ROS; mitochondria-targeted antioxidant

Mitoquinone (MitoQ) is an antioxidant that targets mitochondria. During four hours of cold storage (CS), dose-response tests were used to determine the ideal dosages of mitoquinone (MitoQ) and DecylTPP therapy. In order to investigate the potential protective impact of mitoquinone treatment on CS damage, MitoSOX Red—a fluorescent dye that targets and detects mitochondrial superoxide production—was first used. When CS was applied to normal rat kidney (NRK) cells, mitochondrial superoxide caused the cells' fluorescence to increase by about two times when compared to the control group. When it came to CS-induced mitochondrial superoxide production, quinone from mitochondria significantly defended against it, while DecylTPP, the control chemical, had no such effect. Treatment for mitochondrial superoxide greatly decreased the generation of mitochondrial superoxide, and kidneys treated with DecylTPP had levels of mitochondrial superoxide that were on par with kidneys receiving CS alone [1].

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Protective Effect of MitoQ on Mitochondrial Superoxide Generation during CS. [1]
The optimal doses for MitoQ and DecylTPP treatment were selected from dose-response experiments during 4-h CS. For the cellular-based studies, 1 μM was chosen from a range of 0.5, 0.75, 1.0, 1.5, and 2 μM, whereas the most efficacious dose for the ex vivo studies was 100 μM from a dose-response study of 50, 100, and 500 μM (data not shown). The potential protective benefits of MitoQ treatment against CS injury were tested initially using MitoSOX Red, a mitochondrial-targeted fluorescent dye that measures mitochondrial superoxide generation. As shown in Fig. 2A, NRK cells exposed to CS resulted in a ∼2-fold increase in fluorescence due to mitochondrial superoxide compared with untreated cells. MitoQ offered significant protection against CS-induced mitochondrial superoxide generation; whereas the control compound DecylTPP did not offer any protection. This was further confirmed using a spectrofluorometric-based assay detecting MitoSOX Red fluorescence excitation at 396 and 510 nm, where 396 nm is a specific indicator of mitochondrial superoxide, and 510 nm detects nonspecific oxidant generation (Robinson et al., 2006) (Fig. 2B). In addition, kidneys exposed to CS alone displayed increased mitochondrial superoxide generation compared with control kidneys (Fig. 2C). MitoQ treatment markedly decreased mitochondrial superoxide generation, whereas kidneys treated with DecylTPP had comparable levels of mitochondrial superoxide to kidneys exposed to CS alone (Fig. 2C).

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MitoQ Attenuates Nitrotyrosine Adduct Formation during CS. [1]
Immunocytochemistry and immunohistochemistry were used to evaluate nitrotyrosine protein adducts during CS. As shown in Fig. 3A, NRK cells exposed to CS alone had a significant increase in nitrotyrosine staining (red fluorescence) compared with untreated cells. MitoQ attenuated nitrotyrosine formation ∼2-fold, whereas the control compound DecylTPP did not decrease CS-mediated nitrotyrosine formation (Fig. 3A). Peroxynitrite-treated cells had intense nitrotyrosine formation (positive control) and was blocked when the nitrotyrosine antibody was preabsorbed with excess 3-nitrotyrosine (negative control; ONOO− + block). Rat kidney immunohistochemistry data were consistent with the in vitro study findings regarding the effect of MitoQ and nitrotyrosine. Figure 3B shows an increase in nitrotyrosine (brown staining) in the distal and proximal tubules and to a lesser extent in the glomeruli of CS kidneys compared with control kidneys. Kidneys treated with MitoQ had less nitrotyrosine formation. In contrast, DecylTPP-treated kidneys had similar amounts of nitrotyrosine compared with CS kidneys. The specificity of nitrotyrosine staining was also confirmed using antibody preabsorbed with excess 3-nitrotyrosine (CS + block).

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MitoQ Prevents Mitochondrial Respiratory Complex Inactivation during CS. [1]
Mitochondrial respiratory complex activity was evaluated in both the in vitro and ex vivo renal models to investigate whether 4-h CS alters mitochondrial respiratory function. Complexes I and II were significantly inactivated after CS of NRK cells compared with untreated cells (Fig. 4). MitoQ completely prevented complex I and II inactivation, whereas DecylTPP had no significant effect on complex activity. Complexes III and IV were not assessed in this study because we have previously shown both complex activities to be unchanged with 24-h CS (Mitchell et al., 2010); therefore evaluation of these complex activities was not warranted. Consistent with the in vitro findings, CS of rat kidneys led to partial inactivation of complexes I and II activity and had no effect on complexes III and IV (Fig. 4). MitoQ protected against complex I and II inactivation of CS kidneys, whereas DecylTPP did not have any effect.

