Coenzyme Q10 ameliorates cerebral ischemia reperfusion injury in hyperglycemic rats

Accepted Manuscript Title: Coenzyme Q10 Ameliorates Cerebral Ischemia Reperfusion Injury in Hyperglycemic Rats Authors: Cui-Jie Lu, Yong-Zhen Guo, Yang Zhang, Lan Yang, Yue Chang, Jing-Wen Zhang, Li Jing, Jian-Zhong Zhang PII: DOI: Reference:

S0344-0338(16)30774-9 http://dx.doi.org/doi:10.1016/j.prp.2017.06.005 PRP 51823

To appear in: Received date: Revised date: Accepted date:

17-12-2016 10-5-2017 4-6-2017

Please cite this article as: Cui-Jie Lu, Yong-Zhen Guo, Yang Zhang, Lan Yang, Yue Chang, Jing-Wen Zhang, Li Jing, Jian-Zhong Zhang, Coenzyme Q10 Ameliorates Cerebral Ischemia Reperfusion Injury in Hyperglycemic Rats, Pathology - Research and Practicehttp://dx.doi.org/10.1016/j.prp.2017.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Coenzyme Q10 Ameliorates Cerebral Ischemia Reperfusion Injury in Hyperglycemic Rats

Cui-Jie Lua1, Yong-Zhen Guo a1 Yang Zhanga1 ,Lan Yanga, Yue Changa, Jing-Wen Zhanga, Li Jing*, Jian-Zhong Zhang*

a

School of Basic Medical Science, Ningxia Key Laboratory of Cerebrocranial

Diseases-Incubation Base of National Key Laboratory, Ningxia Medical University, Yinchuan 750004, China.

*Corresponding authors: * Li Jing, Current Address: Department of Pathology, Ningxia Medical University, Yinchuan 750004, China. [email protected] * Jian-Zhong Zhang, Current Address: Department of Pathology, Ningxia Medical University, Yinchuan 750004, China. [email protected]

1

Cui-Jie Lu, Yong-Zhen Guo, and Yang Zhang have contributed equally to this study and share

first authorship.

Highlights 

The diabetic hyperglycemia aggravates cerebral ischemia/reperfusion injury.



The level of the mitochondrial fusion protein, Mfn2, was decreased and the mitochondrial fission protein, Fis1, was increased in the hyperglycemia group.



Pre-treatment with CoQ10 decrease the infarct volume, the neurological deficit score, and neural karyopyknosis.



CoQ10 reduces the ischemic injury enhanced by hyperglycemia through mediating mitochondrial division/fusion.



CoQ10 may be promising as a neurotherapeutic target for human cerebral ischemia/reperfusion injury under hyperglycemia.

Abstract The purpose of this study is to investigate the effect of coenzyme Q10 (CoQ10) on focal cerebral ischemia/reperfusion (I/R) injury in hyperglycemic rats and the possible involved mechanisms. In this study, we established the transient middle cerebral artery occlusion (MCAO) for 30 min in the rats with diabetic hyperglycemia. The neurological deficit score, 2,3,5-triphenyltetrazolium chloride (TTC) staining and pathohistology are applied to detect the extent of the damage. The expression of Fis1, Mfn2 and Lc3 in the brain is investigated by immunohistochemical and Western blotting techniques. The results showed that the streptozotocin-induced diabetic hyperglycemia and MCAO-induced focal cerebral ischemia were successfully prepared in rats. In the hyperglycemic group, the neurological deficit scores, infarct volumes, and number of pyknotic cells were higher than that in the normalglycemic group at 24 h and/or 72 h reperfusion. Pretreated with CoQ10 (10 mg/kg) for four weeks could significantly reduce the neurological scores, infarct volume, and pyknotic cells at 24 h and/or 72 h reperfusion of the hyperglycemic rats compared with non-CoQ10 pretreated hyperglycemic animals. Immunohistochemistry and Western blotting showed that pretreatment with CoQ10 or insulin could significantly reduce the expression of Fis1 protein in the brain at 24 h and 72 h reperfusion. Inversely, an significantly increased expression of Mfn2 was observed in the rats CoQ10 or insulin pretreated at 24 h and/or 72 h reperfusion when compared with matched hyperglycemic rats. These results demonstrated that hyperglycemia could aggravate ischemic brain injury. Pretreatment with CoQ10 might ameliorate the diabetic hyperglycemia aggravated I/R brain damage in the MCAO rats by maintain the balance between mitochondrial fission and fusion.

