UCP2 silencing aggravates mitochondrial dysfunction in astrocytes under septic conditions.

Uncoupling protein 2 (UCP2) plays a positive role in sepsis. However, the role of UCP2 in experimental sepsis in astrocytes remains unknown. The present study was designed to determine whether UCP2 has a protective effect in an experimental sepsis model in astrocytes asnd to clarify the mechanisms responsible for its neuroprotective effects after sepsis. An experimental astrocyte model mimicking sepsis­induced brain injury was established using lipopolysaccharide (LPS) and interferon (IFN)­Î³. Additionally, UCP2 knockdown in astrocytes was achieved by adenovirus transfection. Tumor necrosis factor (TNF)­α and interleukin (IL)­1ß activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed. The mitochondrial ultrastructure was evaluated, and the expression of UCP2 was determined by western blotting. LPS with IFN­Î³ co­stimulation increased the mRNA and protein expression levels of UCP2 in astrocytes, damaged the mitochondrial structure, and accelerated the release of TNF­α and IL­1ß, resulting in a decrease in the MMP, and the excessive generation of ROS. Moreover, sepsis also caused a reduction in ATP production. The knockdown of UCP2 exacerbated astrocyte injury and mitochondrial impairment. In conclusion, both the function and morphology of mitochondria were damaged in an experimental model of sepsis in astrocytes, and knockdown of UCP2 using shRNA exacerbated this impairment, suggesting that UCP2 has a positive effect on astrocytes as determined in an experimental sepsis model.

Upregulation of UCP2 Expression Protects against LPS-Induced Oxidative Stress and Apoptosis in Cardiomyocytes.

Uncoupling protein 2 (UCP2) has a cardioprotective role under septic conditions, but the underlying mechanism remains unclear. This study aimed at investigating the effects of UCP2 on the oxidative stress and apoptosis of cardiomyocytes induced by lipopolysaccharide (LPS). First, LPS increased UCP2 expression in cardiomyocytes in a time-dependent manner. LPS increased the production of lactate dehydrogenase (LDH), reactive oxygen species (ROS), and malondialdehyde (MDA) and decreased the level of superoxide dismutase (SOD). However, UCP2 knockdown increased the LPS-induced cardiac injury and oxidative stress. In addition, LPS damaged the mitochondrial ultrastructure and led to the disruption of mitochondrial membrane potential (MMP), as well as the release of mitochondrial cytochrome c. UCP2 knockdown aggravated mitochondrial injury and the release of mitochondrial cytochrome c. LPS increased the protein levels of Bax and cleaved-caspase-3, decreased the protein level of Bcl-2, and upregulated the protein level of mitogen-activated protein kinase. However, upon UCP2 knockdown, the protein levels of Bax and cleaved-caspase-3 increased even further, and the protein level of Bcl-2 was further decreased. The protein level of phosphorylated p38 was also further enhanced. Thus, UCP2 protects against LPS-induced oxidative stress and apoptosis in cardiomyocytes.

UCP2 ameliorates mitochondrial dysfunction, inflammation, and oxidative stress in lipopolysaccharide-induced acute kidney injury.

OBJECTIVE: UCP2 is involved in the maintenance of mitochondrial function, immune response and regulation of oxidative stress under physiological or pathological conditions. The aim of this study was to investigate the effects of UCP2 on mitochondrial dysfunction, inflammation, and oxidative stress in septic acute kidney injury (AKI). METHODS: We established LPS-induced AKI model in mice and HK-2 cells. In vivo, the UCP2 inhibitor genipin was used to downregulate UCP2 in mouse kidneys. In vitro, UCP2 overexpression or knockdown was achieved by LV5-UCP2 or si-UCP2 transfection, respectively, to characterize the mechanisms of UCP2 in septic AKI. Indicators of renal injury, cell apoptosis, inflammation, oxidative stress, and mitochondrial dysfunction were assessed. RESULTS: Compared to the control group, LPS treatment increased UCP2 expression in vitro and in vivo. In vitro, UCP2 overexpression protected HK-2 cells from LPS-induced injury by suppression of apoptosis, inflammation, oxidative stress, MMP loss and ROS production, increase of ATP production and mtDNA content, and amelioration of damage to the mitochondrial ultrastructure. Additionally, inhibition of UCP2 expression by si-UCP2 resulted in decreased HK-2 cell resistance to LPS toxicity, as shown by increased apoptosis, inflammation, mitochondrial dysfunction and oxidative stress. In vivo, UCP2 downregulation aggravated the LPS-induced renal injury, inflammation, macrophages infiltration, mitochondrial dysfunction, and oxidative stress. CONCLUSION: UCP2 may protect LPS-induced AKI by ameliorating mitochondrial dysfunction, anti-inflammation, and antioxidative activities, ultimately inhibiting tubule epithelial cell apoptosis, and that increasing the UCP2 content in mitochondria constitutes a new therapeutic approach for septic AKI.

