after partial hepatectomy, attenuation of chronic vasculopathy of small bowel allografts and modulation of neonatal or preterm neonatal cytokine response to endotoxin, etc. Recent studies have demonstrated that ω-3 PUFA can also suppress the activation of HMGB1/TLR4-axis mediated signaling transduction pathway and reduce the expression of proinflammatory gene.Differed from the I/R injury and sepsis, the pathophysiologic alteration will progressed gradually in obstructive jaundice, given that the occlusion has not been removed. However, few studies have focused on the role of ω-3 PUFA administration on the gut mucosa injury in obstructive jaundice. Therefore, the aim of the present research was to investigate the mechanism by which ω-3 PUFA modulate the HMGB1/TLR4 signaling pathway and the consequent inflammatory responses in the progression of obstructive jaundice in a rat model.To sum it up, in this study we established obstructive jaundice by ligation of the bile duct of the Wistar rats, then one of the OJ group was administrated with ω-3 PUFA. Before the animal was sacrificed at 3,7,14 days after the bile duct ligation; the abdominal thoracic duct was cannulated to drain the lymph fluid. Then the peripheral vein blood and the ileum were collected. ELISA was used to determine the TNF-α, IL-1 β, IL-10 and HMGB1 in the blood and lymph fluid, the nitric oxide was also measured. Hematoxylin-eosin and alcian blue-Periodic Acid-Schiff stain (AB-PAS) staining was used to assess the histological alteration and the goblet cell, respectively. The protein of HMGB1、TLR4 and NF-κB on the intestinal epithelial was determined by immunohistochemistry staining and western-blot. The protein of HMGB1, TLR4 and NF-κB mRNA was relatively quantified by qRT-PCR.Part ⅠModification of cannulation of the abdominal thoracic lymph duct and the alteration of cytokines in the blood and lymph fluid after intervention of ω-3 PUFAObjectivesTo modify the catheter procedures of thoracic lymph duct to simplify the drainage of the lymph fluid. Quantify the TNF-α, IL-1β, IL-10, HMGB1 and nitric oxide in the blood and lymph fluid, to investigate the role of lymphatic pathway in the drainage of gut derived inflammatory cytokines; and to explicit the alteration of cytokines in the blood and lymph fluid after ω-3 PUFA intervention.Materials and methodsSeventy two Wistar rats were divided into three groups, the Sham group, the OJ group and the OJPUFA group. Obstructive jaundice were establised by ligation of the bile duct, then only the OJPUFA group were feed with ω-3 PUFA. The animals were sacrificed at 3,7,14 days after the bile duct ligation. As abdominal thoracic duct is closely adjacent to the abdominal aorta, and the lymph duct is only made up of a few layer of epithelial cells and is easily be torn, in addition, there’s almost no connective tissues between these two structures, therefore in this experiment the abdominal thoracic duct was indirectly exposed by directly dissection of the abdominal aorta, and which is different from the previously reported way that extensively dissection of the retroperitoneal tissue to directly find out the lymph duct. Then the lymph fluid was collected with heparin to anticoagulation, the peripheral vein blood was harvest either, then stored at -80℃. ELISA was used to determine the TNF-α, IL-1β, IL-10 and HMGB1 in the blood and lymph fluid, the nitric oxide was also measured with nitrate reductase method. The body weight and the hepatic function were measured either.Data was expressed as mean±SD. All the quantitative data was tested normality with Shapiro-Wilk test, and found that the all the samples was normality. Statistical analysis was performed using the one-way ANVOA or two independent samples t-test, where appropriate. When the data was significant with ANOVA analyses, and meet the requirement of homogeneity of variance (if homogeneity of variance was not achieved by Levene’s test, the Brown-Forsythe test was employed to the equality of group means.), the multiple comparisons was carried out with least significant difference (LSD) test; otherwise, the Dunnett’s T3 test was used. All the statistical graphs were illustrated with SigmaPlot 10.0. A two-tailed P value of<0.05 was considered significant. SPSS software (version 16.0, SPSS, Chicago, IL) was used for all statistical analysis.Results1. The abdominal thoracic duct was indirectly exposed by directly dissection of the abdominal aorta is an effective way to drain the lymph fluid, and the procedure is simple and the success rate is higher.2. The body weight was increased gradually in the sham group, and which was significantly higher than that in the OJ and OJPUFA group; the body weight of the OJ and OJPUFA group was decreased slightly on the 3rd day after bile duct ligation, and then increased slowly. On the 14th day, the OJPUFA group was higher than that in the OJ group (P<0.05).3. There is almost no fluctuating