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MitoQ Decreases Nitrotyrosine Formation and Cell Death during CS Plus Rewarming of NRK Cells. [1]
To test whether MitoQ could potentially offer protection against oxidant production and cell death after reperfusion/transplantation, cells were exposed to CS plus RW. As shown in Fig. 6A, 4-h CS plus overnight (18 h) RW of NRK cells resulted in significant nitrotyrosine formation. Adding MitoQ during CS attenuated nitrotyrosine staining significantly during CS.RW, whereas the control compound DecylTPP had no effect. LDH cytotoxicity revealed a significant increase in cell death of NRK cells exposed to CS.RW compared with untreated cells (Fig. 6B). MitoQ decreased cell death approximately 2-fold during CS alone and CS.RW. DecylTPP did not reduce cell death in either treatment group.

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MitoQ Scavenged ROS Production in Isolated Pancreatic Acinar Cells [2]
Application of 1 mM H2O2 caused a steady rise of ROS in the cells (control group) as reflected by increased intensity of CM-H2DCFDA fluorescence; Cells pretreated with 1 μM MitoQ showed a significantly reduced ROS production compared with cells in the control group, whereas pretreatment of 1 μM dTPP, the lipophilic cation of MitoQ without antioxidant activity was without effect (Figure 1(a)). Neither MitoQ nor dTPP induced ROS production per se (Figure 1(b)).

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MitoQ Did Not Protect against Mitochondrial Depolarisation Caused by AP Precipitants in Isolated Pancreatic Acinar Cells [2]
Neither pretreatment with 1 μM MitoQ nor dTPP caused depolarisation of ΔΨm in isolated pancreatic acinar cells, in contrast to the protonophore CCCP applied at the end of the experiment to elicit complete depolarisation. (Figure 2(a)). However, at 10 μM both MitoQ and dTPP induced a steady decrease of ΔΨm per se that was more profound for MitoQ (Figures 2(b) and 2(c)). CCK (10 nM) induced depolarisation of ΔΨm compared with control cells treated with HEPES alone (Figure 2(d)), an effect that was not significantly affected by pretreatment with either 1 μM MitoQ or dTPP (Figure 2(e)). Similarly, perfusion of TLCS (500 μM) depolarised ΔΨm (Figure 2(f)) that was unaffected by 1 μM MitoQ or dTPP (Figure 2(g)).

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MitoQ Caused Pancreatic Acinar Cell Death and Aggravated CCK-Induced Necrosis [2]
Both 1 μM MitoQ and dTPP caused an increased PI uptake indicative of necrosis; significant differences were observed at 2, 6, and 10 h between cells pretreated with either MitoQ or dTPP and cells in the control group treated with HEPES alone (Figure 3(a)). Addition of 10 nM CCK induced a time-dependent increase of necrosis. However, 1 μM MitoQ did not exert any protection against CCK-induced cell death. Rather both MitoQ and dTPP significantly worsened necrosis compared with CCK alone at 2 h, whereas dTPP, but not MitoQ, aggravated CCK-induced cell death at later time-points (Figure 3(b)).

Treatment with mitoquinone (MitoQ) dramatically decreased neutrophil infiltration and pancreatic edema. Serum amylase is dose-dependently increased by MitoQ; at higher doses, it roughly doubles. When administered at 10 mg/kg (dose 1), MitoQ treatment dramatically raised serum IL-6 levels and nearly doubled Caerulein-induced lung MPO activity [2].

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MitoQ Ameliorated Overall Pancreatic Histopathology in CER-AP but Aggravated Systemic Injury [2]
Figure 4(a) shows representative histopathology slides for control and different treatment groups, with the overall histopathology score and breakdown scores for individual components summarised in Figure 4(b). Intraperitoneal saline injections did not cause any significant histopathological changes of the pancreas, whereas hyperstimulation with caerulein induced typical features of AP; marked oedema, vacuolisation, neutrophil infiltration in the ductal margins, and parenchyma of the pancreas, with focal acinar cell necrosis evident 12 h after the first caerulein injection. The CER-AP was also characterised by significantly increased serum amylase, pancreatic trypsin and MPO activity, and lung MPO activity compared to saline controls.

