Study On Incidence And Mechanisms Of Severe Heatstroke And Rhabdomyolysis

Objectives1To explain the law and possible mechanism of rhabdomyolysis in severe heat stroke, and to find the entry point of treatment of rhabdomyolysis in severe heat stroke in clinic.2To research the therapy of severe heatstroke with RM in vitro and in vivo and explore a new and effective method to find out "second key points" of severe heatstroke with RM in clinic.Methods1Collect clinical data of previous patients with severe heatstroke, and study the clinical indicators and risk factors of patients with severe heatstroke and rhabdomyolysis (RM).①The past10years140cases of severe heatstroke in General Hospital of Guangzhou Military Command of PLA was collected.139cases were male and1female, age is from13-83years old, the average is (27.7±12.9) years. All patients were admitted to hospital within24hours after the occurrence of severe heatstroke. In which20patients died and120cases survival.②By using the univariate analysis(Age, CK, CK-MB, myoglobin protein, rhabdomyolysis, CD14, PCT, C-response protein, WBC, neutral particles percentage, hemoglobin, platelet, Na+, K+, C12-, Ca2+, P3+, BUN, CR, GOT, GST, total bilirubin, direct bilirubin, PT, APTT, GCS score, MRI, coma, ulinastatin used or not, the number of organ failure, of initial temperature, cooling temperature, ICU stay, total hospitalization time, APACHE (acute physiology and chronic health evaluation) II score, physi cal discomfort, blood filtration and etc.) and multivariate logistic regression, risk factors may result in severe heatstroke death in clinic was studied.2Construct damage model by heat stress of Human skeletal muscle cell strain (HMF) and Human umbilical vein endothelial cells(HUVEC) in vitro, and observe and study the changes of skeletal muscle, endothelial cell from molecular and cellular level.2.1Heat against skeletal muscle cells2.1.1Cell culture.2.1.2Cell viability and proliferation of HMF was detected by CCK-8.2.1.3The release of inflammatory factors in the heat against HMF cells was detected by the double antibody sandwich ELISA method.2.1.4Membrane permeabilit in heat to HMF was detected by flow cytometry combined with Fluo-3calcium influx.2.1.5Cytoskeleton in heat fight against HMF was detected by Brilliant Blue R-250staining.2.1.6Cell cycle changes in heat against the HMF was detected by flow cytometry.2.1.7HMF cell proliferation of applying ulinastatin was detected by CCK-8.2.1.8The release of inflammatory factors in the heat against HMF cells by applying ulinastatin was detected by the double antibody sandwich ELISA method.2.2Heat against endothelial cells2.2.1cell culture.2.2.2Cell viability and proliferation of MUVEC was detected by CCK-8.2.2.3The release of inflammatory factors in the heat against HUVEC cells was detected by the double antibody sandwich ELISA method.2.2.4The release of IL-6, TNF-α in the heat against HUVEC cells was detected by the double antibody sandwich ELISA method.2.2.5Cytoskeleton in heat fight against HUVEC was detected by Brilliant Blue R-250staining.2.2.6Cell cycle changes in heat against the HUVEC was detected by flow cytometry.2.2.7HUVEC cell proliferation of applying ulinastatin was detected by CCK-8.2.2.8The release of inflammatory factors in the heat against HUVEC cells by applying ulinastatin was detected by the double ant ibody sandwich ELISA method.3Study the relationship between severe heatstroke rhabdomyolysis (RM) and inflammation factors, kidney, lung and electrolyte changes in vivo.①Glycerol was applied to Wista rats RM model.②Serum electrolytes, inflammatory factor, microscopic pathological examination and electron microscopy performance by applying ulinastatin to rats with rhabdomyolysis and control groups were observed. Histopathological section of rats with rhabdomyolysis by applying Xuebijing was observed.Results1Collect clinical data of previous patients with severe heatstroke, and study the clinical indicators and risk factors of patients with severe heatstroke and rhabdomyolysis (RM).From the univariate analysis, rhabdomyolysis, creatine kinase (CK), Ca2+, PLT, GCS score, the number of organ failure, APECHEⅡ score, initial body temperature may affect prognosis of patients with severe heat stroke. The multivariate logistic regression analysis reveals the existence of rhabdomyolysis, the number of organ failure, APACHE Ⅱ score could determine the prognosis of patients with severe heatstroke.2Construct damage model by heat stress of Human skeletal muscle cell strain (HMF) and Human umbilical vein endothelial cells(HUVEC) in vitro, and observe and study the changes of skeletal muscle, endothelial cell from molecular and cellular level.2.1HMF2.1. IContrast to the control group, the viability of the group which heat on HMF decreased (P<0.001), and it depend on time and temperature.2.1.2Through24hours cell proliferation of HMF, with the extent and time lasting, the survival rate of skeletal muscle cells, at each time compared with the normal control group, was significantly decreased (P <0.001). In lh period, survival rate of the three groups was no significant difference (P>0.05), in3h period, the43℃group survival was significantly decreased (P<0.01) compared with39℃and41℃group, in5h and7h,OD value of41℃and43℃group was close to the negative control, the two groups decreased significantly (P<0.001) compared with the39℃group, showing that the heatstress on the skeletal muscle cells was cytotoxic and the survival rate depend on temperature and time.2.1.3ELISA examination shows the expression is no significant difference of IL-1β, IL-8, IL-10, INF-γ (P>0.05) between heat against groups compared with the control group, and the amount of IL-6and of TNF-alpha is significantly increased (P<0.001) compared with the normal control group;41℃and43℃group rise (P<0.05) compared to39℃group;43℃group and41℃group has no significant difference (P>0.05). IL-6levels in skeletal muscle cells is