in the concentration of TBIL, IBIL, ALT and AST in the sham group. As for the TBIL and IBIL in the OJ and OJPUFA group, the samples amounted to peak value on the 3rd day and then declined sharply, furthermore, there is no difference in these two groups. The AST and ALT increased gradually on the 3rd,7th and 14th day, and the value is somewhat lower in the OJPUFA group, but only on the 14th day, the difference is significant (P<0.05).4. There were less TNF-aα IL-1β, IL-10, HMGB1 and NO in the sham group. The TNF-α in the the OJ and OJPUFA group had similar change tendency, the levels gradually increased until arrived at the maximum value on the 7th day, and then declined. The OJ group is much higher (P<0.05) than that in the OJPUFA group on the 14th day, both in the blood and lymph fluid.Both the IL-10 and HMGB1 in the OJ and OJPUFA group are all kept on ascending, but the value is much lower in the OJ group than that in the OJPUFA group; compare with the OJ group, the concentration of IL-1β in the blood on the 14th day and in the lymph fluid on the 7th and 14th days, the OJPUFA group decreased significantly (P< 0.05); However, the concentration of IL-10 in the blood of 0J group on the 14th day is much higher (P<0.05) than that in the OJPUFA group. As for the HMGB1, the OJ group is much higher in the blood on the 7th and 14th day, and in lymph fluid on the 14th day (P<0.05). The NO is much higher in the blood of OJ group on the 7th and 14th day (P<0.05); but in the lymph fluid, only on the 14th day, the OJPUFA group is much lower.5. Compared the concentration of these cytokines derived from the blood with lymph fluid, we can find from figure(1-5) to figure (1-9) that the TNF-α and IL-1β is much higher in the lymph fluid(P<0.05) than that in the blood; while the IL-10 is much lower in the lymph fluid compared with the blood; and the HMGB1 is much higher in the blood on the 3rd and 7th day then decreased significantly on the 14th day (P< 0.05), the concentration of NO is similarly in the blood and lymph fluid (P>0.05).Conelusions1. The procedure of dissection of abdominal thoracic duct indirectly is an effective and simply way to drain the lymph fluid.2. the ω-3 PUFA can improve the hepatic function in obstructive jaundice, however the impact is limited given that the occlusion has not been removed.3. thew-3 PUFA can improve the immune disturbance in the subjects suffered from obstructive jaundice by alleviating the production of HMGB1, TNF-α and IL-1β, regulating the secretion of IL-10 and decreasing the level of NO.4. The gut may play an important role in the secretion of HMGB1 in the obstructive jaundice rats.Part Ⅱω-3 PUFA attenuates intestinal mucosa injury by modulating HMGB1/TLR4 signaling pathways in obstructive jaundice ratsObjectivesTo investigate the change of the histomorpology and the number of goblet cells in obstructive jaundice rat ileum after ω-3 PUFA intervention; and to explore the mechanism by which ω-3 PUFA modulate the HMGB1/TLR4 signaling pathway in the intestinal epithelial cells of the obstructive jaundice rat model.Materials and methodsHematoxylin-eosin and alcian blue-Periodic Acid-Schiff stain (AB-PAS) staining was used to assess the histological alteration and the goblet cell, respectively. The villous height and the crypt depth were measured. The protein of HMGB1、TLR4 and NF-κB on the intestinal epithelial was determined by immunohistochemistry staining and western-blot. The protein of HMGB1, TLR4 and NF-κB mRNA was relatively quantified by qRT-PCR. Data was expressed as mean±SD. And the specific statistical analysis was detailed in part I. All the statistical graphs were illustrated with SigmaPlot 10.0. A two-tailed P value of <0.05 was considered significant. SPSS software (version 16.0, SPSS, Chicago, IL) was used for all statistical analysis.Results1. There were nearly no histological changes in the ileum mucosal samples of the sham group. In the OJ group, the intestinal mucosa present with hyperemia, vacuolization and infiltrated with inflammatory cells on the 3rd day postoperation. And the damage aggravated progressively, on the 7th and the 14th day after the bile duct ligated, some of the intestinal epithelial cells broke off from the villous, and the ileum mucosa hemorrhage or ulcer can also be found. The histological change in the OJPUFA group is somewhat slightly relieved compared to the OJPUFA group. However, the expression of these pathologic appearances differs highly in individual rats.The villous height, crypt depth and villous height/crypt depth ratio on the 14th day in the sham group is significantly higher than that in the OJ group and OJPUFA group (P>0.05). The villous height and crypt depth were declined significantly in the OJ group than that in the OJPUFA group(P < 0.05). As for the villous height/crypt depth ratio, there’s no significant differce between the OJ group and OJPUFA group (P>0.05).2. The amount of the goblet