Jatonik polyherbal mixture induced rat liver MMPT pore opening in normal Wistar rat: In vitro and in vivo studies / 中草药·英文版

OBJECTIVE@#To assess acute toxicity, the in vitro and in vivo effects of methanol and ethyl acetate extracts (JME and JEE) of Jatonik polyherbal mixture on some mitochondria-related parameters and their effect on the activity of some liver enzymes.@*METHODS@#Acute toxicity of JME and JEE was determined using Lorke's method. In vitro and in vivo opening of the mitochondrial membrane permeability transition pore (MMPT pore) was spectrophotometrically assayed. Production of malondialdehyde (MDA) as an index of lipid peroxidation and the activity of mitochondrial ATPase was evaluated in vitro and in vivo and the effect of JME and JEE on the activity of liver enzymes such as alkaline phosphatase (ALP), aspartate and alanine aminotransferase (AST and ALT) and gamma-glutamyl transferase (GGT) was also investigated.@*RESULTS@#JME had an LD50 of 3 808 mg/kg b.w whereas JEE had an LD50 greater than 5 000 mg/kg b.w. of rats. After the rats have been fed with both extracts, a photomicrograph of a piece of liver tissue showed no apparent symptoms of toxicity. From the in vitro and in vivo studies, both extracts prompted intact mitochondria to open their MMPT pores. When compared to the control, lipid peroxide product release and ATPase activity were significantly increased (P < 0.05) in vitro and in vivo. The activities of AST, ALT, and GGT were all reduced at 50 mg/kg when treated with JME, but the activity of AST was considerably enhanced when treated with JEE (P < 0.05). The results revealed that both JME and JEE of the Jatonik polyherbal mixture had low toxicity, profound MMPTpore induction, and enhanced ATPase activity, but an increased MDA production.@*CONCLUSION@#Jatonik extracts may be a promising target for drug development in diseases where there is dysregulation of apoptosis, however, further studies are needed to better clarify the molecular mechanism involved in these phenomena.

Role of CypD-mediated mPTP in melatonin improving cognitive impairment induced by repeated exposure to sevoflurane / 中国药理学通报

Aim To investigate the involvement of cy-clophilin D ( CypD ) -mediated mitochondrial permeability transition pore ( mPTP) in the neuroprotective effects of melatonin on cognitive impairment induced by repeated exposure to sevoflurane in newborn animals. Methods Mice were randomly assigned into control group, sevoflurane ( Sevo) group, and melatonin pre-treatment + sevoflurane ( Sevo + Mel) group. JC-1 kit was used to assess the mitochondrial membrane potential ( MMP) ; Western blot analysis was used to evaluate the protein expressions of CypD, postsynaptic density protein 95 ( PSD95 ), and Synapsin-1; and behavioral test were employed to measure cognitive function. Results The MMP level in the Sevo group was significantly reduced compared to the control group (P < 0. 01 ), the expression of CypD increased (P <0. 05), whereas the expression of PSD95 and Synapsin-1 decreased ( P < 0. 01) . Furthermore, the new object recognition index and spatial memory ability both exhibited a significant decline (P < 0. 01, P < 0. 05). However, when compared to the Sevo group, Sevo + Mel group could raise the MMP level (P <0. 01), increase the expression of synaptic proteins ( P < 0. 05 ), decrease the expression of CypD (P <0. 01) and elevate the new object recognition index and the spatial memory capacity ( P < 0. 01 ). Conclusions Melatonin could ameliorate cognitive impairment induced by repeated exposure to sevoflurane in newborn mice, and the underlying mechanism may be attributed to the inhibition of mPTP mediated by CypD and the promotion of synaptic protein synthesis.

Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells / 中国中药杂志

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Protective effect and mechanism of mesenchymal stem cell-derived extracellular vesicle on radiation-induced liver injury / 器官移植

Objective To evaluate the protective effect and the underlying mechanism of mesenchymal stem cell-derived extracellular vesicle (MSC-EV) on radiation-induced liver injury and liver cell line injury in mouse models. Methods C57BL/6 mice were randomly divided into the blank group, model group and MSC-EV treatment group (treatment group), with 9 mice in each group. AML12 cells were randomly divided into the control group, irradiation group and MSC-EV intervention group (intervention group). Animal and cell models with radiation-induced injury were established by one-time 15 Gy and 6 Gy X-ray irradiation, respectively. At 48 h after irradiation, liver tissues and serum samples of mice were collected and prepared for subsequent experiments. At 15 h post-irradiation, cell experiment was carried out. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and content of malondialdehyde (MDA) in liver tissues and cells were measured. The relative expression levels of interleukin (IL)-1β, IL-6, transforming growth factor (TGF)-β and CXC chemokine ligand (CXCL)10 messenger RNA (mRNA) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). Liver tissues were prepared for hematoxylin-eosin (HE) staining to calculate liver pathological injury score. The apoptosis of liver tissues and cells was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and propidiumiodide (PI) staining, respectively. The expression levels of glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1) proteins were detected by Western blot. The production level of reactive oxygen species (ROS) was detected by dihydroethidine (DHE) staining. The fluorescence intensity of mitochondrial permeability transition pore (mPTP) was determined. Results Compared with the blank group, serum levels of AST and ALT were up-regulated, and the relative expression levels of IL-1β, TGF-β and CXCL10 mRNA in the mouse liver tissues were up-regulated, and MDA content was increased, liver injury score was elevated, cell apoptosis rate was increased, intracellular ROS level was elevated, and the relative expression levels of GPX4 and FSP1 proteins in the mouse liver tissues were down-regulated in the model group, and the differences were statistically significant (all P<0.05). Compared with the model group, serum levels of AST and ALT were decreased, and the relative expression levels of IL-1β, TGF-β and CXCL10 mRNA in the liver tissues of mice were down-regulated, MDA content was declined, liver injury score was declined, cell apoptosis rate was decreased, intracellular ROS level was decreased, and the relative expression levels of GPX4 and FSP1 proteins in the liver tissues of mice were up-regulated in the treatment group, and the differences were statistically significant (all P<0.05). Compared with the control group, cell apoptosis rate was increased, intracellular ROS level was elevated, the fluorescence intensity of mPTP was weakened, the relative expression levels of IL-1β, TGF-β and IL-6 mRNA were up-regulated, MDA content was increased, and the relative expression levels of GPX4 and FSP1 proteins were down-regulated in the irradiation group, and the differences were statistically significant (all P<0.05). Compared with the irradiation group, cell apoptosis rate was declined, intracellular ROS level was decreased, the fluorescence intensity of mPTP was strengthened, the relative expression levels of IL-1β, TGF-β and IL-6 mRNA were down-regulated, MDA content was decreased and the relative expression levels of GPX4 and FSP1 proteins were up-regulated in the intervention group, and the differences were statistically significant (all P<0.05). Conclusions MSC-EV may effectively alleviate radiation-induced liver injury by reducing ferroptosis of liver cells, enhancing antioxidant level and decreasing the production of lipid peroxide, thereby effectively alleviating radiation-induced liver injury.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside / 中国天然药物

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

Asymmetric Opening of Mitochondrial Permeability Transition Pore in Mouse Brain Hemispheres: A Link to the Mitochondrial Calcium Uniporter Complex

Abstract The prolonged entry of large amounts of calcium into the mitochondria through the mitochondrial calcium uniporter complex (MCUC) may cause the permeability transition pore (mPTP) to open, which contributes to the pathogenesis of several diseases. Tissue-specific differences in mPTP opening due to variable expression of MCUC components may contribute to disease outcomes. We designed this study to determine differential mPTP opening in mitochondria isolated from different regions of mouse brain and kidney and to compare it with the expression of MCUC components. mPTP opening was measured using mitochondria isolated from the left/right brain hemispheres (LH/RH, respectively) and from kidney cortex/medulla, while the expression level of MCUC components was assessed from total cellular RNA. Interestingly, LH mitochondria showed less calcium-induced mPTP opening as compared to RH mitochondria at two different calcium concentrations. Conversely, mPTP opening was similar in the renal cortex and renal medulla mitochondria. However, the kidney mitochondria demonstrated bigger and faster mPTP opening as compared to the brain mitochondria. Furthermore, asymmetric mPTP opening in the LH and RH mitochondria was not associated with the expression of MCUC components. In brief, this study demonstrates thus far unreported asymmetric mPTP opening in mouse brain hemispheres that is not associated with the mRNA levels of MCUC components.

