Metformin mitigates early brain injury after subarachnoid hemorrhage primarily by increasing SIRT1 and inhibiting inflammation factors

doi:10.21203/rs.3.rs-1605033/v1

Clinically, early brain injury (EBI) which refers to the acute injuries to the whole brain in phase of the first 72 h following subarachnoid hemorrhage (SAH), is intensely investigated to improve neurological and psychological function. It had been revealed that metformin (Met) possesses extensive functions, all can be showed in anti-inflammatory, antiapoptotic and anti-tumor activities. Here, to investigate the anti-inflammatory effect of Met, patients with SAH undergoing embolization of intracranial aneurysm in the Neurosurgery Department of the Jinling Hospital, were reviewed for a clinical retrospective analysis from January 2013 to November 2019. Meanwhile, we examined the effects of Met on the inflammatory factors and apoptosis after SAH and the explanation mechanisms of action in vivo. Met administration 2days before SAH ameliorated cerebral inflammation, brain edema, and neuronal death and improved neurologic function. Collectively, these results indicate that Met reduces the inflammatory response and alleviates early brain injury after SAH, primarily by increasing SIRT1 levels and inhibiting inflammation factors.

Metformin

Subarachnoid hemorrhage

Early brain injury

Apoptosis

Inflammation factors

SAH is a destructive subtype of stroke characterized with a high mortality and morbidity rate (45–50%) and a poor neurologic prognosis, including neurological and psychological deficits [1]. Abundant studies show that EBI, which refers to the acute injuries to the whole brain in phase of the first 72 h following SAH, instead of cerebral vasospasm, plays a key role in the prognosis of patients with SAH [2,3]. Therefore, it will be more significant to discovery new therapeutic approaches for EBI treatment to improve prognosis of patients with SAH. Inflammation and apoptosis are involved in the development of EBI, and the levels of inflammatory factors correlate with bleak prognosis. The major inflammatory factors are TNF-α, IL-1β, IL-6 and c reaction protein (CRP) associated with the progress of EBI. We all know that apoptosis and neuroinflammation are main factors in the pathogenesis of EBI following SAH. Therefore, it is hypothesized that anti-apoptotic therapy and brain neuroinflammation reduction are important aspects for SAH treatment.

Met, a biguanide, is a first-line antidiabetic drug with anti-hyperglycaemic effects [4]. It had been revealed that Met possesses extensive functions, all can be showed in anti-inflammatory, antiapoptotic and anti-tumor activities [5,6]. Regarding pharmacological functions above, Met might be a promising approach to treating SAH. Indeed, some studies have demonstrated the neuroprotective function of Met in different central nervous system [6,7]. Nevertheless, EBI due to Met treatment remain unclear, and the molecular mechanisms of Met are unclear. For exploring the possible effect of Met and mechanisms in EBI after Experimental Subarachnoid Hemorrhage, this study is elaborated.

Ethics statement

All experimental protocols for this study were approved by the Ethics Committee of Jinling Hospital and conformed to the principles of Good Clinical Practice and the Declaration of Helsinki. All retrospective clinical data also had been approved by the patients’ relatives on phone.

2.1. Methods

2.1.1. Clinical Data Collection. From January 2013 to November 2019, patients with SAH undergoing embolization of intracranial aneurysm in the Neurosurgery Department of the Jinling Hospital, Nanjing Medical University, were reviewed for a clinical retrospective analysis. Inclusion criteria were as follows: (1) patients who underwent transcatheter arterial coil embolization for SAH caused by ruptured aneurysm which was confirmed by computed tomography angiography or digital subtraction angiography, (2) patients diagnosed with type 2 diabetes, (3) received treatment within 48 hours of ictus, (4) Hunt-Hess graded I-II. Exclusion criteria were as follows: (1) SAH caused by factors other than aneurysm, (2) patients with insufficient data, (3) patients with documented infections prior to admission as well as patients with known autoimmune diseases.

