The Protective Effect And Preliminary Mechanism Of Uncoupling Protein 2 During Mitochondrial Dysfunction In Cardiomyocytes Under Septic Conditions

BACKGROUNDSepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection, which continues to pose serious clinical challenges. Sepsis affects individuals of all ages and is the leading cause of morbidity and mortality for critically ill patients. According to statistics, about 750,000 patients are diagnosed to have severe sepsis in the United States each year. And the incidence is considered to increase by 1.5%/year, rising to millions of cases annually by 2020. Sepsis, which can lead to severe sepsis and septic shock, is an important cause of mortality. The pathogenesis of sepsis is very complicated. It involves a complex process of cellular activation resulting in the activation of neutrophils, monocytes, and microvascular endothelial cells; the triggering of neuroendocrine mechanisms; and cytokine storm, inflammatory mediators falls, bacterial translocation and intestinal endotoxemia, the interaction between the coagulation system and inflammatory systems, micro-circulation and mitochondrial dysfunction, and so on. In recent years, the cellular hypoxia induced by mitochondrial dysfunction has become a hot field of research.Sepsis-induced myocardial dysfunction (SIMD), one of the main predictors of morbidity and mortality associated with sepsis, is present in>40% of cases of sepsis. A number of studies on both patients and experimental animals have indicated that mitochondrial dysfunction seems to be related to the severity and prognosis of sepsis. The heart is rich in mitochondria, and thus the role of mitochondrial damage in SIMD has received much attention. Previous studies have indicated that multiple aspects of mitochondrial dysfunction contribute to SIMD, such as the overproduction of reactive oxygen species (ROS), the altered generation of adenosine triphosphate (ATP) and the disruption of mitochondrial membrane potential.At present, the mechanisms for the occurrence of myocardial injury in patients with sepsis is still not very clear. The possible mechanism may be related to the circulation and microcirculation, mitochondrial dysfunction, apoptosis, inflammation pathway damage, oxidative stress and other factors. As an important role in myocardial injury, Mitochondrial damage receive more and more attention in sepsis.Mitochondrial uncoupling proteins (UCPs) belong to the SLC25 anion carrier family,among which five UCP isoforms have been identified.The closely related UCPs are UCP1-UCP3. UCP1 is mainly expressed in brown adipose tissue and UCP3 in muscle and adipose tissues, whereas UCP2 has been found in several tissues, such as liver, brain,pancreas, adipose tissue, immune cells, spleen, kidney, and the central nervous system.UCP3 gene is located on 11q13, and UCP2 closely linked,separated by only 6000bp. UCP2 and UCP3 have 59 and 57% identity, respectively, with UCP1, and 73% identity with each other. Chemiosmotic hypothesis suggest that hydrogen ions (H+),which generating the electrochemical potential, pump from the mitochondrial matrix first, and then back to the mitochondrial matrix through ATP synthase during which ATP is synthesized in the electron transport chain.However, without ADP mitochondria can also finish mitochondrial respiratory function, during which H+no longer leak throught ATP synthase into the mitochondrial matrix but leak by UCPs. This process, which was confirmed this uncoupling effect is mediated by UCPs the carrier on the mitochondrial, does not produce ATP. At present, studies mainly focused on the UCPs functions related to metabolic disorders, glucose and lipid metabolism regulation and oxidative stress.In vitro and in vivo, by modulating MMP, as well as the ATP and ROS levels, the upregulation of UCP2 plays a neuroprotective role, while UCP2 knockout mice present with increased mitochondrial ROS production. In addition, UCP2 is involved in the regulation of other physiological or pathological events, such as in the formation of atherosclerotic plaque, food intake and metabolic diseases. Previous studies have shown that UCP2 can inhibit the production of ROS by reduces △μH+ When the mitochondrial membrane potential rises.And Derdak study found that ROS may be the bridge between NF-κB and UCP2, and UCP-2 inhibited NF-kB production by reduced ROS production, thereby protected the heart cells from oxidative stress injury.However, the exact role of UCP2 in myocardial cells under septic conditions remains to be determined. It remains unclear as to whether UCP2 plays a protective role in myocardial cells under septic conditions by regulating MMP and the generation of mitochondrial ROS. We hypothesized that UCP2 may regulate MMP and mitochondrial function under septic conditions. In this study, we measured the levels of ROS and ATP production, as well as the extent of MMP and other relative indicators following the silencing of UCP2 and UCP2-overexpression in H9C2 cardiomyocytes under septic conditions.OBJECTIVEThe role of UCP2 