Kangxian Yixin Prescription Interferes with Mitochondrial Permeability Transition Pore to Inhibit Cardiomyocyte Apoptosis / 中国实验方剂学杂志

Objective:To explore the mechanism of Kangxian Yixin prescription in regulating mitochondrial permeability transition pore（mPTP）and inhibiting cardiomyocyte apoptosis. Method:H9c2 cardiomyocytes were cultured routinely. After 8 h of starvation，the cells were divided into the normal group，model group，Kangxian Yixin prescription（0.25 g·L^-1） group，and cyclosporin A（CsA，10 μmol·L^-1） group and treated with the corresponding drugs for 24 h for follow-up experiments. The H9c2 cardiomyocyte hypertrophy model was induced by norepinephrine（NE），whose optimal concentration was determined by real-time polymerase chain reaction （Real-time PCR）. The degree of mPTP opening was detected by flow cytometry， followed by the measurement of mRNA and protein expression levels of apoptosis-related factors cyclophilin D（Cyp-D），cytochrome C（Cyt-C），and cysteine aspartate-specific protease-3（Caspase-3） after mPTP opening and the quantification of mitochondrial membrane potential. Result:When the concentration of NE was 200 μmol·L^-1， the mRNA expression levels of atrial natriuretic peptide（ANP） and brain natriuretic peptide（BNP） were the highest， implying that it was the optimal concentration to induce H9c2 cell hypertrophy. Compared with the normal group，the model group exhibited excessive opening of mPTP，weakened relative fluorescence intensity in mitochondria， decreased mitochondrial membrane potential（<italic>P</italic><0.05，<italic>P</italic><0.01），and elevated mRNA and protein expression of Cyp-D，Cyt-C，and Caspase-3（<italic>P</italic><0.05）. Compared with the model group，both Kangxian Yixin prescription and CsA inhibited mPTP opening，enhanced the relative fluorescence intensity of mitochondria， increased mitochondrial membrane potential（<italic>P</italic><0.05，<italic>P</italic><0.01），and lowered the mRNA and protein expression of Cyp-D，Cyt-C，and Caspase-3 （<italic>P</italic><0.05）. Conclusion:Kangxian Yixin prescription inhibits cardiomyocyte apoptosis possibly by regulating mPTP opening and inhibiting the expression of apoptosis-related factors Cyp-D，Cyt-C， and Caspase-3.

The mechanism of tilianin against ischemia-reperfusion injury of H9c2 cells / 药学学报

Z-VAD-FMK was combined with hypoxia-reoxygenation (H/R) injury to establish a necroptosis model of H9c2 cells to mimic the pathological changes of myocardial ischemia reperfusion injury (MIRI) in vitro and to study the effect and mechanism of tilianin against myocardial ischemia-reperfusion injury. A cell counting kit-8 (CCK-8) was used to detect cell viability, and commercial kits were used to detect lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant. Hoechst 33342/PI immunofluorescence staining was used to detect cell death. DCFH-DA, BBcellProbeTMM61, and JC-1 probes were used to detect reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), and mitochondrial membrane potential (MMP), respectively. An enzyme-linked immunosorbent assay (ELISA) method was used to detect the release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The results show that the cell viability, SOD activity, and MMP of the model group induced by H/R injury decreased, as compared with control group, but the necroptosis rate, LDH level, and ROS release increased significantly. Furthermore, mPTP of the model group cells opened, and TNF-α, IL-1β, and IL-6 levels were significantly higher. Molecular docking modeling showed that tilianin can bind to calmodulin-dependent protein kinase II (CaMKII), and Western blot results showed that compared with control group, the expression levels of p-CaMKII and phospho-mixed lineage kinase domain-like protein increased in the model group, and tilianin could decrease the expression level of these proteins. The above results indicate that tilianin can protect H9c2 cells by inhibiting the phosphorylation of CaMKⅡ at threonine 287, protecting mitochondrial function, and inhibiting the opening of mPTP to prevent necroptosis. This study has value for research on new methods to treat H/R injury.