Keywords: ischemia; coenzyme Q10; hyperglycemia; mitochondria; fission 1; mitofusin 2

Introduction Epidemiological studies demonstrate that diabetes is a well-known risk factor for increasing mortality and morbidity in stroke patients, and hyperglycemia enhances the brain’s vulnerability to ischemia injury, which ultimately results in worse prognosis [1,2]. Various clinical and experimental studies have shown that the occurrence of ischemic stroke in the presence of hyperglycemia can aggravate cerebral ischemia/reperfusion (I/R) injury, expand the infarct volume, delay neurological function recovery, and increase the neuronal degeneration and death [3,4]. The mechanisms underlying hyperglycemia-aggravated brain I/R damage are not yet recognized and the effective preventive or control measures are still not approached. During this pathological process, oxidative stress is a common culprit in the pathophysiology of both diabetes and ischemic brain injuries [5]. Hyperglycemia increases the radical generation of excessive superoxide anions during reperfusion following the cerebral ischemia and exacerbates cerebral damage with oxidative stress [6,7]. Accordingly, the modulation of some detrimental pathways can offer promising opportunities for the management of ischemic injuries in diabetes. Indeed, it has been previously reported that reactive oxygen species (ROS) or oxidative stress plays a detrimental role in cerebral I/R injury in diabetic rats [5,8]. Moreover, the cell death or apoptosis induced by I/R is obviously increased by hyperglycemia and acidosis, in which the cell death be associated with mitochondrial dysfunction and mitochondrial calcium overload [9,10]

Coenzyme Q10 is a potent antioxidant and free radical scavenger. In addition to its antioxidant properties, CoQ10 plays a key role in mitochondrial oxidative phosphorylation and ATP production and is found in the membranes of many organelles in humans [9]. Some previous evidences suggest that the protective effect of CoQ10 is associated with its ability to decrease ROS production, to affect antioxidants and to generate ATP, to stabilize mitochondrial membrane potential, to improve mitochondrial respiration, to inhibit mitochondria-mediated cell death pathway and to activate mitochondrial biogenesis [10,11]. CoQ10 mainly decreases oxidative stress and lipid peroxidation, promotes inflammatory response, and protects neurovascular tissues after the reperfusion following focal cerebral ischemia [12].

Additionally, in the previous study, CoQ10 pretreatment was shown to reduce the production of ROS and apoptosis so that plays a neuroprotective role in cultured cell [10]. Unfortunately, whether CoQ10 has a neuroprotective effect on streptozotocin (STZ)-induced diabetic rats after cerebral I/R and the possible underlying mechanism are still under investigation. It is our speculation that CoQ10 might ameliorate hyperglycemia-aggravated I/R brain damage through maintain mitochondrial fission and fusion balance. In this study, Sprague-Dawley rats were injected with STZ to induce type I diabetes. The focal cerebral I/R model was induced by transient middle cerebral artery occlusion (MCAO) using the intraluminal filament technique. The present study was designed to observe the effects of CoQ10 pretreatment on hyperglycemia-aggravated I/R brain damage of the rats with respect to neurological deficit, infarction, pathohistology and the expression of Fis1, Mfn2 and Lc3.

Materials and methods Animals and reagents Healthy SPF male Sprague-Dawley rats with body weights of 200-220 g were provided by the Experiment Animal Center of Ningxia Medical University (Yinchuan, China). Animals were housed with three animals per cage. The rats were kept in a room under standard laboratory conditions with free access to food and water, controlled room temperature (22 ± 1°C), and a cycle of 12 h light / dark. Procedures were performed with the approval of the Ethics Committee for Animal Experimentation. All efforts were made to minimize animal stress and to reduce the number of rats used for this study. Drugs and reagents were as follows: polyclonal anti-Lc3 II/I antibody (Abcam, USA), polyclonal anti-Fis1 antibody (Abcam, USA), and polyclonal anti-mitofusin2 antibody (Abcam, USA); anti-β-tubulin mouse monoclonal antibody (CWBIO, Beijing, China); horseradish peroxidase (HRP)-conjugated secondary antibody (ZSGB-BIO, Beijing, China); streptozotocin (Calbiochem, Germany); and CoQ10 (C59H90O4,

