This study aimed to explore the mechanism of resveratrol(RES) pretreatment in improving mitochondrial function and alleviating myocardial ischemia-reperfusion(IR) injury by inhibiting stromal interaction molecule 2(STIM2) through microRNA-20 b-5 p(miR-20 b-5 p). Ninety rats were randomly assigned into sham group, IR group, IR+RES(50 mg·kg~(-1) RES) group, IR+RES+antagomir NC(50 mg·kg~(-1) RES+80 mg·kg~(-1) antagomir NC) group, and IR+RES+miR-20 b-5 p antagomir(50 mg·kg~(-1) RES+80 mg·kg~(-1) miR-20 b-5 p antagomir) group, with 18 rats/group. The IR rat model was established by ligation of the left anterior descending coronary artery. Two weeks before the operation, rats in the IR+RES group were intraperitoneally injected with 50 mg·kg~(-1) RES, and those in the sham and IR groups were injected with the same dose of normal saline, once a day. Ultrasonic instrument was used to detect the left ventricular internal diameter at end-diastole(LVIDd) and left ventricular internal diameter at end-systole(LVIDs) of rats in each group. The 2,3,5-triphenyte-trazoliumchloride(TTC) method and hematoxylin-eosin(HE) staining were employed to detect the myocardial infarction area and histopathology, respectively. Real-time quantitative PCR(qRT-PCR) was carried out to detect the expression of miR-20 b-5 p in myocardial tissue. Oxygen glucose deprivation/reoxygenation(OGD/R) was performed to establish an OGD/R model of H9 c2 cardiomyocytes. CCK-8 assay was employed to detect H9 c2 cell viability. H9 c2 cells were assigned into the control group, OGD/R group, OGD/R+RES group(25 μmol·L~(-1)), OGD/R+RES+inhibitor NC group, OGD/R+RES+miR-20 b-5 p inhibitor group, mimic NC group, miR-20 b-5 p mimic group, inhibitor NC group, and miR-20 b-5 p inhibitor group. Flow cytometry was employed to detect cell apoptosis. Western blot was employed to detect the expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-cysteine proteinase 3(cleaved-caspase-3), and STIM2 in cells. The mitochondrial membrane potential(MMP) assay kit, reactive oxygen species(ROS) assay kit, and adenosine triphosphate(ATP) assay kit were used to detect the MMP, ROS, and ATP levels, respectively. Dual luciferase reporter gene assay was adopted to verify the targeting relationship between miR-20 b-5 p and STIM2. Compared with the sham group, the modeling of IR increased the myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and down-regulated the expression of miR-20 b-5 p(P<0.05). These changes were alleviated in the IR+RES group(P<0.05). The IR+RES+miR-20 b-5 p antagomir group had higher myocardial infarction area, LVIDd, LVIDs, and myocardial pathology and lower expression of miR-20 b-5 p than the IR+RES group(P<0.05). The OGD/R group had lower viability of H9 c2 cells than the control group(P<0.05) and the OGD/R+RES groups(25, 50, and 100 μmol·L~(-1))(P<0.05). Additionally, the OGD/R group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved caspase-3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the control group(P<0.05) and the OGD/R+RES group(P<0.05). The OGD/R+RES+miR-20 b-5 p inhibitor group had higher H9 c2 cell apoptosis rate, protein levels of Bax and cleaved-caspase 3, and ROS level and lower Bcl-2 protein, MMP, and ATP levels than the OGD/R+RES group(P<0.05). miR-20 b-5 p had a targeting relationship with STIM2. The expression of STIM2 was lower in the miR-20 b-5 p mimic group than in the mimic NC group(P<0.05) and lower in the inhibitor NC group than in the miR-20 b-5 p inhibitor group(P<0.05). RES pretreatment can inhibit the expression of STIM2 by promoting the expression of miR-20 b-5 p, thereby improving the function of mitochondria and alleviating myocardial IR damage.
Animals ; Rats ; Adenosine Triphosphate ; Antagomirs/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Glucose/metabolism* ; MicroRNAs/metabolism* ; Mitochondria, Heart/drug effects* ; Myocardial Infarction/drug therapy* ; Myocardial Reperfusion Injury/drug therapy* ; Myocytes, Cardiac ; Oxygen/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism* ; Resveratrol/therapeutic use* ; Stromal Interaction Molecule 2/metabolism*

The organosulfur compound sulforaphane (SFN; C₆H₁₁NOS₂) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
Animals ; Antioxidants/pharmacology* ; Apoptosis/drug effects* ; Brain/ultrastructure* ; Carbon Monoxide Poisoning/metabolism* ; Cytoprotection ; Humans ; Isothiocyanates/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Mitochondria/metabolism* ; Sulfoxides