Shexiang Tongxin Dropping Pill () Reduces Coronary Microembolization in Rats via Regulation of Mitochondrial Permeability Transition Pore Opening and AKT-GSK3β Phosphorylation / 中国结合医学杂志

OBJECTIVE@#To investigate the protective effects of Shexiang Tongxin Dropping Pill (, STDP) following sodium laurate-induced coronary microembolization (CME) in rats.@*METHODS@#Forty rats were divided into 4 groups: the control (sham) group, CME group, low-dose STDP pretreatment group (20 mg·kg@*RESULTS@#The rats in the CME group showed a significant increase in the fibrinogen-like protein 2 expression level and mitochondrial dysfunction and a decrease in the expression level of antioxidant biomarkers (superoxide dismutase and catalase, P<0.01 for all). In contrast, the rats in the low- and high-dose STDP pretreatment groups showed a significant decrease in coronary microthrombi (P<0.05); moreover, STDP restored the antioxidant-related protein activities and mitochondrial function, inhibited mPTP opening, decreased AKT-Ser473 phosphorylation, and increased GSK3β-Ser9 phosphorylation (P<0.05 or P<0.01).@*CONCLUSION@#STDP may be useful for treatment of CME, possibly via regulation of mPTP opening and AKT/GSK3β phosphorylation.

Chrysophanol localizes in mitochondria to promote cell death through upregulation of mitochondrial cyclophilin D in HepG2 cells / 中草药·英文版

Objective: Chrysophanol (Chry) displays potent anticancer activity in human cancer cells and animal models, but the cellular targets of Chry have not been fully defined. Herein, we speculated whether mitochondria were a target involved in Chry-induced cytotoxicity. Methods: Human liver cancer cell line HepG2 was incubated. The cytotoxicity was evaluated by MTT assay. Mitochondria localization was evaluated by a confocal microscopy. Mitochondrial membrane potential ΔΨm was detected by TMRE staining and determined by the flow cytometer. The levels of ATP, mitochondrial superoxide anions, and GSH/GSSG were determined according to the assay kits. The apoptosis were evaluated through Hoechst33342/PI and Annexin V/PI staining, respectively. The expression of cyclophilin D (CyPD) was determined by immunoblot method, and the interaction between CyPD and Chry was analyzed by molecule docking procedure. Results: Chry itself mainly localized in mitochondria to cause mitochondrial dysfunction and cell death in HepG2 cells. As regard to the mechanism, cyclosporin A as the inhibitor for the formation of mitochondrial permeability transition pore (mPTP) moderately suppressed cell death, indicating mPTP involved in the process of cell death. Further, Chry enhanced the protein expression of Cyclophilin D (CyPD) which is a molecular componentry and a modulator of mPTP, while antioxidant N-acetyl-L-cysteine inhibited the expression of CyPD. Molecule docking procedure disclosed two hydrogen-bonds existed in CyPD-Chry complex with −11.94 kal/mol of the binding affinity value. Besides, the mtDNA-deficient HepG

Effect of Wuzi Yanzong Wan on Sperm Apoptosis by Intervening with Mitochondrial Permeability Transition Pore / 中国实验方剂学杂志

Objective:To study the effect of Wuzi Yanzong Wan on the expressions of voltage-dependent anion channel 1 （VDAC1）， adenine nucleotide transposase （ANT）， cyclophilin D （CypD） and other proteins， and analyze its mechanism in intervening with sperm mitochondrial permeability transition pore （mPTP） opening. Method:Forty rats were randomly divided into 4 groups， namely normal group， model group， positive group （Shengjing capsule， 1.6 g·kg-1·d-1）， and Wuzi Yanzong Wan group （4.0 g·kg-1·d-1）， with 10 rats in each group. Except for the normal group， tripterygium wilfordii glycosides （GTW， 30 mg·kg-1） was intragastrically administered for 8 weeks to establish the oligozoospermia model. After the 4th week， each group was given drugs through intragastric administration for 4 weeks， and fasted for 12 h after the last administration. These rats were anesthetized with 3% chloral hydrate， and their testis and epididymis tissues were collected. Western blot was used to determine the protein expressions of VDAC1，ANT，CypD，B-cell lymphoma/leukemia2 （Bcl-2）， Bcl-2 associated X protein （Bax）， Caspase-3， Caspase-9 in rat testis， Testicular tissue and its ultrastructure were observed under electron microscopy. The apoptosis in spermatogenic cells was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL）. Result:Western blot results showed that compared with the normal group， the expressions of VDAC1， CypD， Caspase-3， Caspase-9 and Bax/Bcl-2 in the model group were increased （P<0.01）. The expressions of VDAC1， CypD， Caspase-3， Caspase-9 and Bax/Bcl-2 were significantly decreased in the positive group and the Wuzi Yanzong Wan group compared with the model group （P<0.01）. There was no significant change in the expressions of ANT and Bcl-2 protein between the groups. Testicular ultrastructural evaluation showed different sizes and disordered arrangement of sperm mitochondria and a large number of swelling and vacuoles in the model group， while complete structure and neat arrangement of sperm mitochondria and much less swelling and vacuole in positive group and Wuzi Yanzong Wan group. TUNEL results showed that the apoptosis rate of spermatogenic cells in the model group was significantly higher than that of the normal group （P<0.01）， while the apoptosis rate in the positive drug and Wuzi Yanzong Wan group was significantly lower than that of the model group （P<0.01）. Conclusion:Wuzi Yanzong Wan may resist germ cell apoptosis by inhibiting the expressions of VDAC1， CypD and Bax， reducing the permeability of mPTP， and preventing the cascade activation reaction of the Caspase family of apoptosis proteins.