Sigma,

USA);

Insulin

(Novo

Nordisk,

Tianjin,

China);

2,3,5-triphenyltetrazolium chloride (TTC, Amresco, Solong, CA, USA) was purchased from Sigma-Aldrich (Shanghai, China). Experimental groups Rats were randomly divided into four groups (Fig. 1), 1) Normoglycemic rats with MCAO (NG), saline pretreated and MCAO; 2) Diabetic hyperglycemic rats with MCAO (HG), STZ-induced and MCAO; 3) Hyperglycemic rats with MCAO pretreated with CoQ10 (CoQ10), STZ-induced, CoQ10 10 mg/kg.d (CoQ10 1% aqueous solution of Tween 80) injected intramuscularly for 4 weeks [11,13,14], and MCAO; 4) Hyperglycemic rats with MCAO pretreated with insulin (insulin), STZ-induced, insulin (2 U/d) injected subcutaneously for 4 weeks, and MCAO. Each group was divided into sham, reperfusion at 24 h, and reperfusion at 72 h subgroups (n=10, per subgroup).

Diabetic hyperglycemia model The rats were fasted overnight and injected intraperitoneally with STZ (60 mg/kg) that was freshly dissolved in 0.1 M citrate buffered saline (pH 4.5). Age-matched rats receiving the same volume of 0.1 M citrate-buffered saline served as normoglycemic controls. Diabetes was confirmed by the measurement of blood glucose levels 2 days after STZ injection using a One Touch glucometer. Animals with a blood glucose level higher than 16.8 mmol/L were designated the diabetic rats. Ischemia and reperfusion model Focal cerebral ischemia was induced by transient MCAO using the intraluminal filament technique. The rats were anaesthetized, and a midline incision in the ventral side of the neck was made to expose the right and left common carotid arteries. During the period of surgery, the body temperature of the rats was maintained at approximately 37°C with a heating pad and lamp and was monitored by a rectal thermometer. The left common carotid artery, internal carotid artery and external carotid artery were then exposed through a midline incision in the neck, and a monofilament nylon suture (external diameter 0.28-0.38 mm) with a silicone-coated tip was inserted into the internal carotid artery approximately 16 to 18 mm from the

bifurcation through the external carotid artery stump and gently advanced to occlude the middle cerebral artery [15]. The sham group received only an incision of the cervical skin to expose the common carotid artery, without occlusion of blood flow. After 30 min of MCAO, we gently removed the intraluminal filament and regional cerebral blood flow was restored to normal. Then, the external carotid arteries were permanently ligated. After the surgery, the animals were returned to their cages, were allowed free access to food and water, and closely monitored. The neurological deficit scores, body weight and blood glucose were assessed 24 h and 72 h after reperfusion following focal cerebral ischemia 30 min. Upon 24 and 72 h of reperfusion after 30 min of ischemia, rats were weighed and then euthanized with an intraperitoneal injection of chloral hydrate. For TTC staining 2 mm thick coronal sections were taken at the level of -0.3 mm from the bregma. The brain samples were sectioned at 5 μm for Haematoxylin＆Eosin (HE) staining, histopathology, and immunohistochemistry. The brains from the ischemic caudoputamen and the cortex were stored at -80 °C for Western blotting analysis. Evaluation of neurological deficits The animals were subjected to a neurological examination after 24 h reperfusion using an established scoring system (Feeney score) [15,16]; the observer was blinded to the identity of the treatment groups. To test beam walking ability, the rat was made to walk on a 2.5 cm wide, 80 cm long, 10 cm high beam. The neurological deficit was scored on a 6-point scale: 0, walking without falling; 1, walking with a fall rate of less than 50%; 2, walking with a fall rate of greater than 50%; 3, Affected side completely paralyzed and moving forward with the help of contralateral hind; 4, sitting on the balance bean and unable to walk on it; 5, falling from the beam. Scores 2 to 5 was considered as the indicator of MCAO model. Infarct volume analysis The animals were decapitated and the brains were rapidly collected, sectioned into coronal slices of 2 mm thickness and stained with 2% TTC at 37°C for 30 min in the dark to evaluate the infarct area. The slices were fixed in 4% formalin overnight and then photographed with a digital camera. Unstained areas (pale color) were