MitoQ treatment at both doses tested significantly reduced pancreatic oedema and neutrophil infiltration. However, pancreatic necrosis was not prevented, with a trend toward greater necrosis at the higher dose although this did not attain significance. MitoQ dose-dependently increased serum amylase with an approximate doubling at the higher dose (Figure 5(a)). Pancreatic trypsin activity and MPO activity were not significantly affected by MitoQ at either dose (Figures 5(b) and 5(c)). In addition, MitoQ treatment nearly doubled lung MPO activity induced by caerulein (Figure 5(d)) with a significant increase of serum IL-6 levels also evident at dose 1 (Figure 5(e)).

The nonantioxidant analogue dTPP significantly reduced oedema, neutrophil infiltration, and necrosis at both doses, resulting in an overall reduction of the histopathological score. Serum amylase was not significantly affected (Figure 5(a)), although dTPP reduced pancreatic trypsin and MPO activity (Figures 5(b) and 5(c)). Similar to the results obtained with MitoQ, dTPP also significantly increased caerulein-induced lung MPO activity and serum IL-6 levels (Figures 5(d) and 5(e)).

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MitoQ Did Not Protect against TLCS-AP [2]
Sham operation only induced mild oedema of the pancreatic acinar cells without discernible signs of inflammation and necrosis. Infusion of 3 mM TLCS into the pancreas via the pancreatic duct resulted in marked histopathological changes of the head of the pancreas at 24 h, characterised by significantly increased oedema, inflammation, necrosis and thus, overall histopathology score (Figure 6(a)(i–iv)). However, the body and tail of the pancreas were much less affected (data not shown). The TLCS-AP was associated with increased serum amylase, pancreatic MPO activity, and serum IL-6 levels compared to the sham group (Figures 6(b)–6(d)).

Neither MitoQ nor dTPP at the lower dose induced histopathological changes of the pancreas, with oedema, inflammation, necrosis, and overall histopathological score unaltered (Figure 6(a)(i–iv)). Similarly, there were no significant changes of serum amylase and pancreatic MPO activity when TLCS-AP mice were treated with either MitoQ or dTPP (Figures 6(b) and 6(c)). Serum IL-6 levels were marginally increased by MitoQ or dTPP treatment but this did not attain statistical significance (Figure 6(d)). Application of MitoQ or dTPP at both doses alone to mice in the absence of caerulein injections or TLCS infusion showed that both MitoQ and dTPP significantly increased lung MPO activity per se (data not shown).

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Biphasic Effects of MitoQ on PMA-Induced ROS Production in PMNs [2]
Figure 7(a) illustrates the effect of 1 μM MitoQ or dTPP on PMA-induced ROS production in isolated PMNs. The NAD(P)H oxidase stimulator PMA (50 ng/mL) induced a dramatic increase of ROS in the extracellular solution around PMNs within the first few minutes that peaked at 8 mins and declined to a plateau after approximately 20 mins. Application of the NAD(P)H oxidase inhibitor DPI reduced the peak phase and completely inhibited the ROS plateau (Figure 7(c)). MitoQ treatment caused a biphasic effect on ROS production in PMNs. Thus, a concentration-dependent inhibition of the initial ROS peak induced by PMA was observed, with the peak time delayed to 10 minutes (Figures 7(a)–7(c)). Application of 1 μM dTPP had no significant effect on the peak of PMA-induced ROS production in PMNs compared to cells treated by PMA alone. Interestingly, MitoQ caused a concentration-dependent potentiation of PMA-induced ROS production at 40 mins (Figure 7(c)), an action shared by dTPP only at the higher concentration.

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CS Induced Renal Injury and Cell Death Is Decreased with MitoQ Treatment. [1]
Periodic acid-Schiff staining was performed to examine histopathological changes during CS of rat kidneys. Significant widespread tubular damage such as dilation, brush border loss, and cellular debris/cast formation occurred with CS exposure (Fig. 5A). MitoQ improved renal histology significantly, whereas DecylTPP treatment did not reverse renal injury. TUNEL staining, indicated by brown staining of the nuclei, was used as a marker of cell death. As shown in Fig. 5B, CS led to a significant increase in cell death (red arrows) compared with control kidneys. MitoQ treatment decreased cell death by ∼2-fold compared with CS kidneys, whereas DecylTPP offered no protective benefits against cell death.