The protective effects of cyclosporin A on vascular permeability in sepsis rats / 药学学报

The protective effects of cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPTP), on vascular permeability in sepsis rats were investigated. Cecal ligation and puncture (CLP)-induced sepsis rats were used for in vivo studies, and the effects of CsA (1 and 5 mg·kg-1) on vascular permeability of lung, kidney, and intestine, mitochondrial respiratory control ratio, and the survival of the sepsis rats were observed. Lipopolysaccharide (LPS) was used for stimulating vascular endothelial cells (VECs) in vitro, and the effects of CsA on leakage of microvascular, immunofluorescence of zonula occludes-1 (ZO-1), and transendothelial electrical resistance (TER) were observed. All the animal welfare and experimental procedures are in accordance with the regulations of the Animal Ethics Committee of the Army Medical University. Compared with sham-operated group, the vascular permeability of lung, kidney, and intestine in sepsis rats increased significantly (P<0.05). Compared with conventional treatment group, CsA could significantly decrease the vascular permeability of lung, kidney, and intestine (P<0.05 or P<0.01), and prolong the survival period. The results of microcirculation also showed that CsA could significantly reduce the permeability of mesenteric venules in sepsis rats. At the cellular level, LPS stimulation significantly increased the permeability of vascular endothelial cells, including the decrease of transmembrane resistance and protein expression of ZO-1 (P<0.05). CsA can significantly reduce the increase of permeability of vascular endothelial cells induced by LPS stimulation (P<0.01). The function of mitochondria in the kidneys and intestines of sepsis rats was obviously impaired, and the respiratory control ratio of mitochondria was decreased. LPS significantly increased MPTP opening of VECs, while CsA significantly inhibited MPTP opening and improved mitochondrial function. CsA may protect mitochondrial function by inhibiting the opening of MPTP and play a protective role in the vascular permeability of sepsis rats. This study will provide an insight for the treatment of sepsis vascular leakage.

The role of mitochondrial membrane permeability transition pore in apoptosis / 医学研究生学报

Mitochondria play a central role in various apoptotic pathways within cells, changes of mitochondrial permeability transition(MPT) is the key point, which is mainly regulated by mitochondrial permeability transition pore(mPTP). So far, the structural model of mPTP, the way of mPTP participating in apoptosis and the regulatory nodes are still unclear. Our aim is to summarize mechanism of mitochondria mediate apoptosis, currently recognized structural model of mPTP, the pathological changes of all components of mPTP during apoptosis and the possible damage mechanisms underneath. mPTP is closely related to apoptosis, inhibit the opening of mPTP will reduce apoptosis. Therefore, interventions acting on inhibitomg mPTP opening can be helpful to prevent and treat diseases.