defined as infarct area. The hemispheric and infarct areas of each section were traced using ImageJ software (NIH, Maryland, USA). The infarct area was measured by subtracting the intact area in the ischemic hemisphere from the total area in the contralateral hemisphere. Immunohistochemistry The sections were rehydrated through a series of dewaxing washes and were then submerged in citrate solution and heated in a microwave. The sections were treated with 3% H2O2 for 30 min at room temperature to quench endogenous peroxidase activity. The sections were then submerged in citrate solution and heated in a microwave for antigen retrieval before nonspecific binding sites were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min. The sections were incubated with anti-Fis1 (1:200), or anti-Mfn2 (1:200) antibodies at 4°C overnight. The sections were then incubated for 1 h at room temperature. The sections were washed and then incubated with HRP-conjugated secondary antibody. The reaction was visualized with DAB, and the sections were counter-stained with hematoxylin. The sections were then mounted in cover slips and analyzed under a light microscope. Western blotting After reperfusion, the brain tissues were isolated from ischemic penumbra cortices and homogenized in ice-cold buffer, and the protein concentration of the supernatants was measured by a Bio-Rad protein assay kit. Equal amounts of the protein samples were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gels, separated by electrophoresis, and transferred onto a polyvinylidene fluoride membrane (PVDF). After being blocked with 3% BSA, the membranes were incubated for 24 h at 4°C overnight using an anti-Lc3 antibody (1:1000), an anti-Fis1 antibody (1:2000) or an anti-Mfn2 antibody (1:1000). The membranes were washed and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The membranes were then washed again, and the immunoreactive bands were detected using the ECL method. Optical densities of the bands were scanned and quantified using image analysis systems (Bio-Rad, USA). β-tubulin served as an internal control.

Statistical analysis All data were expressed as the means ± SD and analyzed using the SPSS19.0 software (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance (ANOVA) was used for statistical comparisons between the different groups. For nonparametric data, comparisons were made by Mann-Whitney’s U test. Statistical significance was considered to exist at P<0.05.

Results Body weight and blood glucose Compared with NG group, the body weight of 4 weeks of STZ-injection HG group, including CoQ10 and insulin group, was significantly decreased (P<0.05, Fig. 2A). There were no significant differences of body weight among HG, CoQ10 and insulin groups (P>0.05, Fig. 2A). The blood glucose in HG, CoQ10, and insulin groups were significantly increased compared to NG group (P<0.05, Fig. 1B). The CoQ10 intervention group exhibited lower blood glucose in the second week of the treatment compared to HG group (P<0.05, Fig. 2B).

The neurological deficit scores In this study, we showed the neurological changes after CoQ10 pretreatment using the neurological deficit scores. As shown in Fig. 3, compared with the NG group, the neurological deficit scores were higher in HG, CoQ10 and insulin groups at reperfusion of 0 h, 24 h and 72 h after cerebral ischemia. When the animals were pretreated with 10 mg/kg.d CoQ10 for 4 weeks, a significant decreased neurological deficit scores was observed at I/R of 72 in the rats with hyperglycemia (P<0.05, Fig. 3).

Infarct volumes The cerebral ischemic infarct was showed by TTC staining in NG, HG, CoQ10 and insulin groups (Fig. 4A). A significantly increased infarct volume was detected in

the HG group compared to NG group at 24 h reperfusion (Fig. 4B, P<0.05). Pretreatment with CoQ10 significantly reduced infarct volume at 24 h reperfusion in the diabetic hyperglycemia rats with 30 min of MCAO compared to the matched HG group (P<0.05, Fig. 4B).

Histological changes Histological changes were investigated in frontal cortex and caudate nucleus under I/R (Fig. 5A). In the sham group, there was normal brain structure, no clearly swelling or necrosis, and no detectable abnormalities (Fig. 5A). In the NG group, cerebral edema (widened gaps around cells and vessels) and karyopyknosis/pyknosis were observed at 24 h and 72 h of reperfusion (Fig. 5A). In the corresponding time points of reperfusion, the hyperglycemia group showed more visible brain edema and pyknotic neurons (Fig. 5A). At I/R of 24 h and 72 h, the edema and pyknotic cells seem to be reduced by CoQ10 and insulin treatment (Fig. 5A). The number of pyknotic cells were significantly increased in HG group compared to the matched normal blood glucose group (P<0.05, Fig. 5B). In the rats pretreated with CoQ10 or insulin, the numbers of pyknotic cells were significantly decreased at 24 h of reperfusion (P<0.05, Fig. 5B).