2.1.2. Clinical Outcome Measures. We collected data on demographic variables (age, sex etc.), Hunt-Hess grade, American society of Anesthesiologists (ASA) physical status classification, operation time, laboratory data (neutrophil, lymphocyte, CRP, blood glucose) on admission and in the first three days after surgery. Continuous variables are presented as the mean ± standard deviation (SD). Categorical variables are described as the number of individuals (percentage). According to the treatment of diabetes, the abovementioned patients were divided into two groups：the Met group (A group)，the non-Met group (B group). We hypothesized that analyzing for the relationship between Met to inflammatory response are involved, might be effective for predicting clinical outcome and animal experiment.

2.1.3. Animal, Groups, and Sample Collection

Adult male C57BL/6 (22–24g) mice were purchased from the Animal Center of Nanjing Medical University. Mice were housed in a temperature (23 ± 2 ℃), humidity of 40% and a reversed 12 hour (h)/12 h light/dark cycle. Mice were supplied with food and water ad libitum. All procedures were followed with the guidelines of the National Institutes of Health on the care and use of animals. After arriving at the vivarium, the mice were accustomed to the location for at least one week before being used in the experiment. Mice were randomly allotted to seven groups: (a) Sham group, (b) SAH + 0.9% normal saline (NS) 24h group, (c), SAH + Met24h, (d)SAH + NS48h group, (e) SAH+ Met48h group, (f) SAH + NS72h group, (g) SAH+ Met72h group. They were injected intraperitoneally with Met (200 mg/kg, Sigma, USA) or an equal volume of the NS 2 days before SAH induction. The Met dose was based on previous researches [8], since they observed beneficial effects of Met in similar models. The experimental SAH model was also performed according to a previous study [9]. Each group of animals were sacrificed at the time point of 24h, 48 h and 72h post-SAH except those for Morris Water Maze（MWM）test. A schematic of the experiment is shown in Supplemental Fig. 1a

2.1.4. Neurologic scoring and body weight

Two researchers blinded to the grouping method using the neurological severity score (NSS) evaluated neurological impairment. The NSS includes tasks on spontaneous activity, symmetry in the movement of all four limbs, forepaw outstretching, climbing, body proprioception, and response to whisker stimulation[10]. A lower score shows better neurological function. Body weight change was expressed as a ratio of body weight after surgery to body weight before surgery[11]

2.1.5. MWM test

MWM (XinRuan Science and Technology Company, Shanghai) test referred to the protocol [12] was used to assess cognitive and memory function after SAH. In brief, mice were trained four times per day for five days during the acquisition phase. In each acquisition experiment, the mice were given 60 second(s) to find a platform 2 cm below the water surface. Once finding and arriving at the platform, mice could stay on the platform for 15s. If they missed the mission, mice were led to the place and stayed there for 30 s. A probe trail was performed on the next day after five consecutive acquisition phases.

2.1.6. SAH severity evaluation

Two investigators blinded to the experiment group using the Sugawara's grading scale. Briefly, six parts of the basal brain were scored ranging from 0 to 3, based on the amount of subarachnoid blood clot. Grade 0: no subarachnoid blood, grade 1: minimal subarachnoid blood, grade 2: moderate clot, grade 3: blood clot and recognizable arteries.

2.1.7. Evaluation of the brain edema

After the mice were sacrificed by using 10% chloral hydrate (0.35 ml/100 g), the whole brain was divided into the hemispheres and cerebellum, and weighed as wet weight. Brain specimens were dried at 110℃ for 3 days and weighted again as dry weight. The water percentage is calculated as: % H2O = (wet weight-dry weight)/wet weigh* 100% [13].

2.1.8. Enzyme-linked immunosorbent assay (Elisa) and the blood glucose

The blood samples were collected from the left ventricle of mice before cardiac perfusion. The samples were centrifuged at 12,000 rpm for 30 min at 4°C. The levels of IL-1β, IL-6, TNF-a and CRP were measured with ELISA kits according to the instructions (Multisciences, Zhejiang, China). The blood glucose was measured by glucometer (Contour TS, Bayer, German).