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Mitochondrial dysfunctions induced by oxidative stress could play a pivotal role in the development of testicular damage and degeneration, leading to impaired fertility in adulthood. MitoQ as mitochondria-targeted antioxidant has been used in many diseases for a long time, but its therapeutic effects on testicular injury 'have not been reported yet. Here, we examined the protective action mechanism of MitoQ on testicular injury from oxidative stress induced by triptolide (TP). Mice were orally administrated with MitoQ (1.3, 2.6 and 5 .2mg/kg, respectively) in a TP-induced model of testicular damage for 14 days. And then testis injuries were comprehensively evaluated in terms of morphological changes, spermatogenesis assessment, blood-testis barrier (BTB) integrity, and apoptosis. The results demonstrated MitoQ effectively increased testicular weight, maintained the integrity of BTB, protected microstructure of testicular tissue and sperm morphology by inhibition of oxidative stress. Further mechanism studies revealed that MitoQ markedly activates the Keap1-Nrf2 antioxidative defense system characterized by increasing the expression of Nrf2 and its target genes HO-1 and NQO1. Meanwhile, MitoQ upregulated the expression of mitochondrial dynamics proteins Mfn2 and Drp-1and exerted a protective effect on mitochondria. On this basis, the results from pharmacokinetic study indicated that the MitoQ could enter into testis tissues after oral administration in despite of the low absolute bioavailability, which provided the material basis for MitoQ in the treatment of testicular damage. More importantly, MitoQ reached mitochondria quickly and had an outstanding feature of mitochondria targeting in Sertoli cells. Therefore, these results provide information for the application of MitoQ against testicular injury diseases [3].

Cold Storage In Vitro Model. [1]
Normal rat kidney proximal tubular cells were maintained in six-well 100 or 150-mm, or 150-mm plates in a humidified incubator gassed with 5% CO2 and 95% air at 37°C in DMEM containing 5% fetal calf serum (FCS). Cells were grown to 60% confluence and divided into four treatment groups: 1) untreated (Untx), 2) CS, 3) CS + MitoQ, and 4) CS + DecylTPP. Untreated cells remained at 37°C in DMEM containing 5% FCS (group 1). CS was initiated by washing cells with cold PBS twice and storing them in UW/Viaspan solution alone (4 h at 4°C) (group 2), CS + MitoQ (1 μM) (group 3), or CS + DecylTPP (1 μM) (group 4). In separate experiments, cells were exposed to CS plus RW by replacing UW solution alone or UW solution containing MitoQ or DecylTPP with DMEM containing 5% FCS overnight (18 h at 37°C).

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Measurement of ROS Production [2]
Real-time ROS production and redox changes in pancreatic acinar cells were measured with the probe 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl (CM-H2DCFDA) using a Zeiss LSM510 confocal microscope as previously described. Freshly isolated murine pancreatic acinar cells were incubated with either 1 μM MitoQ or dTPP and simultaneously loaded with 10 μM CM-H2DCFDA for 30 minutes. The cells were then perfused with H2O2 to induce ROS. The fluorescence of CM-H2DCFDA was excited at 488 nm and the emission was collected at 505–550 nm.

For ROS measurement in PMNs, a peroxidase-enhanced luminol chemiluminescent assay was employed using a POLARstar Omega Plate Reader. Cells were plated at a density of 500,000 per well and pretreated with 1 μM MitoQ or dTPP for 10 minutes, before adding 50 μM luminol and 75 units/mL horseradish peroxidase. Activation of NAD(P)H oxidase was induced by 50 ng/mL phorbol myristate acetate (PMA) while inhibition was achieved by using 1 μM diphenylene iodonium (DPI). The luminescence emission at 440 nm for the ROS dye was recorded for 40 min. The chemiluminescence intensity was normalised to negative controls for each mouse/run.

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Measurement of ΔΨm in Pancreatic Acinar Cells [2]
In separate experiments, ΔΨm of pancreatic acinar cells was determined by tetramethyl rhodamine methyl ester assay as described previously. Briefly, the cells were loaded with 40 nM TMRM for 30 minutes prior incubation with either 1 μM or 10 μM of MitoQ or dTPP. Cholecystokinin-8 (CCK-8, 10 nM) or bile acid taurolithocholic acid 3-sulphate (TLCS, 500 μM) was used to induce ΔΨm depolarisation. At the end of the perfusion, the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 10 μM) was added to induce complete depolarisation of ΔΨm. The fluorescence of TMRM was excited at 543 nm and the emission was collected at 560–650 nm.