in myocardial cells under septic conditions remains unclear. We hypothesized that UCP2 may regulate MMP and mitochondrial function under septic conditions. To reveal the protective effect and mechanism of UCP2 in mitochondria. In this study, we measured the levels of ROS and ATP production, as well as the extent of MMP and other relative indicators, and mitochondrial morphology,mitochondrial swelling were observed by TEM following the silencing of UCP2 and UCP2-overexpression in H9C2 cardiomyocytes under septic conditions, which was induced by mixture of lipopolysaccharide and peptidoglycan. This experiment will provide the basis theoretical support for the treatment of myocardial mitochondrial injury in sepsis.METHODS1. Septic cell model and groups(1)Preliminary experiments groups in RNA interference:To filter effectively UCP2 gene fragment, H9C2 cardiomyocytes were randomly divided into five groups, namely: ①Control group received normal saline; ②Lipofectamine group received the same amount of Lipofectamine 2000; ③SiRNAl group, transfected with siRNAl; ④siRNA2 group, transfected with siRNA2;③ncRNA group, transfected with negative control siRNA (ncRNA). Each of the final concentration of siRNA was 80 nmol/1.(2) Cell groups in RNA interference experiments:H9C2 were randomly divided into four groups:①control group, cells without any treatment;②LPS/PepG group, cells treated with LPS and PepG, respectively the concentration of LPS is 2 μg/ml and PepG is 20 μg/ml;③LPS/PepG+siRNA group,cells transfected with siRNA2 and 24 hours later treated with LPS plus PepG as described above;④LPS/PepG +ncRNA group, cells transfected with ncRNA and 24 hours later treated with LPS plus PepG as described above.(3) The experiments groups in UCP2 overexpression:H9C2 were randomly divided into four groups: ①ontrol group, cells received saline stimulation; ②LPS/ PepG group, cells treated with LPS and PepG;③PHBLV group, cells transfected with empty vector and then stimulated by administration with LPS and PepG; ④PHBLV-UCP2 group, UCP2-overexpression cells were stimulated with LPS and PepG. The final concentration of LPS was 2 μg/ml, PepG was 20 μg/ml.2. Small interfering RNA transfection①According to literature and preliminary experimental results, we designed two fragments of RNA interference. The first preliminary experiment was to determine the effective siRNA; then this interference was used as effective siRNA fragments for thr further RNA interference experiments; ②The effective siRNA was transfected to cardiomyocytes, then mitochondrial morphology and function were observed under septic conditions.3. Construction of lentiviral vector with UCP2 overexpression and stable myocardial strain screening.①Total RNA from rat was Extracted, then it was reversed transcribed into cDNA. And then cDNA was amplified by PCR with specific primers of UCP2, pMD19-T vector (cloning vectors) was connected to the UCP2 gene later. At last, ligation product was transfected into E. coli, and positive E. coli clones were screened for sequencing.②UCP2 which was amplified from the target gene T plasmid by PCR, was connected to shuttle vector lentiviral pHBLV-IRES-ZsGreen-PGK-puro. And then the ligation product was transformed、amplified and sequenced as described above. ③To reconstruct the effective virus vector,293T cells were co-transfected with UCP2-overexpressiong shuttle vector and secondary packaging plasmid vector. And then cell supernatants which was rich in lentivirus particles was collected and filtered. At last the virus was concentrated by ultracentrifugation and the concentration of virus was titrated.④H9C2 cell lines were infected with lentivirus obtained above, then puromycin was used to screen cells which was infected successfully. After several generations, the stable H9C2 cell lines with UCP2-overexpression was obtained successfully. Because of the cells can synthesize anti-drug protein, it survived with the puromycin. At last, WB and PCR were used to confirmed that UCP2 overexpression H9C2 cell line which was used in the latter experiments, is constructed successfully.4. Measurement of lactate dehydrogenase (LDH), creatine kinase (CK), IL-6 andTNF-a①Measurement of LDH, CK:Their activity were measured by using commercially available kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacture’s instructions. Absorbance were respectively read at 660 nm and 440 nm in a multifunctional microplate reader.②ELISA assay for detecting IL-6 and TNF-a:48 hours after cell treatment, the culture supernatant was collected for the subsequent measurement of IL-6 and TNFa. The culture supernatants were measured using commercially available ELISA kits. All procedures were performed as the protocol instructions strictly. The samples were analyzed in triplicate.5. Analysis of mitochondrial morphology by TEM and FCM.①Mitochondrial morphology observed byTransmission electron microscopy:H9C2 were collected and fixed in a solution containing 3.0% formaldehyde,1.5% glutaraldehyde in 100 mM cacodylate containing 2.5% sucrose (pH 7.4). H9C2 cells were stained with 4% aqueous uranyl acetate, dehydrated, infiltrated and embedded in epoxyresin. Ultrathin sections (80 nm) were cut and imaged with an Hitachi transmission electron microscope.