cell in the sham group is a bit less than that in the OJ group and OJPUFA group on the 3rd day after operation, but there’s no significant difference(P>0.05). Subsequently, the number of goblet cell decreased sharply in the OJ group and OJPUFA group. On the 7th day, the quantity of the goblet cell in the sham group surpassed the OJ group and OJPUFA group, however compared with the OJPUFA group, there’s no significant difference (P>0.05). On the 14th day after bile duct ligation, the goblet cells in the OJ group diminished significantly than that in the sham group and OJPUFA group(P<0.05).3. Immunohistochemical staining for HMGB1. In sham group, HMGB1 immunoreactivity staining weakly in the nuclei of the intestinal epithelial cells, and only few cells were positively stained. In the OJ group, especially on the samples harvest on the 14th day after bile duct ligation, besides the nuclei, the cytoplasm of of the intestinal epithelial cells, some nonparenchymal cells and the inflammatory cells were all strongly stained. In the OJPUFA group, the distribution of HMGB1 immunoreactivity is similar to that in the OJ group with obvious nuclear staining in the epithelial cells, nonparenchymal cells and the infiltrated inflammatory cells. But the staining is somewhat weakly than that in the OJ group.Relative HMBGl protein expression by Western blotting. HMBG1 protein in the sham group keeps at a low level at any time point, and which was apparently lower than that in OJ and OJPUFA group (P<0.05). Compared with the OJ group, the expression of HMBG1 protein in the OJPUFA group is less than that in the OJ group, furthermore, the difference reached significant on the 14th day(P<0.05).Relative HMBGl mRNA expressions by qRT-PCR. The mRNA levels of HMBG1 were significantly up-regulated in the 0J and OJPUFA group compared with the sham group (P<0.05). On the 3rd and the 7th day after bile duct ligation, the mRNA levels were slightly higher in the 0J group, but there is no significant difference. On the 14rd after bile duct ligation, the level of mRNA in the OJPUFA group was significantly down-regulated (P<0.05).4. Immunohistochemical staining for TLR4. In sham group, there’s almost no TLR4 immunoreactivity staining on the intestinal epithelial cells. TLR4 immunoreactivity stained weakly in the cytomembrane of the intestinal epithelial cells on the 3rd day in the OJ group. While, on the 7th day and the 14th day of the OJ group, the cell membrane was strongly stained, especially on the epithelial cells of villus tip; and some of the inflammatory cells were also positively stained. The staining in OJPUFA group is slightly inferior to the OJ group at any time point.Relative TLR4 protein expression by Western blotting. TLR4 protein in the sham group was significantly lower than that in OJ and OJPUFA group (P<0.05). The expression of TLR4 protein in the OJPUFA group is lower than that in the OJ group, while the levels were not significant between these two groups except for the value on the 14th day(P<0.05).Relative TLR4 mRNA expressions by qRT-PCR. The mRNA levels of TLR4 were significantly up-regulated in the OJ and OJPUFA group than that in the sham group (P<0.05). Though the expression was higher in the OJ group than that in the OJPUFA group, only on the 14th day there was a significant difference between these two groups (P<0.05).5. Immunohistochemical staining for NF-κB p65. In sham group, only a few nuclei of the intestinal epithelial cells were weakly stained. In the 0J group, besides the nuclei, the cytoplasm of the intestinal epithelial cells and some of the inflammatory cells were all strongly stained. In the OJPUFA group, the distribution of positive staining is similar to that in the 0J group with apparently nuclear staining in the epithelial cells and the infiltrated inflammatory cells, but the staining is weakly than that in the OJ group.Relative NF-κB p65 protein expression by Western blotting. The protein expression in the sham group was much lower than that in OJ and OJPUFA group (P<0.05). Compared with the 0J group, the expression of NF-κB p65 protein in the OJPUFA group is slightly down-regulated. However, significant decline was found in the OJPUFA group on the 14th day after bile duct ligation was found (P<0.05).Relative NF-κB p65 mRNA expressions by qRT-PCR. The mRNA levels of NF-κB p65 were significantly up-regulated in the 0J and OJPUFA group compared with the sham group (P<0.05). Except for the 14th day after bile duct ligation(P<0.05), there was no significant difference between the 0J group and the OJPUFA group (P>0.05).Conclusions1.ω-3 PUFA can ameliorate the damage of the intestinal mucosa and the decreasing of the goblet cells caused by obstructive jaundice.2. Modifying the expression of HMGB1 and TLR4 may serve as the mechanism by which the ω-3 PUFA attenuate the intestinal injury result from obstructive jaundice. |
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