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In Vitro Cell Death Assay for Pancreatic Acinar Cells [2]
Pancreatic acinar cell death was detected as the intensity of fluorescent dye propidium iodide (PI) taken up by the nuclei of necrotic cells. For cell death induced by CCK-8, a time-course fluorescent plate reader method was used. Briefly, cells were isolated from one murine pancreas, centrifuged, and resuspended into 1 mL solution. Cells were carefully pipetted into individual wells to ensure homogeneity. Cells were treated with CCK-8 (10 nM) alone, or in the presence of 1 μM of either MitoQ or dTPP. For normal control groups, cells treated with equal volume of extracellular solution. After 5 min, PI (50 μM) was then added to all wells mixed by automated agitation. The microplate was then placed in the POLARstar Omega Plate Reader (preheated to 37°C), and fluorescence determined by excitation 543 nm and emission 620 nm with bottom reading. The assay was set to run with a cycle time of 600 seconds. All fluorescence measurements are expressed as changes from basal fluorescence (F/F 0 ratio), where F 0 represents the initial fluorescence recorded at the start of the experiment, and F the fluorescence recorded at specific time points.

Cold Storage Ex Vivo Model. [1]
Male Fischer 344 inbred rats weighing between 250 and 300 g were anesthetized with ethrane, followed by shaving and prepping with betadine. A 2-ml bolus of 0.9% (w/v) NaCl was administered intravenously, and an incision was made 1 cm superior to the symphysis pubis to the tip of the xiphoid process. Bulldog clamps were placed on the aorta and vena cava (proximal and distal to the renal vessels) to prevent blood flow to the kidneys. A 22-gauge surgical needle was used to puncture the rat's aorta to flush the renal grafts with saline (10 ml per kidney) using a small catheter. Once the kidneys started to turn light brown (perfusion), another vent was formed in the vena cava to allow blood flow from the kidneys. Once both kidneys were completely flushed, the right kidney was recovered and served as a control (group 1), and the left kidney was exposed to CS alone for 4 h at 4°C (group 2). In additional experiments, kidneys were flushed with saline through the aorta using a small catheter followed by flushing the right kidney with saline containing MitoQ (100 μM) and the left kidney with saline containing DecylTPP (100 μM) (10 ml per kidney). Tissues were recovered and stored in CS + MitoQ (100 μM; right kidney) or CS + DecylTPP (100 μM; left kidney) for 4 h at 4°C (groups 3 and 4, respectively). A thin middle section from all of the kidneys were cut and immediately fixed in 10% formalin before being embedded in paraffin for sectioning (4 μm) and histological evaluation. The remaining portion of the kidneys were quickly frozen in liquid nitrogen and stored in −80°C until needed for biochemistry analyses.

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Experimental AP Models [2]
Seven intraperitoneal injections of a supramaximal dose (50 μg/kg) of caerulein, a CCK-8 analogue, were given on an hourly basis to induce hyperstimulation acute pancreatitis (CER-AP). Control mice received equal volumes of PBS injection. In the MitoQ treatment groups, MitoQ at 10 mg/kg (dose 1) or 25 mg/kg (dose 2) was given at the first and third injections of caerulein. Similarly, dTPP at 9.6 mg/kg (dose 1) or 24 mg/kg (dose 2) was given for the dTPP treatment group. MitoQ and dTPP were at the same molar concentration at doses 1 and 2. Mice were sacrificed at 12 h after the first caerulein injection to collect samples.

Bile acid-induced AP was achieved by retrograde infusion of TLCS into the pancreatic duct (TLCS-AP). After induction of anesthesia, TLCS applied using a mini infusion pump at a speed of 5 μL/min for 10 minutes. Successful infusion of TLCS into pancreas was demonstrated by a diffuse light blue colour (methylene blue) appearing in the pancreatic head. Control mice received sham surgery without TLCS infusion. In the treatment groups, MitoQ (10 mg/kg) or dTPP (9.6 mg/kg) was given at 1 h and 3 h after TLCS infusion. Mice were sacrificed at 24 h after the TLCS infusion or sham surgery. In both animal models, analgesia was achieved by administration of 0.1 mg/kg buprenorphine hydrochloride.