②Degree of mitochondrial swelling detected by FCM:To determine large amplitude swelling of H9C2, the isolation of mitochondria and cytosol was performed using the Cell Mitochondria Isolation Kit (Beyotime Institute of Biotechnology, China). Size (forward scatter, FSC) and structure (side scatter, SSC) of mitochondrion were detected. The degree of mitochondrial swelling was quantified as the FSC/SSC ratio.6. mitochondrial membrane potential qualitative observated by LSCM and quantitatively analyzed by FCM.①MMP analyzed by FCM:H9C2 cells were stained with JC-1 according to the manufacture’s instructions. The fluorescence was read at 488 nm excitation and 530 nm emission for green, and at 540 excitation and 590 emission for red. The ratio of aggregated JC-1 (red fluorescence) and monomeric JC-1 (green fluorescence) represented △ψm of H9C2 cells.② MMP observated by LSCM:MMP was assessed by laser scanning confocal microscope with JC-1 staining according to the manufacture’s instructions. H9C2 cells were stained with JC-1 for 20 minutes at 37℃ after 24 h incubation with LPS/pepG. Cells on 35mm Confocal special dish were scanned with LSCM. The ratio of red fluorescence and green fluorescence represented △ψm of H9C2 cells.7. UCP2 gene expression levels were detected by RT-PCR and WB.①UCP2 protein detected by WB:H9C2 cells were lysed and the protein concentrations were measured using BCA protein assay.30 mg protein from each sample were subjected to SDS-PAGE gels and then transferred to PVDF filter membranes. The membranes were blocked with non-fat dried skimmed milk powder in wash buffer for 1 hour and subsequently incubated with primary antibodies overnight at a dilution recommended by the suppliers. Membrane were washed three times with wash buffer and then incubated with corresponding horseradish peroxidase-conjugated secondary antibodies. Protein signal was developed by ECL substrate. The immunoreactive protein bands were visualized by the system of In-Vivo Imagine System F. The bands intensity were quantified by the software of Gel-Pro Analyzer 4.0.②mRNA levels of UCP2 detected by RT-PCR:Total RNA was extracted from H9C2 cells using TRIzol and then reverse transcribed to synthesize cDNA using reverse transcription RT-PCR kits. The threshold cycle (Ct) was obtained from triplicate samples and averaged. Calculations were based on the "Delta-Delta method" using the equation R (ratio)= 2-△△Ct and standardized by the house-keeping gene 18s-RNA.8. Assay of SOD、MDA and GSH.Oxidative stress factor SOD, MDA, GSH were detected with the assay kit according to the manufacture’s instructions. Brieflily, mitochondrial protein concentration was detected by Coomassie brilliant blue assay, and then OD values was read by microplate reader, lastly the activity or concentration was calculated.9. Representative of Mitochondrial function such as mitochondrial ATP, mitochondrial ROS, and mtDNA were detected.① ROS:Production of ROS in H9C2 cells was fluorometrically monitored using DCFH-DA according to the manufacture’s instructions. DCFH-DA passively diffuses into cells and is deacetylated to turn into fluorescent compoundDCFH. DCFH reacts with ROS to form the fluorescent product DCF which is trapped inside the cells. Then DCF fluorescence was read by multifunctional microplate reader at an excitation wavelength of 488 nm and at an emission wavelength of 525 nm. To visually observe the changes of ROS, fluorescence photograph were taken by fluorescence microscopy with 470nm (excitation) and 520nm (emission) filters.② ATP:The cellular amount of ATP were measured using a firefly luciferase-based ATP assay kit according to the manufacturer’s proposes. Luminance (RLU) was measured by a multifunctional microplate reader. Standard curve of ATP concentration was prepared from a know amount (0.01 μM-10 μM) and the protein concentration of each group was detected using the Bradford Protein assay. The ATP levels were expressed as nmol/mg protein.③mtDNA:Total genomic DNA was extracted with a DNA extraction kit, and DNA concentration was measured by optical density. The mtDNA copy number was obtained according to manufacture’s protocol. The threshold cycle (CT) is the cycle at which the first significant increase in the fluorescent signal is detected. Relative values for Cytb and 18s-RNA in samples were used to obtain a ratio of mtDNA to nuclear DNA in that sample.10. Statistical Analysis:Experimental values were obtained from three independent experiments with a similar pattern and expressed as means±SD. For statistical differences between control and treatments groups we used ANOVA, followed by post-hoc pairwise comparison (LSD) tests for analysis. Statistical analysis was carried out using SPSS software package 18.0. Significance was set at P<0.05.RESULTS1. Construction of septic cell model.Following treatment with LPS/PepG, the levels of CK, LDH, TNF-a and