The Mechanism Of Hydrogen Sulfide Inhibiting The Formation Of Neutrophil Extracellular Traps In Hyperhomocysteinemia Rats

Background and PurposeHyperhomocysteinemia（HHcy）belongs to the category of metabolic syndrome.It is a systemic metabolic disease and an independent risk factor of cardiovascular and cerebrovascular diseases.Homocysteine（Hcy）is produced by the metabolism of the methionine which is the essential amino acid.The accumulation of abnormal metabolism in the body can lead to HHcy which induces thrombotic disease and the disease is a major reason of high mortality in clinical patients.Therefore,the mechanism,the treatment and the prevention of the thrombotic diseases which induced by HHcy become important contents in the field of medical research.Studies found that hydrogen sulfide（H₂S）,an endogenous gas signal molecule,inhibits platelet activation and aggregation under HHcy conditions and exerts antioxidative effects under a variety of physiological and pathological conditions.It becomes an important role in the course of prevention and treatment of hyperhomocysteinemia.However,the specific mechanism of hydrogen sulfide on the HHcy is still unclear.In the project,we utilize the HHcy animal model,the electron microscopy,western blotting,flow cytometry and other experimental methods to study platelets of the important factors and neutrophil extracellular traps（NETs）during the course of thrombotic diseases developing,which explore and elucidate the effect of H₂S inhibiting platelet activation and NETs formation,and establish theoretical foundation for the prevention and treatment of thrombotic diseases caused by HHcy.MethodsMale Wistar rats were purchased from Animal Experiment Center of Harbin Medical University,weighing 150-200g,after fed on a regular diet for 1-2 weeks,the rats were randomly divided into the following three groups:the control group（Control）:gastric tube in the feeding method,rats were orally infused with 2.5%starch paste（2.0g/kg/d）daily,and intraperitoneal injection of 0.9%Na Cl（0.1ml/kg/d）;the HHcy model group:gastric tube in the feeding method,the rats were orally infused with 2%methionine（2.0g/kg/d）daily,and intraperitoneal injection of 0.9%Na Cl（0.1ml/kg/d）;the HHcy+Na HS model group:the rats were orally infused with2%methionine（2.0 g/kg/d）daily,and then intraperitoneally injected with Na HS（Sodium Hydrosulfide,H₂S donor,5.6mg/kg/d）.After two weeks,the serum Hcy level was measured and the level of Hcy was equal or greater than 16.0μmol/L,proved that the model was successfully prepared.At the end of 4,8,and 12w,peripheral blood of abdominal aorta of rat was taken and divided into two parts:neutrophils were acquired from one part,the purity and activity of the cells were determined by Wright-Giemsa staining and Trypan Blue staining,Flow cytometry experimental method combined with DCFH probe to detected neutrophils ROS levels,and ELISA kit used to detect SOD activity.Sytox Green and MPO detected neutrophil extracellular traps formation which induced by PMA（phorbol ester）and platelet-rich plasma（PRP）stimulation.Expression of TLR4,PAD4 and phospho-p38 MAPK in neutrophils were measured by Western blotting.The other part was used to isolate the serum and platelets,the ELISA kit was used to measure the serum HMGB1 level,ds DNA Kits were used to measure ds DNA level of rat serum and supernatants of PMA or PRP stimulated neutrophils in vitro.The morphology and ultrastructure of platelets were observed by transmission electron microscopy and the coagulation parameters（MPV,PDW,PCT）were also measured.ResultsActivation of platelets in HHcy rats is characterized by gradual increasing of platelets pseudopodia and surface area.The concentrations of serum HMGB1 and ds DNA of supernatant liquid by platelet-rich plasma stimulated neutrophils are increased;SOD activity and ROS level of neutrophils are also increased.Western blotting showed that compared to the control group,the expression of TLR4,PAD4and phospho-p38 MAPK of neutrophils in the HHcy group increase.However,Na HS reduces serum HMGB1 level and SOD activity of neutrophils,ROS level and ds DNA concentration and TLR4,PAD4 and phospho-p38 MAPK expression in neutrophils which compared to the HHcy groups.ConclusionsExogenous H₂S inhibits neutrophil activation and NETs formation which induced by platelet activation under HHcy conditions.Exogenous H₂S inhibits the level of HMGB1,the expression of TLR4,and p38 MAPK phosphorylation and ROS production in molecular signaling pathway（HMGB1/TLR4/p38 MAPK/ROS）.