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Pharmacokinetic and tissue distribution study in mice [3]
Diet was prohibited for 12 h before the experiment while water was taken freely. All mice were randomly assigned to two groups for intravenous or oral administration of 2.6 mg/kg MitoQ, respectively. Blood sample was collected into heparinized tubes at 0 (pro-drug), 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h after tail injection (i.v.) administration. Meanwhile, blood sample was collected into heparinized tubes at 0 (pro-drug), 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h after orally administration. Blood samples of each time point were taken from 6 mice. Blood sample was immediately centrifuged at 3000 ×g for 10 min at 4 °C, and plasma was transferred into a new 1.5 mL Eppendorf tube and then stored at −80 °C until analysis. The pharmacokinetic parameters of MitoQ were calculated by DAS Software (version 3.0, China State Drug Administration) using non-compartmental methods. Absolute bioavailability was calculated by comparing the area under the curve (AUC) of oral administration with AUC of the same drug following intravenous administration at the same dose, as a previous report (Ma et al., 2014). The unit of the concentration (nmol/L) of MitoQ derived from the calibration curve was converted to ng/mL by multiplying relative molecular mass.Eighteen mice were randomly assigned to three groups (6 mice/group) to carry out tissue distribution study. Mice in three groups were sacrificed at 10 min, 1 h and 2 h respectively after intravenous injection 2.6 mg/kg pure MitoQ. Subsequently, testes were immediately removed, washed in normal saline and blotted dry with filter paper. An accurately weighed amount of the soft tissue samples (0.2 g) was individually homogenized with normal saline (0.5 ml) and stored at −80 °C until analysis. The unit of the concentration (nmol/L) of MitoQ derived from the calibration curve was converted to ng/mL by multiplying relative molecular mass.

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Dosage information/dosage regimen [3]
Animals were randomly assigned to five groups (n = 10 per group). Control group: mice were given vehicle by gavage and intraperitoneal (i.p.) injection orderly once a day (ethanol/0.9% saline =1:9, vol); TP model group: mice were given an intraperitoneal injection with TP at a dose of 120 μg/kg after pre-treatment with above-mentioned vehicle; MitoQ group: mice were orally administrated with MitoQ at the dose of 1.3, 2.6, 5.2 mg/kg, respectively,for 1 h and then an i.p. injection with TP (120 μg/kg)every day. After the continuous treatment of MitoQ for 14 days, mice were euthanized, testicular tissues were removed and weighted to calculate the testis index (testis weight/body weight) and stored in liquid nitrogen for future studies.

Pharmacokinetic Study of MitoQ in Mice [3]
The validated LC-MS/MS method was successfully applied to the pharmacokinetic study of mice after a single oral or tail injection of 2.6 mg/kg MitoQ. The main pharmacokinetic parameters calculated by the non-compartmental model are listed in Figure 6F. The mean plasma concentration-time curves of MitoQ are shown in Figures 6D and 6E.
The time to peak concentration (Tmax) was observed approximately 0.74 h after oral administration, indicating that MitoQ can be rapidly absorbed into the bloodstream. However, the peak plasma concentration (Cmax) was low, only 9.77 ± 2.76 ng/ml. The half-life (T1/2) of MitoQ after intravenous and oral administration was 1.066 ± 0.319 h and 0.74 ± 0.49 h, respectively. The absolute bioavailability of MitoQ was low after both intravenous and oral administration (17.95% ± 5.98%). The apparent volumes of distribution after intravenous and oral administration were 76.268 ± 13.534 L/kg and 324.52 ± 241.44 L/kg, respectively. The distribution of MitoQ in testicular tissue is shown in Figure 6G. Ten minutes after oral and intravenous administration, the highest concentrations of MitoQ in testicular tissue were 35.82 ± 8.233 ng/g and 194.55 ± 8.59 ng/g, respectively. However, MitoQ was not detected in any tissue samples two hours after administration. The data indicate that MitoQ does not accumulate in tissues. The results also show that MitoQ can cross the blood-testis barrier, providing a material basis for testicular injury. Mitoquinone (MitoQ10 mesylate) is a mitochondrial-targeting antioxidant currently under development for the treatment of neurodegenerative diseases. This study aimed to establish and validate a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of mitochondrial quinones in rat plasma after oral administration of MitoQ10, and to detect and identify its metabolites. After a simple protein precipitation step, reversed-phase liquid chromatography with a gradient elution using acetonitrile/water/formic acid as the mobile phase was employed to analyze plasma samples. Mitochondrial quinones were analyzed using positive ion mode electrospray ionization multiple reaction monitoring (MRM) with a deuterated compound (d3-MitoQ10 methanesulfonate) as an internal standard. The calibration curve for mitochondrial quinones showed a linear relationship in the concentration range of 0.5–250 ng/mL, with a correlation coefficient >0.995. This method exhibited high sensitivity (limit of quantitation of 0.5 ng/mL), acceptable accuracy (relative error <8.7%), and acceptable precision (intra-day and inter-day coefficients of variation <12.4%). The recoveries of mitochondrial quinones at concentrations of 1.5, 20, and 200 ng/mL ranged from 87% to 114%. This method has been successfully applied to pharmacokinetic studies of rats after a single oral administration, and four metabolites of MitoQ10 have been preliminarily identified: hydroxylated MitoQ10, demethylated MitoQ10, and MitoQ10 quinone glucuronide and sulfate conjugate. [4]

[1]. The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renaltubular cells and rat kidneys. J Pharmacol Exp Ther. 2011 Mar;336(3):682-92.