Effects of interleukin-6 and IL-6/AMPK signaling pathway on mitochondrial biogenesis and astrocytes viability under experimental septic condition.

OBJECTIVE: Interleukin-6 (IL-6) is a neuromodulation factor with extensive and complex biological activities. IL-6 has been reported to activate AMPK, while AMPK regulates mitochondrial biogenesis and autophagy. The aim of this study was to investigate the role of IL-6 in mitochondrial biogenesis using astrocytes under experimental septic condition and examined how IL-6/AMPK signaling pathway affected this process. METHODS: The primary cultures of cerebral cortical astrocytes were randomly allocated into six groups: control group, LPS+IFN-γ group, IL-6 group (LPS+IFN-γ+IL-6), C group (LPS+IFN-γ+IL-6+Compound C), siRNA group (LPS+IFN-γ+IL-6+IL-6R siRNA) and siRNA+C group (LPS+IFN-γ+IL-6+IL-6R siRNA+ Compound C). All groups were stimulated for 6 h. Cytokines and reactive oxygen species (ROS) analyses, detection of adenosine triphosphate (ATP), mtDNA content and cell viability, evaluation of the mitochondrial ultrastructure and volume density, western blots of proteins associated with mitochondrial biogenesis and phospho-adenosine monophosphate activated protein kinase (p-AMPK) were performed respectively. RESULTS: Compared with LPS+IFN-γ group, IL-6 group had milder ultrastructural damage of mitochondria, higher mtDNA content and mitochondrial volume density, higher expression of proteins associated with mitochondrial biogenesis (PGC-1α, NRF-1 and TFAM) and p-AMPK, and thus higher cell viability, whereas blocking IL-6/AMPK signaling pathway, the protective effect of IL-6 has been diminished, compared with IL-6 group. CONCLUSION: IL-6 enhances mitochondrial biogenesis in astrocytes under experimental septic condition through IL-6/AMPK signaling pathway.

[Correlation between uncoupling protein 2 expression and myocardial mitochondrial injury in rats with sepsis induced by lipopolysaccharide].

OBJECTIVE: To investigate the correlation between uncoupling protein 2 (UCP2) expression and myocardial mitochondria injury in rats with sepsis induced by lipopolysaccharide (LPS). METHODS: The rat model of sepsis was established through an intraperitoneal injection of LPS. Forty male Sprague-Dawley rats were randomly and equally divided into control group (an intraperitoneal injection of normal saline), sepsis 6 h group (LPS-6 h group), sepsis 12 h group (LPS-12 h group), sepsis 24 h group (LPS-24 h group), and sepsis 48 h group (LPS-48 h group). The serum and heart tissues were harvested at corresponding time points and myocardial mitochondria was extracted. The microplate reader was applied to measure creatine kinase (CK), creatine kinase-MB (CK-MB), and reactive oxygen species (ROS). Flow cytometry was applied to measure the degree of mitochondrial swelling and mitochondrial membrane potential (MMP). Western blot was used to measure the expression level of UCP2. Electron microscopy was applied to observe the morphological changes in heart tissues and myocardial mitochondria. RESULTS: Compared with the control group, the LPS groups had significantly increased serum levels of CK, CK-MB, and myocardial ROS, as well as a significantly increased degree of mitochondrial swelling (P<0.05), and these values reached their peaks at 24 hours after LPS injection. The LPS groups had a significant decrease in MMP (P<0.05), which reached the lowest level at 24 hours after LPS injection. Western blot showed that the LPS groups had a significant increase in the expression level of myocardial UCP2 compared with the control group (P<0.05), which reached its peak at 24 hours after LPS injection. The results of electron microscopy showed mitochondrial swelling, partial rupture of the mitochondrial membrane, and cavity formation in rats in the LPS groups. The most severe lesions occurred in the LPS-24 h group. In rats with LPS, the ROS level in the myocardial mitochondria and the degree of mitochondrial swelling were positively correlated with the expression level of UCP2 (r=0.796 and 0.893, respectively; P<0.05), while MMP was negatively correlated with the expression level of UCP2 (r=-0.903, P<0.05). CONCLUSIONS: In the rat model of sepsis, the myocardium and myocardial mitochondria have obvious injuries, and the expression level of UCP2 is closely correlated with mitochondrial injury. Therefore, UCP2 might play an important role in myocardial mitochondrial injury in sepsis.

