Role Of Hydrogen Sulfide On Mesenteric Lymph Drainage Alleviating Multiple Organ Injury Induced By Hemorrhagic Shock In Rats And Its Mechanism

Numerous studies have shown that mesenteric lymph duct ligation and mesenteric lymph drainage alleviates the multiple organ dysfunction and structural damage following hemorrhagic shock. The gut-derived infection through lymphatic pathway is an important factor which induces multiple organ injury after severe shock. Hydrogen sulfide (H2S), a gaseous signal molecule, aggravates inflammatory response following hemorrhagic shock, while suppression of H2S lessens the degree of organ injury. Moreover, whether H2S involved in the mechanisms underlying the deleterious effect of the post-shock mesenteric lymph (PSML) on the organ injury, it remains unknown.Therefore, in the present study, using D, L-propargylglycine (PPG), the inhibitor of cystathionine-y-lyase (CSE), and sodium hydrosulfide hydrate (NaHS), the donor of H2S, to observe the effects of H2S on PSML drainage ameliorating the organic function, structure and the contents of Toll-like receptor4(TLR4), interleukin10(IL-10), IL-12, tumor necrosis factor α (TNFα), and investigating the role and mechanism of H2S in PSML drainage alleviating the organ injury following hemorrhagic shock in rats.Thirty male Wistar rats were randomly selected as sham, shock, shock+drainage, shock+drainage+PPG, shock+drainage+NaHS groups. Before bleeding30min, the rats in shock+drainage+PPG and shock+drainage+NaHS groups were performed intraperitoneal injection with PPG (45ml.kg-1) and NaHS (28μmol.kg-1), respectively; the equal account of saline was injected into the other groups. All of the animals were anesthetized by intramuscular injection with pentobarbital. The right femoral vein was separated and cannulated for anticoagulation with heparin sodium. A minimally heparinized polyethylene catheter was introduced into the right femoral artery for continuously monitoring the animals’mean artery pressure (MAP) through the experiment. Another catheter was inserted into the left femoral artery for bleeding by a syringe pump. A laparotomy was carried out to separate the mesenteric lymph duct from surrounding connective tissues. After a30-min stabilization period, the animals were subjected to SS, HS and HS plus mesenteric lymph drainage. In the shock, shock+drainage, shock+drainage+PPG, shock+drainage+NaHS groups, blood was withdrawn from the right femoral artery at an even speed. The MAP was reduced to40mmHg within10min and maintained at this level by withdrawing or reperfusing shed blood as needed, thus, the hemorrhagic shock model was established. After60min of hypotension, the ejective blood and the equal Ringer’s solution were reperfused within30min through right femoral vein. The MAP was observed for3h at the end of infusion. In the sham group, the rats were anesthetized, cannulated and operated as described above, but no blood was withdrawn or infused. In shock+drainage, shock+drainage+PPG, shock+drainage+NaHS groups, after infusion finished, the mesenteric lymph duct was cannulated and mesenteric lymph was drained up to3hours after shock.Blood samples were collected from the abdominal aorta in3hours after infusion or at corresponding time point in each group; the plasma was prepared for the further determination of creatine phosphokinase isoenzyme (CK-MB), lactic acid dehydrogenase-1(LDH-1), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid (TBA), urea, creatinine (Cre), total protein and H2S. Among them, the CK-MB was the indicator to reflect myocardial cell damage; the AST, ALT and TBA reflect the liver function; and the urea, Cre reflect the renal function. The left lobe of lung was taken out for the preparation of bronchoalveolar lavage fluid (BALF) for the determination of cell counting and protein quantification, and the lung permeability index (LPI) was calculated. Meanwhile, the superior lobe of right lung was taken out for the measurement of ratio in dry/weight. The lung, heart, kidney and liver tissue specimens from a fixed location were prepared. Parts of these tissues were fixed in paraformaldehyde for the determination of morphological changes. The remaining parts were homogenized for the measurement of H2S content and CSE activity, and the contents of TLR4, IL-10,IL-12and TNFα.Results showed that PSML drainage increased the MAP at time points in the shock group, NaHS decreased the effect of PSML drainage on MAP. The observations of histomorphology changes showed that the lung, heart, kidney and liver had nearly normal structure in rats of sham group, these tissues had morphological damages in shock group and less damage in shock+drainage and shock+drainage+PPG group, moreover, NaHS aggravated the degree of multiple organ injury. The numbers of cells and LPI in shock group were higher than those in sham group; these indices in shock+drainage group were apparently lower than those in shock group; these indices in shock+drainage+PPG group and shock+drainage+NaHS group were lower or higher than those in shock+drainage group, respectively. However, there was a reverse change in ratio of D/W as above. Meanwhile, the contents of AST, ALT, TBA, LDH-1, CK-MB, Urea and Cre in shock group were significantly increased compared with those in sham group; the contents of AST, ALT, TBA, Urea and Cre in shock+drainage group were significantly decreased than those in shock group; the contents of AST, ALT, TBA and Cre in shock+drainage+PPG group were lower than those in shock+drainage group; the contents of AST, ALT, Urea and Cre in shock+drainage+NaHS group were higher than those in shock+drainage group.Meanwhile, it showed that the contents of H2S in shock group the plasma and the pulmonic, hepatic, renal and myocardial tissue were remarkable increased; PSML drainage decreased the level of H2S; the levels was decreased by PPG and increased by NaHS. Moreover, the levels of TLR4, IL-10, IL-12and TNFa in pulmonic, hepatic, renal and myocardial tissue in shock group were higher than those in sham group, and the IL-10in hepatic homogenate, IL-12in pulmonic and hepatic homogenate, TNFα in pulmonic, hepatic, renal and myocardial homogenate were lessened by PSML drainage. The PPG, an inhibitor of CSE, reduced the contents of TLR4in pulmonic, hepatic, renal and myocardial homogenate, IL-10in pulmonic, renal and myocardial homogenate, IL-12in pulmonic homogenate, TNFα in pulmonic, hepatic, renal and myocardial homogenate. At the same time, the NaHS, a donor of H2S, increased the level of TLR4in pulmonic, hepatic, renal and myocardial homogenate, IL-10and IL-12in pulmonic, hepatic and renal homogenate, and TNFα in pulmonic, hepatic, renal and myocardial homogenate.Results of the experiment in this study demonstrated that negative effect of H2S is involved in the mechanism of PSML drainage reducing lung, liver, kidney and myocardium etc. major organs dysfunction and structure injury. And its mechanism may be related to the TLR4-mediated inflammatory response. These findings add and perfect the theory of "mesenteric lymphatic pathway in pathogenesis of multiple organic injuries", and provide the experimental basis for clinical prevention and treatment of critically ill patients, with a focus on lymphatic pathway and the regulation of H2S.