[2]. Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis. Mediators Inflamm. 2015;2015:901780.

[3]. MitoQ ameliorates testis injury from oxidative attack by repairing mitochondria and promoting the Keap1-Nrf2 pathway. Toxicol Appl Pharmacol. 2019 May 1:370:78-92.

[4]. Quantitation and metabolism of mitoquinone, a mitochondria-targeted antioxidant, in rat by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2007;21(13):1958-64.

Mitoquinone is based on a novel technology—targeting lipophilic cations—that transports and concentrates antioxidants into mitochondria (cellular organelles that provide energy for life processes), achieving concentrations up to a thousand times higher than those in the submitochondrial membrane. A 2004 genomic study of inherited early-onset Parkinson's disease revealed a direct molecular link between mitochondrial dysfunction and the pathogenesis of Parkinson's disease. The study also indicated that mitochondrial dysfunction is a key early event in the pathogenesis of sporadic Parkinson's disease. Clinical studies by the Parkinson's Disease Research Group suggest that high doses of the antioxidant coenzyme Q (which mitochondrial quinone effectively targets mitochondria) appear to slow the progression of Parkinson's disease symptoms. Drug Indications It has been investigated for the treatment of hepatitis (viral, hepatitis C) and Parkinson's disease. Mechanism of Action Mitoquinone targets mitochondria through covalent binding to the lipophilic triphenylphosphine cation. Due to their high mitochondrial membrane potential, these cations can accumulate up to 1000 times more in cellular mitochondria compared to non-targeted antioxidants such as Coenzyme Q or its analogues, allowing the antioxidant component to block lipid peroxidation and protect mitochondria from oxidative damage. By selectively blocking mitochondrial oxidative damage, it can prevent cell death. Mitoquinones may help prevent neuronal cell damage that leads to Parkinson's disease. It is designed to slow or halt the progression of Parkinson's disease at its root by addressing cell damage caused by mitochondrial dysfunction. MitoQ is expected to slow or halt the progression of Parkinson's disease symptoms. Hepatitis C virus can directly alter mitochondrial function, leading to increased production of reactive oxygen species (free radicals), which in turn leads to liver scarring and cirrhosis. Even without a sustained virological response, mitoquinones can be used to halt or reduce the progression of liver inflammation and fibrosis. Mitoquinones act directly on mitochondria through two steps: the targeting component guides the drug to the mitochondria; the antioxidant component helps prevent cell damage. Most kidneys used for transplantation come from deceased donors. These kidneys must be cryopreserved/stored before transplantation to maintain tissue quality and allow time for recipient selection and transport. However, cryopreservation (CS) can lead to tissue damage, kidney discarding, or long-term renal dysfunction post-transplantation. We have previously identified mitochondrial superoxide and other downstream oxidants as important signaling molecules that cause cryopreservation-plus-warming (RW) damage in rat proximal tubular cells. Therefore, the aim of this study was to determine whether the addition of the mitochondrial-targeting antioxidant mitochondrial quinone (MitoQ) to the University of Wisconsin (UW) preservation solution could prevent cryopreservation damage. CS exposure was induced by placing kidney cells or isolated rat kidneys in UW solution (4°C, 4 hours) or in UW solution containing MitoQ or its control compound Decyltriphenylphosphine bromide (DecylTPP) (1 μM in vitro; 100 μM in vitro). Following CS exposure, changes in oxidant production, mitochondrial function, cell viability, and kidney morphology were assessed. CS induced a 2–3-fold increase in mitochondrial superoxide production and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or kidney damage. MitoQ treatment reduced oxidant production by about 2-fold, completely halted mitochondrial dysfunction, and significantly improved cell viability and/or kidney morphology, while DecylTPP treatment provided no protection. These findings suggest that MitoQ may have therapeutic uses to help reduce damage and kidney discarding during organ preservation and may improve kidney function after transplantation. [1]