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

Atractylenolide III (ATL-III), a sesquiterpene compound isolated from Rhizoma Atractylodis Macrocephalae, has revealed a number of pharmacological properties including anti-inflammatory, anti-cancer activity, and neuroprotective effect. This study aimed to evaluate the cytoprotective efficiency and potential mechanisms of ATL-III on corticosterone injured rat phaeochromocytoma (PC12) cells. Our results demonstrate that ATL-III increases cell viability and reduces the release of lactate dehydrogenase (LDH). The results suggest that ATL-III protects PC12 cells from corticosterone-induced injury by inhibiting the intracellular Ca overloading, inhibiting the mitochondrial apoptotic pathway and modulating the MAPK/NF-ΚB inflammatory pathways. These findings provide a novel insight into the molecular mechanism by which ATL-III protected the PC12 cells against corticosterone-induced injury for the first time. Our results provide the evidence that ATL-III may serve as a therapeutic agent in the treatment of depression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Survival ; drug effects ; Corticosterone ; toxicity ; Inflammation Mediators ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactones ; pharmacology ; Mitochondria ; drug effects ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; NF-kappa B ; metabolism ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Phosphorylation ; drug effects ; Rats ; Sesquiterpenes ; pharmacology ; Signal Transduction ; drug effects

Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.
Adult ; Aged ; Aged, 80 and over ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; therapeutic use ; Cofilin 1 ; metabolism ; Drug Resistance, Neoplasm ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Mitochondria ; drug effects ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; pathology ; Tumor Cells, Cultured

Ageratina adenophora is a noxious plant and it is known to cause acute asthma, diarrhea, depilation, and even death in livestock (Zhu et al., 2007; Wang et al., 2017). A. adenophora grows near roadsides and degraded land worldwide (He et al., 2015b). In the areas where it grows, A. adenophora is an invasive species that inhibits the growth of local plants and causes poisoning in animals that come in contact with it (Nie et al., 2012). In China, these plants can be found in Yunnan, Sichuan, Guizhou, Chongqing, and other southwestern areas (He et al., 2015a) and they have become a dominant species in these local regions. It threatens the native biodiversity and ecosystem in the invaded areas and causes serious economic losses (Wang et al., 2017). It has been reported that A. adenophora can grow in the northeast direction at a speed of 20 km per year in China (Guo et al., 2009). Because of the damage caused by A. adenophora, it ranks among the earliest alien invasive plant species in China (Wang et al., 2017).
Adenosine Triphosphatases/metabolism* ; Ageratina/toxicity* ; Animals ; Biodiversity ; Chemical and Drug Induced Liver Injury/pathology* ; China ; DNA, Mitochondrial/genetics* ; Ecosystem ; Introduced Species ; Liver/drug effects* ; Mice ; Microscopy, Electron, Transmission ; Mitochondria, Liver/pathology* ; Plant Extracts/toxicity*

ObjectiveTo investigate the effects of Zhibai Dihuang Decoction (ZDD) on sperm mitochondrial permeability transition (MPT) in the rat model of ureaplasma urealyticum (UU) infection (UUI).

METHODSNinety male SD rats were randomly divide into five groups: normal control, UUI model control, ZDD, doxycycline, and ZDD + doxycycline. The UUI model was established in the latter four groups of rats by UU injection into the bladder. On the second day after modeling, the animals of the normal control and UUI model control groups were treated intragastrically with 0.9% sodium chloride solution and those in the other groups with corresponding drugs, all for 21 consecutive days. At 24 hours after drug withdrawal, epididymal samples were obtained for detection of the protein and mRNA expressions of VDAC2 and ANT4 in the sperm mitochondria by RT-PCR and Western blot respectively and determination of the contents of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and energy charge (EC) in the sperm mitochondria by high-performance liquid chromatography.