Fis1 and Mfn2 expression results by immunohistochemistry Immunohistochemistry of Fis1 was performed at 24 h or 72 h of reperfusion after 30 min of cerebral ischemia. In the NG group, visible Fis1 immunoreactive cells were detected (Fig. 6A). An obviously increased Fis1-labelled cells were observed at I/R 24 h and 72 h of HG, CoQ10 and insulin groups (Fig. 6A). In the reperfusion of 24 h group, the most Fis1 immunoreactive cells were observed. Significant differences was detected in the Fis1 positive cells at 24 h and 72 h of reperfusion between the NG and HG group (P<0.05, Fig. 6B). Fis1-labelled cells at I/R of 24 h and 72 h were significantly decreased in CoQ10 and insulin groups compared to those of HG group (P<0.05, Fig. 6B).

As shown in Fig. 7, a lot of Mfn2-stained cells were found in sham groups. A scattered Mfn2-positive cells were demonstrated in HG group (Fig. 7A). Compared with normoglycemic rats, a decreased Mfn2-positive cells was demonstrated in HG group at reperfusion of 24 h and 72 h (Fig. 7B). The rats in CoQ10 or insulin groups were significantly revealed increase of Mfn2-immunopositive cells at 72 h of reperfusion compared with the HG group (Fig. 7B, P<0.05).

Western blot To further delineate the neuroprotective effects of CoQ10 to ischemic brain damage in diabetic rats, we examined the expression of Fis1, Mfn2, and Lc3-II/I by Western blotting (Fig. 8A). The bands corresponding Fis1, Mfn2, and Lc3-II/I were revealed in NG, HG, CoQ10, and insulin groups (Fig. 8A). Compared with NG group, the Fis1 expression at 72 h of reperfusion was significantly increased in HG group (Fig. 8B). The relative values of Mfn2 at 24 h and 72 h reperfusion were obviously decreased in HG group compared to matched NG group, respectively (Fig. 8B). The ratio of Lc3-II/Lc3I in HG group was significantly increased at reperfusion of 24 h compared to the NG group (Fig. 8B). Pretreatment with CoQ10 or insulin was significantly decreased the ratio of Lc3-II/Lc3I (Fig. 7B).

Discussion Cerebral ischemic injury is one of the most serious medical problems that can occur in the population [17]. Diabetes has been reported to be the most important risk factor for cerebral ischemia and reperfusion injury because it can induce and aggravate ischemic brain damage, resulting in a series of serious adverse effects [18]. The mechanisms of hyperglycemia aggravated cerebral ischemic injury is still under investigation, in which the effective preventions of hyperglycemia aggravated damage have not yet been confirmed [19]. The results of this study show that the STZ-induced diabetic hyperglycemia and MCAO-induced focal brain ischemia were successfully prepared in rats. Hyperglycemia increased neurological deficit scores, infarct volumes and numbers of pyknotic cell at I/R of 24 h and/or 72 h. These results were consistent

with the previous reports [20] and further demonstrated that hyperglycemia can aggravate the brain injury under I/R [8]. CoQ10 is a natural ubiquinone, and it plays a key role in mitochondrial energy (adenosine

triphosphate)