Opening of liver mitochondrial permeability transition pore in streptozotocin-induced diabetic rats and its inhibition by methanol fraction of Ficus mucoso (Welw) root bark / 中西医结合学报

OBJECTIVE@#Several pathologies arise from the inappropriate opening of the mitochondrial permeability transition (mPT) pore. In this regard, inhibition of mPT pore represents a cytoprotective approach to preserve mitochondrial function for treatment of diseases characterized by excessive tissue wastage such as diabetes mellitus. The aim of this study, therefore, was to study the effects of fractions of Ficus mucoso, a medicinal plant used in the traditional treatment of diabetes, on mPT pore in normal and streptozotocin (STZ)-induced diabetic rat liver.@*METHODS@#Different solvent fractions of the crude methanol extract of F. mucoso were obtained by vacuum liquid chromatography and were tested on the mPT pore. Of all the fractions tested, methanol fraction of F. mucoso (MFFM) was the most potent and was used for in vivo studies. Diabetes mellitus was induced by a single intraperitoneal injection of 60 mg/kg STZ, while treatment lasted for 14 d. In vivo, 30 male Wistar rats were divided into five groups: A, normo-glycemic control (distilled water); B, STZ (65 mg/kg; diabetic control); C, STZ + MFFM (25 mg/kg); D, STZ + MFFM (50 mg/kg); E, STZ + glibenclamide (5 mg/kg). The mPT, mitochondrial ATPase activity, lipid peroxidation and cytochrome c release were assessed spectrophotometrically while blood glucose levels were monitored using glucometer.@*RESULTS@#In vitro, the solvent fractions of F. mucoso, at all concentrations tested, had no effect on the mPT pore, in the absence of calcium, with no significant release of cytochrome c. Interestingly, calcium-dependent pore opening was inhibited by all solvent fractions of F. mucoso, with the MFFM having the highest inhibitory effect of 83% at 3 mg/mL. Induction of opening of the mPT pore, significant (P < 0.001) enhancement of mitochondrial ATPase activity and elevated malondialdehyde (MDA) levels in STZ-induced diabetes were significantly (P < 0.001) reversed by MFFM and were comparable with the effects of glibenclamide, a standard antidiabetic drug. Also, treatment with MFFM at different doses significantly (P < 0.001) reduced high serum blood glucose compared to the diabetic control.@*CONCLUSION@#F. mucoso could be useful in therapeutic management of diabetes mellitus given its ability to prevent excessive tissue wastage via inhibition of pore opening, and reduction in levels of MDA and serum blood glucose.

Sevoflurane reduces cerebral injuries in rats resuscitated from cardiac arrest through down regulating the expression of Bax and Bak / 中华急诊医学杂志

Objective To investigate the neuroprotective mechanism of sevoflurane in rats resuscitated from cardiac arrest (CA).Methods A ventricular fibrillation-induced CA model was established.Forty Wistar rats were randomly divided into the sham group,sevoflurane group and control group.Apoptosis-related proteins were measured by Western blot at 24 h after restoration of spontaneous circulation (ROSC).The status of mitochondrial permeability transition pore (MPTP) were measured using a spectrophotometer,and the mitochondrial membrane potential (MMP) were measured with JC-1 fluorescent probe.At 72 h after ROSC,the apoptotic index of neurons in hippocampal CA1 region was counted by TUNEL staining.Results The protein expression of Bax,Bak,cleaved-caspase 9,cleavedcaspase 3 and cytosolic cytochrome c were lower in the sevoflurane group (all P＜0.05),the protein expression of Bcl-2 was higher in the sevoflurane group compared with the control group (P＜0.05).The sevoflurane group had a less opening status of MPTP and a higher MMP compared with the control group (all P＜0.05).The sevoflurane group had less apoptotic neurons compared with the control group (P＜0.05).Conclusion By up-regulating the expression of Bcl-2,down-regulating Bax and Bak,sevoflurane could reduce the apoptosis of neurons and decrease the opening of MPTP,eventually reduce cerebral injuries.

The characteristics of myocardial injury in rats resuscitated from cardiac arrest / 中华急诊医学杂志