RESULTSThe protein expressions of VDAC2 and ANT4 in the rat sperm mitochondria were 0.626 ± 0.074 and 0.527 ± 0.096 in the normal control group, 0.039 ± 0.011 and 0.044 ± 0.011 in the UUI model control group, 0.101 ± 0.037 and 0.127 ± 0.040 in the ZDD group, 0.236 ± 0.070 and 0.253 ± 0.054 in the doxycycline group, and 0.475 ± 0.064 and 0.367 ± 0.086 in the ZDD + doxycycline group, significantly lower in the UUI model control than in the normal control group (P<0.05 and P<0.01), but remarkably higher in the doxycycline and ZDD + doxycycline groups than in the UUI model control (P<0.01) and the ZDD group (P<0.05 and P<0.01), and the expression of VDAC2 was markedly higher in the ZDD + doxycycline than in the doxycycline group (P<0.01). The mRNA expressions of VDAC2 and ANT4 were 0.008 ± 0.001 035 and 0.026 50 ± 0.003 401 in the normal control group, 0.000 79 ± 0.000 226 and 0.001 64 ± 0.000 205 in the UUI model controls, 0.002 06 ± 0.000 861 and 0.005 04 ± 0.002 537 in the ZDD group, 0.003 34 ± 0.000 229 and 0.008 57 ± 0. 000 690 in the doxycycline group, and 0.004 85 ± 0.000 495 and 0.013 13 ± 0.000 826 in the ZDD + doxycycline group, significantly lower in the UUI model control than in the normal control group (P<0.05 and P<0.01), but remarkably higher in the ZDD, doxycycline and ZDD + doxycycline groups than in the UUI model controls (P<0.01) as well as in the doxycycline and ZDD + doxycycline groups than in the ZDD group (P<0.01) and in the ZDD + doxycycline than in the doxycycline group (P<0.01). The levels of ATP, ADP, AMP and EC in the sperm mitochondria were (203.41 ± 13.16) mg/L, (129.87 ± 14.68) mg/L, (149.05 ± 5.65) mg/L and 0.56 ± 0.01 in the normal control group, (96.22 ± 12.55) mg/L, (99.87 ± 3.28) mg/L, (212.53 ± 19. 43) mg/L and 0.36 ± 0.03 in the UUI model control group, (101.99 ± 5.97) mg/L, (104.99 ± 16.40) mg/L, (183.97 ± 12.43) mg/L and 0.40 ± 0.01 in the ZDD group, (159.44 ± 33.16) mg/L, (118.51 ± 12.99) mg/L, (160.64 ± 14.19) mg/L and 0.50 ± 0.06 in the doxycycline group, and (194.07 ± 9.36) mg/L, (121.62 ± 9.41) mg/L, (150.21 ± 12.87) mg/L and 0.55 ± 0.01 in the ZDD + doxycycline group. The levels of ATP and EC were significantly lower and that of AMP higher in the UUI model control than in the normal control group (P<0.01), while the former two were remarkably higher and the latter one lower in the doxycycline and ZDD + doxycycline groups than in the UUI model controls (P<0.05 and P<0.01). Compared with the ZDD + doxycycline group, the ZDD group showed significantly decreased ATP and EC but increased AMP, while the doxycycline group exhibited decreases in both ATP and EC (P<0.05 and P<0.01).

CONCLUSIONSZDD can upregulate the decreased protein and mRNA expressions of VDAC2 and ANT4 in the sperm mitochondria and improve sperm mitochondrial permeability transition and mitochondrial energy metabolism in rats with UU infection, which may be one of its action mechanisms in the treatment of UU infection-induced male infertility.

Animals ; Anti-Bacterial Agents ; therapeutic use ; Doxycycline ; therapeutic use ; Drugs, Chinese Herbal ; metabolism ; therapeutic use ; Energy Metabolism ; Epididymis ; Humans ; Infertility, Male ; Male ; Mitochondria ; drug effects ; Permeability ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatozoa ; drug effects ; Ureaplasma Infections ; drug therapy ; Ureaplasma urealyticum ; Voltage-Dependent Anion Channel 2 ; metabolism

ObjectiveTo explore the antagonistic effect of vitamin E (VE) on male reproductive toxicity induced by di-2-ethylhexyl phthalate (DEHP) in pubertal SD rats and its underlying mechanisms.

METHODSThirty 5-week-old male SD rats were randomly divided into five groups of equal number, corn oil control, low-dose (10 mg/kg/d), medium-dose (100 mg/kg/d) and high-dose DEHP exposure (500 mg/kg/d), and VE intervention (high-dose DEHP + VE ［100 mg/kg/d］), and treated respectively for 30 successive days. At 3 days after treatment, the testes of the animals were harvested for determination of the oxidative stress index, serum reproductive hormone levels, cauda epididymal sperm parameters, and expressions of cell apoptosis-related genes and proteins.