[A preliminary study of long-term mitochondrial dysfunction in rat brain caused by lipopolysaccharide-induced sepsis].

OBJECTIVE: To preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis. METHODS: Wistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria. RESULTS: The sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria. CONCLUSIONS: In the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.

Sepsis-induced brain mitochondrial dysfunction is associated with altered mitochondrial Src and PTP1B levels.

Sepsis-induced brain dysfunction (SIBD) is often the first manifestation of sepsis, and its pathogenesis is associated with mitochondrial dysfunction. In this study, we investigated the roles of the tyrosine kinase Src and protein tyrosine phosphatase 1B (PTP1B) in brain mitochondrial dysfunction using a rat model of lipopolysaccharide (LPS)-induced sepsis. We found that there was a gradual and significant increase of PTP1B levels in the rat brain after sepsis induction. In contrast, brain Src levels were reduced in parallel with the PTP1B increase. Sepsis led to significantly reduced tyrosine phosphorylation of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III. Pretreatment of mitochondrial proteins with active PTP1B significantly inhibited complexes I and III activities in vitro, whereas Src enhanced complexes I, II, and III activities. PTP1B and Src were each co-immunoprecipitated with OXPHOS complexes I and III, suggesting direct interactions between both proteins and complexes I and III. Src also directly interacted with complex II. Furthermore, pretreatment of mitochondrial proteins with active PTP1B resulted in overproduction of reactive oxygen species and decreased mitochondrial membrane potential. Pretreatment with active Src produced the opposite effect. These results suggest that brain mitochondrial dysfunction following LPS-induced sepsis in rats is partly attributed to PTP1B and Src mediated decrease in mitochondrial protein tyrosine phosphorylation.

Silencing of uncoupling protein 2 by small interfering RNA aggravates mitochondrial dysfunction in cardiomyocytes under septic conditions.

Uncoupling protein 2 (UCP2) regulates the production of mitochondrial reactive oxygen species (ROS) and cellular energy transduction under physiological or pathological conditions. In this study, we aimed to determine whether mitochondrial UCP2 plays a protective role in cardiomyocytes under septic conditions. In order to mimic the septic condition, rat embryonic cardiomyoblast-derived H9C2 cells were cultured in the presence of lipopolysaccharide (LPS) plus peptidoglycan G (PepG) and small interfering RNA (siRNA) against UCP2 (siUCP2) was used to suppress UCP2 expression. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR), western blot analysis, transmission electron microscopy (TEM), confocal microscopy and flow cytometry (FCM) were used to detect the mRNA levels, protein levels, mitochondrial morphology and mitochondrial membrane potential (MMP or ΔΨm) in qualitative and quantitative analyses, respectively. Indicators of cell damage [lactate dehydrogenase (LDH), creatine kinase (CK), interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the culture supernatant] and mitochondrial function [ROS, adenosine triphosphate (ATP) and mitochondrial DNA (mtDNA)] were detected. Sepsis enhanced the mRNA and protein expression of UCP2 in the H9C2 cells, damaged the mitochondrial ultrastructure, increased the forward scatter (FSC)/side scatter (SSC) ratio, increased the CK, LDH, TNF-α and IL-6 levels, and lead to the dissipation of MMP, as well as the overproduction of ROS; in addition, the induction of sepsis led to a decrease in ATP levels and the deletion of mtDNA. The silencing of UCP2 aggravated H9C2 cell damage and mitochondrial dysfunction. In conclusion, our data demonstrate that mitochondrial morphology and funtion are damaged in cardiomyocytes under septic conditions, while the silencing of UCP2 using siRNA aggravated this process, indicating that UCP2 may play a protective role in cardiomyocytes under septic conditions.

[Effect of autophagy and mitochondrial coenzyme Q on exocrine function of pancreas in rats with acute sepsis].

