Protective Effects Of Insulin On Lipopolysaccharide-induced Impairments Of H9C2 Cells And Corresponding Mechanism

ObjectiveSepsis is defined as the presence (probable or documented) of infection together with systemic manifestations of infection, its essence is inflammatory mediators and cytokines mediated by host immune response. Sepsis is one of the most common critical diseases in pediatric intensive care unit (PICU), and it has high morbidity and mortality. Sepsis progresses rapidly and its further development can often lead to sever sepsis, septic shock and multiple organ dysfunction (MODS). Sever sepsis, septic shock and multiple organ dysfunction can often affect various organs due to the failure of the microcirculation and mitochondrial dysfunction, the heart is easily vulnerable to damage. Sepsis-induced myocardial dysfunction (SIMD) is common in clinic, its features can be represent as a myocardial contractile function damage, biventricular reversible dilatation, ejection fraction decreased, and falling response on the fluid resuscitation and vasoactive drugs. It is reported that reversible myocardial depression can occur in 40% of septic patients and myocardial dysfunction can occur in 7% of septic patients. Septic patients with myocardial dysfunction had a high mortality rate of 50% than those patients without myocardial dysfunction. Septic patients with myocardial dysfunction had such a high mortality rate due to its complicated pathogenesis. It has been found that a variety of mechanisms are involved such as immune system activation, oxidative stress reaction, inflammatory injury, apoptosis, toll like receptor, adrenergic signal abnormalities and myocardial mitochondrial dysfunction, and so on. However, the exact pathogenesis of Sepsis-induced myocardial dysfunction has not been fully understood. Recently, increasing evidences have revealed that excessive production of mitochondrial reactive oxygen species (ROS) and oxidative stress play important role in the pathogenesis of sepsis-induced myocardial dysfunction.Sepsis-induced myocardial dysfunction has no specific treatment. Clinical study have found that to strictly control the blood glucose-levels of septic patients with intensive insulin therapy can significantly reduce the mortality of patients. Moreover, intensive insulin therapy can be helpful for the septic patients with myocardial injury and promote the recovery of cardiac function. Insulin is a protein hormone secreted by pancreatic β-cell and its main physiological role is to regular the body’s metabolism. Insulin can promote the tissues and cells glucose uptake and utilization, promote glycogen synthesis, inhibit glycogen degradation and gluconeogenesis, and finally decrease blood glucose levels. Insulin can promote the synthesis and storage of fatty acid and reduce fat decomposition. Insulin can also promote the body’s use of amino acid and increase the synthesis of protein. Recent studies show that Insulin play important roles in regulation of anti-inflammation response, inhibiting oxidative, immune response, as well as blood glucose control, but its mechanisms are not clear. It has been found that Insulin may increase uncoupling protein 2 (UCP2) expression, and reduce the production of ROS which play a protective role against oxidative stress damage. UCP2 is a proton anion transport protein which is located in the mitochondrial inner membrane. UCP2 is involved in the process of uncoupling of mitochondrial oxidative phosphorylation, ATP synthesis, regulation of cell energy generation, regulation of intracellular inflammation, inhibition of ROS, maintaining calcium balance within mitochondria and mitochondrial membrane potential, participating in regeneration of mitochondria and so on. In early experiments, we found that high levels of UCP2 protein expression in septic myocardial tissue induced by lipopolysaccharide, so we speculate that UCP2 may be associated with myocardial injury of sepsis. In addition, silencing of UCP2 aggravates mitochondrial injury in H9C2 cells and increases the levels of inflammatory cytokines and ROS, which indicates that UCP2 may play a protective role in cardiomyocytes by regulating inflammatory response and oxidative stress. However, there are-few researches about the-protective effect of Insulin on myocardial injury of sepsis and the possible role of UCP2. In this paper, we speculate that Insulin may regulate intracellular inflammatory response and the oxidative stress by regulating the expression of UCP2, which may play a protective role in reducing myocardial injury.Lipopolysaccharide (endotoxin) is a major cell wall component of gram negative bacteria which is commonly used in the study of models of sepsis. Therefore, H9C2 cells are used in this experiment as the research object. Lipopolysaccharide is used to induce sepsis in H9C2 cells which mimics models of sepsis in vitro. By observing the protective effects of insulin on lipopolysaccharide-induced impairments of H9C2 cells and the possible roles of UCP2, the study aims at investigating the effect of insulin on the expression of UCP2. In addition, this study also attempt to provide experimental and theoretical foundations for the clinical application of insulin treatment of sepsis.Methods1. Models of sepsis and groupingAfter cultured for 24 h, H9c2 cells were randomly divided into four groups as follows:control group (CON), LPS stimulation group (LPS), LPS+70 IU/L Insulin group (IN 70 IU/L), LPS+350IU/L Insulin,group (IN 350 IU/L), and LPS+700 IU/L Insulin group (IN 700 IU/L). H9c2 Cells in Insulin group were treated with 70 IU/L,350 IU/L or 700 IU/L insulin 15min before LPS stimulation, and H9c2 cells in control group were treated with an equal volume of saline. After that, cells in group