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In summary, this is the first report showing that mitochondrial superoxide increases significantly during early CS and leads to mitochondrial and kidney damage. We found that the mitochondrial-targeting antioxidant MitoQ significantly protected proximal tubular cells and isolated rat kidneys from CS-mediated oxidative stress, mitochondrial dysfunction, cell death, and kidney damage. These findings suggest that pre-transplant infusion of MitoQ into the kidneys may have therapeutic uses to reduce CS damage, improve transplant recipient outcomes, and increase the number of donated organs available for transplantation, all of which could potentially reduce healthcare costs. [1]

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Although oxidative stress is closely associated with the occurrence of acute pancreatitis (AP), the use of antioxidant therapy in patients has been less than ideal to date. This study aimed to evaluate the potential protective effect of the mitochondrial-targeting antioxidant MitoQ in experimental acute pancreatitis (AP) using in vitro and in vivo methods. MitoQ blocked H₂O₂-induced intracellular reactive oxygen species (ROS) responses in mouse pancreatic acinar cells, while the control analog dTPP did not have this effect. MitoQ did not reduce cholecystokinin (CCK) or bile acid TLCS-induced mitochondrial depolarization and itself induced depolarization at a concentration of 10 µM. Both MitoQ and dTPP increased basal cell death and CCK-induced cell death in ELISA readings. In the TLCS-induced AP model, MitoQ treatment had no protective effect. In the secretin overstimulation-induced AP (CER-AP) model, MitoQ exhibited a mixed effect. Therefore, a partial improvement in histopathological scores was observed, similar to the effect of dTPP, but without reducing biochemical markers such as trypsin or serum amylase. Interestingly, in the CER-AP model, MitoQ simultaneously increased the levels of pulmonary myeloperoxidase and interleukin-6. MitoQ had a biphasic effect on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase in ROS but subsequently increasing its levels. Our results suggest that MitoQ is not suitable for the treatment of AP, consistent with previous assessments of antioxidants for this disease. [2] In summary, these results further highlight the inapplicability of antioxidant therapy to AP, as previously emphasized in a randomized, double-blind, placebo-controlled clinical trial. MitoQ had no protective effect against experimental TLCS-AP and a mixed effect, including an increase in inflammatory markers, was observed in a milder CER-AP model. These results are consistent with previous studies suggesting that inhibition of reactive oxygen species (ROS) can enhance pancreatic acinar cell necrosis by inhibiting a protective apoptosis mechanism that promotes local pancreatic damage in acute pancreatitis (AP). [2] In conclusion, this study confirms a novel beneficial effect of MitoQ on TP-induced in vivo testicular damage. In a TP-induced mouse model of testicular injury, MitoQ effectively restored the microstructure of testicular tissue, restored the integrity of the blood-testis barrier (BTB), and maintained spermatogenesis. The mechanism of these effects may involve protecting testicular tissue in two ways. On the one hand, MitoQ exerts its antioxidant effect by regulating mitochondrial dynamics. On the other hand, MitoQ reduces oxidative stress by activating the Nrf2/Keap1 signaling pathway. Its potency, efficacy, and pharmacokinetic characteristics make it a potential candidate drug for clinical application in the treatment of mitochondrial-related testicular injury and male infertility. [3]

C37H44O4P

583.716631889343

583.297

444890-41-9

845959-50-4 (mesylate);845959-52-6 (beta-CD complex);444890-41-9 (cation);336184-91-9 (bromide);

11388332

Typically exists as solid at room temperature

9.4

0

4

16

42

886

0

[P+](C1C=CC=CC=1)(C1C=CC=CC=1)(C1C=CC=CC=1)CCCCCCCCCCC1C(C(=C(C(C=1C)=O)OC)OC)=O

OIIMUKXVVLRCAF-UHFFFAOYSA-N

InChI=1S/C37H44O4P/c1-29-33(35(39)37(41-3)36(40-2)34(29)38)27-19-8-6-4-5-7-9-20-28-42(30-21-13-10-14-22-30,31-23-15-11-16-24-31)32-25-17-12-18-26-32/h10-18,21-26H,4-9,19-20,27-28H2,1-3H3/q+1

10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)decyl-triphenylphosphanium

MitoQ; MitoQ10; Mito Q; MitoQ 10; mitoquinone; Mitoquinone cation; UNII-47BYS17IY0; MITOQUINONE [WHO-DD]; 10-(6'-ubiquinonyl)decyltriphenylphosphonium bromide; 444890-41-9; Mitoquinone ion; Mito-Q; Mito-Q; MitoQ-10

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)