relatively obvious.2.1.4With the time of heat last, compared with the control group, intracellular calcium influx increase in skeletal muscle cells by applying Fluo-3calcium influx for fluorescence detection and flow cytometry2.1.5With time and degree of heat, compared with the control group, cytoskeleton of skeletal muscle cells gradually appears thicker, shorter, the stress fibers.2.1.6Compared with the control group, the cell cycle of HMF of heat to combat block in G0/G1phase, in about18h or so back to normal.2.1.7Ulinastatin has no inhibitory effect on the HMF proliferation (P>0.05) in vitro by giving a variety of doses(0-7000U/ml).2.1.8After HMF cells under heat stress for1h, Oh group, IL-6of43℃heat against groups was significantly increased (P<0.001) than the37℃control group and43℃heat against+ulinastatin group;TNF-alpha was significantly increased (P<0.01), but concentration of IL-1β, IL-8, IL-10, IFN-γ in this experiment did not detect significant differences (P>0.05). IL-6of6h group increased significantly, but IL-6of43℃heat against groups was significantly increased (P<0.05) than the37℃control group and43℃heat against+ulinastatin group. The37℃control group was no significant difference (P>0.05) compared with43℃heat against+ulinastatin group, but IL-8of43℃heat against groups was significantly increased (P<0.05) than the37℃control group and43℃heat against+ulinastatin group.37℃control group also had difference (P<0.05) with43℃heat against+ulinastatin group. TNF-α of43℃heat against groups was significantly increased (P<0.05) than the37℃control group and43℃heat against+ulinastatin group, other value of inflammatory factors is too low to have no obvious practical significance. It is noteworthy that, the level of inflammatory factors at37℃control group is almost no significant difference (P>0.05) with43℃heat against+ulinastatin group at6h group.2.2HUVEC2.2.1Contrast to the normal control group, the viability of the group which heat on HUVEC decreased (P<0.001), and it depend on time and temperature.2.2.2Through24hours cell proliferation of HUVEC, the cells were placed in normal culture environment for24h, CCK-8test showed that,39℃group1h,3h and5h period,24hours cell proliferation of three groups was no significant difference (P>0.05),7h period decreased significantly (P<0.01).41℃and43℃group24h proliferation rate compared with39℃group was significantly decreased (P<0.001),43℃group3h,5h and7h period24h proliferation rate compared with41℃group decreased significantly (P<0.01)2.2.3ELISA examination showed the expression is no significant different of IL-1β, IL-8, IL-10, INF-γ (P>0.05) between heat against groups compared with the control group, and the amount of IL-6and of TNF-alpha significantly rise (P<0.001).41℃and43℃group compared with39℃group increased significantly (P<0.01), the groups of43℃and41℃were no significant difference (P>0.05).2.2.4IL-6and TNF-α expression levels increased significantly in endothelial cells with heat against compared with normal control group by applying PCR.2.2.5With time and degree of heat last, compared with the control group, cytoskeleton of HUVEC gradually appears thicker, shorter, the stress fibers.2.2.6Compared with the control group, the cell cycle of HUVEC of heat to combat block in G0/G1phase, in about12h or so back to normal.2.2.7Ulinastatin had no inhibitory effect on the HUVEC proliferation (P>0.05) in vitro by giving a variety of doses(0-7000U/ml) ulinastatin incubated with HUVEC.2.2.8After HUVEC cells under heat stress for1h, Oh group, IL-6of43℃heat against groups was significantly increased (P<0.001) than the37℃control group and43℃heat against+ulinastatin group, TNF-α was significantly increased (P<001), but concentration of IL-13, IL-8, IL-10, IFN-γ in this experiment did not detect significant differences (P>0.05). IL-6of6h group increased signi ficantly, but IL-6of43℃heat against groups was significantly increased (P<0.05) than the37℃control group and43℃heat against+ulinastatin group. However,43℃heat against groups was significantly increased (P<0.05) than43℃heat against+ulinastatin group. At6h group, TNF-α of43℃heat against groups was significantly increased (P<0.05) than the37℃control group and43℃heat against+ulinastatin group. Concentrations of IL-1β, IL-8,IL-10IFN-γ is too low to have no obvious practical significance.3Study the relationship between severe heatstroke rhabdomyolysis (RM) and inflammation factors, kidney, lung and electrolyte changes in vivo.3.1According to the relevant literature, rat model with rhabdomyolysis was successfully made by applying glycerin.3.2Compared with the control group, IL-6, TNF-α, CK, CR, BUN, of the glycerol-induced rat model with rhabdomyolysis is increased (P<0.05). The level of IL-6, TNF-α, CK, CR, BUN, of Wista rats with rhabdomyolysis may decrease (P<0.05)3.3From the biopsy, compared with the control group, rhabdomyolysis, kidney damage and lung damage is alleviated by applying intravenous ulinastatin and Xuebijing.ConclusionFrom the univariate analysis, rhabdomyolysis, CK, Ca2+,PLT, GCS score, the number of organ failure, APECHEⅡ score, initial body temperature may affect patients with severe heat stroke prognosis. Multivariate logistic regression analysis reveales the existence of rhabdomyolysis, the number of organ failure, APACHE11score could determine the prognosis of patients with severe heatstroke.There are striated muscle cells, endothelial cells and other kind of cell in muscle tissue. Endothelial cells are target cells and effector cells. Therefore, we study the impact of heat on endothelial cells in vitro at the same time. Heat has cytotoxic effects on skeletal muscle cells and endothelial cells, it can inhibit cell proliferation, promotes the release of cytokines IL-6and TNF-alpha, resulting in increased cellular calcium influx(one of the incentives in Rhabdomyolysis), cytoskeleton change, cell cycle stagnation.UTI has no proliferation effects to two kind of cell, and it can...