production

in

the

respiratory

chain

and

in

extra-mitochondrial redox activity in the cell membrane [21,22], which it is able to transfer electrons from Complex I or II to Complex III [23]. Importantly, it is also known to enhance the availability of other antioxidants. CoQ10 is able to reduce protein oxidation and improve antioxidant potential [10,11]. Supplementation of exogenous CoQ10 can increase the level of mitochondrial coenzyme Q10, decrease the ratio of acetyl CoA / CoA, enhance the activity of succinate-cytochrome reductase, enhance the utilization of oxygen in oxygen-free state, and promote the oxidation of glucose [24]. In energy metabolism disorders, the direct supply of energy for the production of ATP is increased, and reperfusion after ischemia has a protective effect on the cells [9]. In the present study, Pretreated with CoQ10 (10 mg/kg) for four weeks resulted in a significant reduction in the neurological scores, infarct volumes and pyknotic cells at I/R of 24 h and/or 72 h. It is suggested that pretreatment with CoQ10 might ameliorate the I/R brain damage aggravated by diabetic hyperglycemia in the MCAO rats. We already observed in vitro that treatment with CoQ10 before the cells exposure to ultraviolet B could obviously increase the astrocytes viability [10]. Recent studies suggest that CoQ10 can reduce insulin resistance and suppress β-cell failure to reduce blood sugar and delay cardiovascular complications in diabetes [25,26]. CoQ10 can effectively reduce the production of ROS, thereby reducing ischemic neuronal damage [27]. CoQ10 might be promising as a candidate therapy for human cerebral I/R injury under hyperglycemia. Studies have shown that hyperglycemia increases mitochondrial fragmentation and inhibits mitochondrial fusion, leading to hyperpolarization of mitochondria and increasing the production of ROS, which increases in cerebral ischemic injury [28,29]. Mitochondria are highly dynamic organelles that undergo constant fission and fusion to maintain mitochondrial structure and function. When the balance is broken, the morphology of mitochondria can form a new individual and network structure,

seriously affecting the function of mitochondria and regulating the apoptosis of cells [30]. Recent researches are indicated that mitochondrial fission and fusion events are mainly regulated by fission 1 (Fis1) for fission, as well as mitofusin 1, 2 (Mfn1, 2) for fusion [31,32]. Mitochondrial division would be restrained if Fis1 were absent [33]. In the present study, significant increase of Fis1-labelled cell and decrease of Mfn2-stained cell at 24 and 72 h reperfusion in the hyperglycemic group was detected compared with that of the non-hyperglycemic group. The results was further conformed by Western blotting. These findings suggest that diabetic hyperglycemia enhance

the

mitochondrial

fission/fusion

imbalance,

induce

mitochondrial

fragmentation and inhibits mitochondrial fusion, and aggravate cerebral ischemic injury [19]. Pretreatment with coenzyme Q10 can reduce cerebral I/R injury in diabetic hyperglycemia,

but

the

mechanism

underlying

CoQ10

ameliorating

the

hyperglycemia-aggravated cerebral ischemic injury is still unclear. In the present study, the results showed that the rats pretreated with CoQ10 significantly reduced Fis1-labelled cells and the expression of Fis1 protein at I/R of 24 h and 72 h. Inversely an increased Mfn2-immunostained cells at 72 h reperfusion and high expression of Mfn2 protein at I/R of 24 h and 72 h were detected in the CoQ10 pretreated group when compared to without CoQ10 group. These findings suggested that CoQ10 decrease the fission (Fis1) and increase the fusion (Mfn2), maintain or improve the balance between the mitochondrial fission and fusion, and reduce cerebral I/R injury under diabetic hyperglycemia. Recent study indicated that CoQ10 activates mitochondrial biogenesis to protect the astrocytes with I/R [10]. It is reported that CoQ10 obviously up-regulates the expression of peroxisome proliferator-activated receptor (PPAR) coactivator-1α (PGC-1α), by which the mitochondrial biogenesis is activated [34]. Studies have shown that CoQ10 participate in several aspects of cell signaling, inhibit oxidative stress, and affect the expression of genes associated with metabolism, cell signaling transduction, and mitochondrial function [35].

In the present study, Western blotting showed that the expression of LC3II was up-regulated in the rats with diabetic hyperglycemia and further up-regulated at 24 h of reperfusion compared to non-hyperglycemia group. Pretreatment with CoQ10 and insulin may significantly reduce the expression of LC3II at 24 h and 72 h of reperfusion after MCAO. It suggests hyperglycemia participate in modulating autophagy following the cerebral I/R. Autophagy plays a central role in many neurodegenerative diseases, such as stroke and brain injury [36]. LC3-II is a widely used marker for autophagosome, which correlates with increased levels of autophagic vesicles. However, it is not known, whether enhanced autophagy is protective or destructive in the ischemia process. In recent studies, it has been found that autophagy plays an important role in the development and progression of diabetes mellitus, which may be related to the number of β-cells and the secretion of insulin. It is a self-protecting mechanism of cells, which the maintenance of the structure and function of β-cells requires autophagy to participate in regulation [37]. Cerebral I/R injury promotes protein kinase B and glycogen synthase kinase-3 phosphorylation, which in turn activates autophagy and accelerates the formation and activation of lysosomal enzymes that can encapsulate autophagosomes [38]. However, our study showed that insulin (2U/d) treatment could not reduce blood glucose of diabetic hyperglycemia rats. We are not sure if it was relating with the rats or drugs we used in this study. Some reports shown that the blood glucose level could not be decreased by insulin therapy in some diabetic patients and the possible mechanism might be associated with PI 3-kinase-dependent regulation of Akt [39,40]. However, the limitation of this study is objective. In order to understand the role of mitochondrial dynamic balance in hyperglycemic ischemia/reperfusion and the mechanism involved in CoQ10 pretreatment, future study could be carried out in more therapeutic models and more factors associated with mitochondrial dynamic. In summary, in the present study the STZ-induced diabetic hyperglycemia and MCAO induced focal brain ischemia were successfully prepared in rats. Hyperglycemia could increase neurological deficit scores, infarct volumes, and number of pyknotic cells at 24 h and/or 72 h of I/R. Pretreatment with CoQ10 could