RESULTSCompared with the control group, the rats of the medium- and high-dose DEHP groups showed significant decreases in the levels of such serum reproductive hormones as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T), sperm parameters as average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), linearity (LIN) and wobble (WOB), and the activities of superoxide dismutase (SOD) and glutathione peroxide (GSH-Px), but significant increases were observed in the latter two groups in the content of malondialdehyde (MDA)(［3.32±0.87］ nmol/mg pro vs ［2.13±0.49］ nmol/ mg pro), mRNA expressions of Bad, Bax, Cytochrome C, Caspase-3 and the Bax/Bcl-2 ratio, and protein expressions of Cytochrome C and Caspase-3. In comparison with the high-dose DEHP group, the VE intervention group exhibited remarkably increased serum LH and T levels, sperm VAP, VSL, VCL, STR and WOB, and activities of SOD and GSH-Px, but markedly decreased mRNA expressions of Bad, Bax, Cytochrome C, Caspase-3 and the Bax/Bcl-2 ratio as well as the protein expressions of Cytochrome C and Caspase-3 in the testis tissue (P<0.05).

CONCLUSIONSExposure to DEHP induces androgen secretion disorders, causes oxidative damage to the testicular tissue, activates the mitochondrial apoptosis pathway in the testis, and ultimately reduces the quality of epididymal sperm, while VE can protect the rat testis from DEHP-induced reproductive toxicity.

Animals ; Antioxidants ; pharmacology ; Apoptosis ; genetics ; Autophagy-Related Protein 5 ; metabolism ; Caspase 3 ; metabolism ; Diethylhexyl Phthalate ; antagonists & inhibitors ; Epididymis ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Malondialdehyde ; metabolism ; Mitochondria ; drug effects ; Oxidative Stress ; drug effects ; Oxidoreductases ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reproduction ; Spermatozoa ; drug effects ; physiology ; Superoxide Dismutase ; metabolism ; Testis ; drug effects ; Testosterone ; blood ; Vitamin E ; pharmacology

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

To explore the effects of honokiol (HKL) on pulmonary microvascular endothelial cells in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) and the underlying mechanisms.
 Methods: In animal experiment, a total of 40 C57BL/6J mice were randomly divided into a control group (Con group), a LPS intervention group (LPS group), a LPS+honokiol (HKL) intervention group (HKL group) and a LPS+HKL+nicotinamide (NAM) intervention group (NAM group) (n=10 in each group). In the cell experiment, the experiment cells were divided into a control group (Con group), a LPS intervention group (LPS group), a LPS+HKL intervention group (HKL group), a LPS+HKL+NAM intervention group (NAM group), and a LPS+HKL+compound C (CMC) intervention group (CMC group). The pathological changes of the lung tissues were evaluated by hematoxylin and eosin (HE) staining; the protein concentration, total cells and neutrophils in the bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung tissues were detected; the changes of pulmonary microvascular permeability were determined by Evans blue assay; the effect of HKL on the vitality of human pulmonary microvascular endothelial cells were examined by cell counting kit-8 (CCK-8); the inhibitors including NAM and CMC were applied to explore the molecular mechanism of the protective effects of HKL. The expression levels of Sirt3, caspase-3, cleaved caspase-3, Bcl-2, Bax, p-adenosine monophosphate activated protein kinase (p-AMPK) and AMPK in lung tissues or cells were detected by Western blot.
 Results: In animal models, compared with the Con group, the mice in the LPS group displayed typical ARDS pathological changes, and the ratio of lung wet/dry weight (W/D) and MPO activity in the lung tissues, protein concentration, total cells and neutrophils in BALF, Evans blue leaking index (ELI), expression levels of cleaved caspase-3 were significantly increased (all P<0.05), while the expression levels of Sirt3 was obviously decreased (P<0.05). Compared with the LPS group, the above changes in the LPS group were significantly improved in the HKL group (all P<0.05); Compared with the HKL group, the curative effect of HKL intervention could be partly inhibited in the NAM group (P<0.05). In cell experiments, compared with the LPS group, the HPMECs viability in the HKL group was markedly improved (P<0.05), while the expression levels of Bcl-2 and Sirt3 were significantly upregulated (P<0.05), and the expression levels of Bax and cleaved caspase-3 were significantly downregulated (P<0.05), accompanied by the activation of AMPK pathway (P<0.05) in the HKL group. Compared with the HKL group, the curative effect of HKL intervention was partly inhibited in the CMC group (P<0.05).
 Conclusion: HKL can significantly attenuate LPS-induced lung injury and inhibit the apoptosis of pulmonary microvascular endothelial cells through regulation of Sirt3/AMPK pathway.
AMP-Activated Protein Kinases ; metabolism ; Acute Lung Injury ; chemically induced ; drug therapy ; Animals ; Biphenyl Compounds ; pharmacology ; therapeutic use ; Humans ; Lignans ; pharmacology ; therapeutic use ; Lipopolysaccharides ; Lung ; Mice ; Mice, Inbred C57BL ; Mitochondria ; drug effects ; metabolism ; Signal Transduction ; drug effects ; Sirtuin 3 ; metabolism