IL-6 in the cell supernatant of LPS/PepG group notably increased compared with those in the control group. And the ultrastructure of the mitochondria was normal in the control group, while mitochondrial swelling and vacuolization, loss of matrix and the disruption of crests were detected in the LPS/PepG-treated cells. All these indicated that the H9C2 cell model of sepsis had been successfully constructed.2.Successful transfection of siRNA and knockdown efficiency of siUCP2.2 siRNAs against UCP2 were used to specifically suppress UCP2 mRNA expression in this study. The mRNA level of UCP2 was reduced by up to nearly 69% following transfection with siRNA2 and by 59% following transfection with siRNAl compared with the control group, And the difference was statistically significant. siRNA2 was thus selected for use in further experiments.3. The expression of UCP2 gene.①Interference experiments showed:Compared with the control group, siRNA induced a 0.22-fold decrease in UCP2 protein expression and a 0.59-fold decrease in UCP2 mRNA expression.②Overexpression experiments showed:Compared with the control group, UCP2 protein relative density and relative UCP2-mRNA copy numbers were significantly higher in PHBLV-UCP2 group,and the difference was statistically significant. These shown that interference and overexpression experiments were successful.4.The impact of UCP2 overexpression and silencing on CK, LDH, and IL-6, TNF-α levels.①Interference experiments showed:Supernatants from the cells treated with LPS/PepG+siRNA had markedly higher CK, LDH, IL-6 and TNF-a concentrations compared with the cells treated with LPS/PepG, which indicated that siRNA aggravate cell injury and the further release of cytokines.②Overexpression experiments showed:compaired with LPS/PepG group, the CK, LDH, IL-6 and TNF-a concentrations were much lower in PHBLV-UCP2 group, and the difference was statistically significan. It indicated that UCP2-overexpression inhibit the cell injury and further release of cytokines induced by sepsis.5.The degree of mitochondrial swelling.①Interference experiments showed:Compared with the LPS/PepG group, the FSC/SSC ratio was significantly increased in LPS/PepG+siRNA group, in which mitochondrial swelling was much significantly, matrix density decreased significantly, ridge intimal rupture and fracture were much frequently, vacuolar degeneration was more obvious.These indicated that the mitochondrial ultrastructure was damaged more severely in LPS/PepG+siRNA group.②Overexpression experiments showed:Compared with Lps/PepG group, the FSC/SSC ratio was significantly lower in PHBLV-UCP2 Group, in which mitochondrial swelling was reduced, matrix density increased significantly, ridge intimal rupture and fracture were less frequently, vacuolar degeneration was less obvious. All these suggesting that UCP2-overexpression help protect mitochondria in sepsis.6. mitochondrial membrane potential.①Interference experiments showed:Compared with Lps/PepG group, red fluorescence/green fluorescence ratio was significantly higher in Lps/PepG+siRNA group, in which MMP was higher also.(DOverexpression experiments showed: compared with Lps/PepG group, red fluorescent/green fluorescence ratio was significantly increased in PHBLV-UCP2 group, in which MMP was higher also.But the increasing range was lower in PHBLV-UCP2 group compaired with Lps/PepG+siRNA group. This suggests:UCP2 can modulate MMP, but the exact extent of the regulation is not clear.7. Mitochondrial ROS and other oxidative stress indicators.①Interference experiments showed:Compared with Lps/PepG group, ROS and MDA contents were significantly higher while SOD and GSH were significantly lower in the Lps/PepG+siRNA group, which indicated that interference UCP2 aggravate cells damage caused by mitochondrial oxidative stress in sepsis.②Overexpression experiments showed:Compared with Lps/PepG group, ROS and MDA content decreased while SOD and GSH increased significantly in PHBLV-UCP2 group, suggesting that UCP2-overexpression can inhibit septic mitochondrial oxidative stress damage.8. Mitochondrial ATP content and mtDNA copy number.①Interference experiments showed:Compared with Lps/PepG group, mtDNA copy number was significantly decreased and ATP was no significant difference in Lps/PepG+siRNA group, suggesting that interference UCP2 can cause further damage on mitochondrial biogenesis. ②Overexpression experiments showed: Compared with Lps/PepG group, mtDNA copy number and ATP were significantly higher in PHBLV-UCP2 group, suggesting that UCP2-overexpression can maintain the function of mitochondrial energy synthesis and mitochondrial biogenesis.CONCLUSIONUCP2 plays a protective role in myocardial mitochondrial damage under septic condiction. The possible mechanisms were:UCP2 can regulate mitochondrial membrane potential through uncoupling effect, change the amount and velocity of mitochondrial ROS synthesis, maintain mitochondrial ATP synthesis and mitochondrial biogenesis function, and ultimately protect the mitochondrial morphology and function in septic condiction.