Objective To investigate the characteristics of myocardial injury and its underlying mechanism in rats resuscitated from cardiac arrest. Methods Forty-two male Wistar rats were randomly(random number) assigned into the post-resuscitation (PR) 4 h, PR 24 h, PR 48 h, and sham groups. Ventricular fibrillation was induced by transcutaneous electrical epicardium stimulation and untreated for 6 min, followed by cardiopulmonary resuscitation (CPR). Myocardial function, glucose metabolism, myocardial ultrastructure, the status of mitochondrial permeability transition pore (MPTP) and mitochondrial membrane potential (MMP) were evaluated at different time points. Results Myocardial dysfunction was found at 4 h after restoration of spontaneous circulation (ROSC). The ejection fraction and cardiac output were decreased (all P<0.01), the diastole left ventricular posterior wall became thicker (P<0.01), and the end-diastolic volume was reduced (P<0.05). However, cardiac function was recovered almost completely at 48 h after ROSC. The PR 4 h group had a higher SUVmax, a more obvious decreased absorbance, and a lower MMP than the sham group (all P<0.01), but no statistically significant differences were noted between the PR 48 h group and the sham group (P>0.05). At 4 h and 24 h after ROSC, the mitochondria was swollen and the mitochondrial crista was sparse, but the myocardial ultrastructure was complete. Conclusions Post resuscitation myocardial dysfunction occurs after ROSC and the myocardial dysfunction is completely reversible at 48 h after ROSC, which may be related to the reversibility of myocardial injury and the gradual recovery of mitochondrial structure and function.

Protective effect of puerarin against anoxia reoxygenation injury in cardiomyocytes based on VDAC1/mPTP regulation mechanism / 中国药理学通报

Aim To study the effect of puerarin( Pue) against myocardial ischemia/reperfusion injury and in-volved mitochondrial mechanism. Methods Anoxia/reoxygenation( A/R) injury model was established in H9c2 cell. Recombinant plasmid pFLAG-VDAC1 was constructed. Cells were randomly divided into 4 groups, normal control group ( Control) , A/R group, puerarin group ( Pue + A/R ) , and pFLAG-VDAC1-Pue group. Real-time PCR was used to investigate the expression of VDAC1 at mRNA level, and the expres-sion of protein level was detected by Western blot. LDH and CK activities were measured by automatic bi-ochemical analyzer. Mitochondrial membrane potential ( Δψm) and cell apoptosis were observed by flow cy-tometry method. Mitochondrial swelling test was used to detect the opening of mitochondria permeability tran- sition pore ( mPTP) . Results Compared with control group, the expression of VDAC1 and mRNA was up-regulated in A/R group, LDH and CK activity were el-evated, and then mPTP opened, Δψm collapsed, cell apoptosis was significantly increased. Puerarin pre-treatment can lower the expression of VDAC1, main-tain Δψm, prevent the opening of mPTP, and reduce apoptosis. However, the protective effect of Puerarin could be cancelled by transfection of pFLAG-VDAC1. Conclusions The cardioprotection of Puerarin against A/R injury is closely related to down-regulation of VDAC1 and prevention of mPTP opening.

Role of mitochondrial permeability transition pore in mediating the inhibitory effect of gastrodin on oxidative stress in cardiac myocytes / 南方医科大学学报

OBJECTIVE@#To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes.@*METHODS@#H9c2 cardiac myocytes were treated with HO, gastrodin, gastrodin+HO, cyclosporin A (CsA), or CsA+gas+HO group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells.@*RESULTS@#Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in HO-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in HO-exposed cells.@*CONCLUSIONS@#Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.

Zhibai Dihuang Decoction improves sperm mitochondrial permeability transition in rats with ureaplasma urealyticum infection / 中华男科学杂志

<p><b>Objective</b>To investigate the effects of Zhibai Dihuang Decoction (ZDD) on sperm mitochondrial permeability transition (MPT) in the rat model of ureaplasma urealyticum (UU) infection (UUI).</p><p><b>METHODS</b>Ninety 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>ZDD 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.</p>

Regulation mechanism and targeted drugs of mitochondrial permeability transition pore on programmed cell death: Research advances / 国际药学研究杂志

Since its discovery in the 1970s, the mitochondrial permeability transition(mPT)has been proposed to be a strategic regulator of programmed cell death(PCD). The mPT denotes an increase in the mitochondrial inner membrane permeability to solutes with molecular masses up to about 1.5×103. It is presumed to be mediated by an opening of a channel, the mPT pore(mPTP), whose molecular nature remains a mystery. Intense research efforts have focused on elucidating the molecular components of the mPTP because it may help to better understand and treat various pathologies ranging from neurodegenerative and cardiac diseases to cancer. This article briefly reviews the new progress of mPTP structural models and its specific molecular mechanisms of regulating PCD, then demonstrates the feasibility of using the mPTP-targeting agents as a potential alternative strategy for effective management of PCD.