LPS and IN were treated with lipopolysaccharide for 24h. And the concentration of LPS in LPS group and Insulin group was 1ug/ml.2. Sample collection and test2.1 Sample collectionCell culture medium and cell specimens were collected after treatment. Cell culture medium was proper packed and placed in -80℃. Cell specimens were collected immediately for protein extraction or RNA extraction.2.2 Detection of lactate dehydrogenase (LDH) level and cell viability LDH and cell viability were measured as instruction manual.2.3 Detection of malondialaldehyde (MDA) and superoxide dismutase (SOD) MDA and SOD were measured as instruction manual. Protein concentration was determined by BCA Protein Assay Kit. After adding to fluid for reaction as instruction manual, OD value was measured by enzyme labeled meter. And the results were calculated according to the instruction.2.4 Detection of reactive oxygen species (ROS)The levels of reactive oxygen species was determinated by colorimetric detection as instruction manual.2.5 Detection of the level of TNF-a and IL-1βThe level of TNF-a and IL-1β were detected by enzyme-linked immune sorbent assay (ELISA) as instruction manual.2.6 Determination of the mRNA expression of UCP2The mRNA expression of UCP2 was detected by transcription polymerase chain reaction (RT-PCT). Total RNA from cell specimens was isolated and reversed using regent kit from Takara and PCR instrument. The relative expression of UCP2mRNA was calculated by using β-actin as the reference gene.2.7 Determination of the protein expression of UCP2The protein of H9C2 cells were extracted by Protein Extraction Kit. Protein concentration was determined by BCA Protein Assay Kit. The protein expression of UCP2 was detected by using Western Blot. By using β-actin as the reference protein, the protein expression of UCP2 was analyzed compared with the gray value.3. Statistical analysisData was analyzed by IBM SPSS 20.0 and all results were reported as mean ±standard deviation (x±s). Differences between groups were analyzed by one-way ANOYA. If there was statistical significance and homogeneity of variance between groups, Least-significant difference (LSD) was used, while if there was statistical significance and heterogeneity of variance between groups, Dunnett T3 was used. P<0.05 was considered statistical significance.Results1. Comparison of LDH level in the supernatant of H9C2 cell culture in each groupThe levels of LDH in the supernatant of H9C2 cell culture in LPS group increased compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly decreased the levels of LDH compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group, the levels of LDH in 350 IU/L insulin group were lower.2. Comparison of cell viability in H9C2 cells in each groupCell viability in H9C2 cells in LPS group decreased compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly increased cell viability compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group, the cell viability in 350IU/L insulin group were higher.3. Comparison of MDA content in H9C2 cells in each groupThe degrees of MDA content in H9C2 cells in LPS group significantly increased compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly decreased the degrees of MDA content compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group, the degrees of MDA content in 350 IU/L insulin group were lower.4. Comparison of SOD activity in H9G2 cells in each groupDegrees of SOD activity in H9C2 cells in LPS group significantly decreased compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly increased the degrees of SOD activity compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group, the degrees of SOD activity in 350 IU/L insulin group were higher.5. Comparison of ROS production in H9C2 cells in each groupThe levels of ROS production in H9C2 cells in LPS group significantly increased compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly decreased the levels of ROS production compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group, the levels of ROS production in 350 IU/L insulin group were lower.6. Comparison of TNF-a level in the supernatant of H9C2 cell culture in each groupThe levels of TNF-a in the supernatant of H9C2 cell culture in LPS group increased compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly decreased the levels of TNF-a compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group, the levels of TNF-a in 350 IU/L insulin group were lower.7. Comparison of IL-1β level in the supernatant of H9C2 cell culture in each groupThe levels of IL-lp in the supernatant of H9C2 cell culture in LPS group increased compared with control group (P<0.05). Insulin intervention decreased the levels of IL-1β compared with LPS group (P>0.05)8. Comparison of mRNA expression of UCP2 in H9C2 cells in each groupThe mRNA expression of UCP2 in LPS group were up regulated compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly increased the mRNA expression of UCP2 compared with LPS group (P<0.05). Compared with 70IU/L and 700IU/L insulin group,-350 IU/L insulin group had a better result.9. Comparison of protein expression of UCP2 in H9C2 cells in each groupThe mRNA protein of UCP2 in LPS group were up regulated compared with control group (P<0.05).350 IU/L and 700 IU/L insulin intervention significantly increased the protein expression of UCP2 compared with LPS group (P<0.05) Compared with 70IU/L and 700IU/L insulin group,350 IU/L insulin group had a better result.Conclusion1. LPS can cause H9C2 cells injury.2.Insulin can attenuates lipopolysaccharide-indeced injury in H9C2 cells, the corresponding mechanism may relate to the decrease of inflammation response and oxidative stress, which is probably mediated through upregulating the expression of UCP2.