alleviate hyperglycemia induced above disorder, and reduce the high-expression of Fis1 and lower-expression of Mfn2 hyperglycemia-induced. It is our conclusion that the hyperglycemia aggravate ischemic/reperfused brain injury. Pretreatment with CoQ10 might ameliorate the I/R brain damage aggravated by diabetic hyperglycemia in the MCAO rats by maintain the balance between mitochondrial fission and fusion.

Funding This work was supported by the National Natural Science Foundation of China (81560208，81360184).

Conflict of interest statement The authors have no conflicts of interest to declare.

Acknowledgments The authors thank Jian-Da Dong, Yi Ma, Feng-Ying Guo, and Wen Yang for work relating to the setup of the MCAO model. Mr. Faisal UL Rehman for editing and proof-reading..

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Figure legends Fig. 1 Summary of groups. 24 or 72: 24 h or 72 h reperfusion; T80: aqueous solution of Tween 80; Q10: 1% CoQ10 aqueous solution of Tween 80; ins: insulin; M: MCAO; WB: Western blot; IHC: immunohistochemistry. Fig. 2 Body weight and blood glucose. (A) Summary of body weights. (B) Summary of blood glucose levels. *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=10, per sub-group). 1W, 2W, 3W, and 4W=the first, second, third, and fourth week of therapy(CoQ10 / insulin). Fig. 3 Summary of the neurological deficits scores. *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=10, per group). Fig. 4 Infarct volumes at 24 h after reperfusion with 30 min of MCAO. (A) Representative TTC-stained sections. An increased infarct in HG group (pale color). (B) Bargragh of infarct volumes. *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=3, per group). Fig. 5 Representative photomicrographs in the cortex. (A) The representative histogragh, no pyknotic cells in sham rats; cerebral edema, pyknotic neurons (arrows) in NG, HG, CoQ10, and insulin group; surviving neurons (empty arrows) in NG, CoQ10, and insulin groups. Scale bar = 100 μm. (B) Summary of pyknotic neurons, *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=4, per sub-group in sham and 24 h reperfusion; n=5, per group in 72 h reperfusion). Fig. 6 Immunohistochemistry of Fis1 in cortex of peri-infarction areas. (A) Representative immunostaining of cortex, a few of Fis1- stained cells (arrows) in NG, CoQ10, and insulin groups; an increased Fis1- stained cells (arrows) in HG group; Scale bar = 50 μm. (B) Summary of Fis1-stained cells, *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=4, per sub-group in sham and 24 h reperfusion; n=5, per group in 72 h reperfusion). Fig. 7 Immunohistochemistry of Mfn2 in cortex of peri-infarction areas. (A) Representative immunostaining of cortex, a lot of Mfn2- stained cells in NG, CoQ10, and insulin groups (arrows); a decreased Mfn2- stained cells in HG group (arrows); Scale bar = 50 μm. (B) Summary of Mfn2-stained cells, *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=4, per sub-group in sham and 24 h reperfusion; n=5, per group in 72 h reperfusion). Fig. 8 Western blot of Fis1, Mfn2, and LC3 II/I. (A) Representative Western blotting of the ischemic hemisphere brain. (B) Summary of the relative values of Fis1, Mfn2,

and ratio of LC3 II/I, , *P<0.05 vs. NG group, respectively; #P<0.05 vs. HG group (n=3, per sub-group).