OBJECTIVE: To investigate the effects of autophagy on exocrine function of pancreas in rats with acute sepsis, and to determine whether the mitochondrial coenzyme Q (Mito Q) can prevent exocrine dysfunction of pancreas mediated by autophagy. METHODS: Experiment I: 30 Sprague-Dawley (SD) rats were randomly divided into three groups, with 10 rats in each group. All the rats were given lipopolysaccharide (LPS, 10 mg/kg) intraperitoneally, and Wortmannin (2 mg/kg), the specific inhibitor of autophagy (LPS + Wortmannin group), Mito Q (6.5 µmol/kg, LPS + Mito Q group), or the same volume of normal saline (LPS group) was respectively injected via the tail vein 1 hour later. Survival rate was assessed within 12 hours after LPS injection. Experiment II: another 100 male SD rats were randomly divided into ten groups with 10 rats in each group: namely control 4, 6 and 12 hours groups, LPS 4, 6 and 12 hours groups, and LPS + Wortmannin 4 hours group, Wortmannin 4 hours group, LPS + Mito Q 6 hours group, and Mito Q 6 hours group. The protocols of model reproduction and drug administration were the same as in the experiment I. Blood samples were collected at each time point, and the amylase content was determined with the velocity method. The levels of reactive oxygen species (ROS) in the pancreases were measured with enzyme-linked immunosorbent assay (ELISA). The expression of the autophagy-related protein LC3 was determined with Western Blot. The pathological changes in the pancreas were observed with microscopy. RESULTS: (1) The survival time in the LPS + Wortmannin group was significantly shorter than that in the LPS group (hours: 7.50±0.64 vs. 11.90±0.13, χ (2) = 19.847, P = 0.001). There was no significant difference in the survival time between LPS + Mito Q and LPS groups (hours: 11.60±0.24 vs. 11.90±0.13, χ (2) = 1.055, P = 0.137). (2) The serum amylase in the LPS 6 hours, LPS + Wortmannin 4 hours, and LPS + Mito Q 6 hours groups were significantly higher than those in the control group at the same time points (U/L: 2 881.00±550.12 vs. 2 099.20±249.57, 3 672.00±779.24 vs. 2 081.36±245.18, 2 975.20±687.03 vs. 2 099.20±249.57, all P < 0.05), and were significantly lowered in LPS 12 hours group (U/L: 794.00±218.71 vs. 2 086.80±261.75, P < 0.01). The pancreatic ROS in the LPS 6 hours and 12 hours groups, LPS + Wortmannin 4 hours group, and LPS + Mito Q 6 hours group were significantly higher than those of the control group at the same time points (kU/L: 3.18±1.06 vs. 1.78±0.37, 3.63±1.08 vs. 1.85±0.41, 3.14±0.98 vs. 1.65±0.34, 3.17±1.03 vs. 1.78±0.37, all P < 0.05). The serum amylase and pancreatic ROS in LPS + Wortmannin 4 hours group were significantly higher than those of the LPS group at the same time points (U/L: 3 672.00±779.24 vs. 2 432.20±442.85, kU/L: 3.14±0.98 vs. 1.87±0.42, both P < 0.05), but there were no differences in above two parameters between LPS + Mito Q 6 hours group and LPS group (U/L: 2 975.20±687.03 vs. 2 881.00±550.12, kU/L: 3.17±1.03 vs. 3.18±1.06, both P > 0.05). Light microscopy showed that obvious pathological changes were found in the pancreas in the LPS 6 hours and 12 hours groups, LPS + Wortmannin 4 hours group, and LPS + Mito Q 6 hours group. Electron microscopy showed that the number of autophagic vacuoles increased 6 hours after LPS administration. There was no difference at any time point in the number of autophagic vacuoles between LPS + Mito Q 6 hours group and LPS 6 hours group, and the autophagic vacuoles were not found after Wortmannin intervention. It was demonstrated by Western Blot that the levels of LC3 protein in the LPS 6 hours and 12 hours groups, and LPS + Mito Q 6 hours group were significantly higher than those of the control group at the same time points (A value: 0.34±0.02 vs. 0.17±0.02, 0.37±0.03 vs. 0.18±0.04, 0.36±0.02 vs. 0.17±0.02, all P < 0.05), but there were no differences between LPS 12 hours group or LPS + Mito Q 6 hours group and LPS 6 hours group (both P > 0.05). CONCLUSIONS: Autophagy prevents exocrine dysfunction of pancreas in septic rats, and the autophagic capacity or autophagosome-formation rate may determine the development of exocrine pancreatic dysfunction. The mitochondria-targeted antioxidant Mito Q does not prevent exocrine dysfunction of pancreas.