2.1.9. Assay of blood brain barrier (BBB) disruption

Evans blue (EB) extravasation method was used to quantitatively evaluate BBB. EB dye (2%, 5 ml/kg, Sigma-Aldrich) was injected by the tail vein and circulated for 1 h. After deep anesthesia, the mice were sacrificed by intracardiac perfusion with saline. The brain hemispheres were weighed and submerged in formamide (10 ml/g, Yatai United Chemical Industry, Wuxi, China), then incubated at 60℃. After 24 h, the extravasations were measured for absorbance of EB at 620 nm by spectrophotometer.

2.1.10. Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining

TUNEL Detection Kit (Beyotime, China) was used for TUNEL staining according to the instructions. Specifically, brain sections (8μm) were incubated with neuron antibody overnight at 4°, then with TUNEL for 1h at 37°C in the dark. After washed with PBS, they were stained with DAPI for 1 min.

2.1.11. Western blot analysis

After being anesthetized with 10% chloral hydrate (5ml/kg, intraperitoneal injection), the bilateral temporal tissues near to the clots (Fig. 1b) were collected for western blotting. Whole protein extraction was conducted according to the previous protocol [14]. Primary antibodies were as follows: SIRT1 (1:20000, MERCK, catalog number: 07-131), Cleaved-Caspase3 (1:1000，Bioss, bs-0081R，lot：BJ03319208)，Bax (1:2000，Proteintech, catalog number:50599-2-2g), β-actin (1:1000，Proteintech, catalog number: 20536-1-AP), Bcl (Affinity, 1:2000, lot#70g9181). HRP conjugated secondary antibodies were goat anti-mouse (1:5000 Proteintech, catalog number: SA00001-1), rat anti-rabbit (1:5000 Proteintech, catalog number: SA00001-2). The protein expression was normalized to β-actin level.

2.2. Statistical analysis

The GraphPad prism (version 8.0) and IBM SPSS (version 25) were used for statistical analysis. The results are expressed as the means ±SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Mann-Whitney test, or Student’s t test were used to analyze MWM. Clinical data was analyzed in the multivariate linear regression analysis in order to identify factors associated with our primary outcome.

3.1. Clinical data. A total of 89 patients were included in this study. Clinical characteristics and hematological parameters of SAH patients are described in Table 1. Factors were included in the multivariate linear regression analysis. There were no significant differences in gender, age, operation time, glucose, ASA classification, Hunt-Hess grade, BMI, smoking and hypertension (P > 0.05). The inflammatory factors variation between on admission and in the first three days after surgery in two groups were listed: A group < B group (P < 0.05), as shown in Table 2.

Table1. Clinical characteristics and hematological parameters of SAH patients

Characteristics	x±s/n(percentage)
age（x±s）	62.29±7.806
Variation of leukocyte（x±s）	1.6155±2.48220
Variation of neutrophil (x±s）	4.8127±7.43214
Variation of CRP（x±s）	3.3403±5.53355
Operation time（x±s）	105.393±37.7433
Blood glucose（x±s）	7.2551±2.76471
BMI（x±s）	24.7491±3.13155
Sex (%)	male（39.3%）female（60.7%）
ASA grade (%)	II (49) III (40)
Hunt-Hess grade (%)	II（55.1%） III（44.9%）
Hypertension (%)	hypertension（77.5%）non (22.5%)
Treatment of diabetes (%)	Met (43.8%) Non-Met (56.2%)
Smoking (%)	smoking（33.7%） non-smoking（66.3%）

Table 2

multivariate linear regression analysis of variation of leukocyte, neutrophil and CRP
Characteristics	Variation of leukocyte		Variation of neutrophil		Variation of CRP
Characteristics	Normalization Coefficient β	P Value	Normalization Coefficient β	p Value	Normalization Coefficient β	p Value
Sex (%)	.156	.424	.199	1.181	− .033	.865
age(x ± s)	.080	.488	.045	.456	− .093	.427
Operation time(x ± s)	− .054	.620	.008	.088	.046	.673
Blood glucose(x ± s)	.064	.560	− .132	-1.391	.139	.213
BMI(x ± s)	.019	.855	− .017	− .188	− .077	.472
ASA grade (%)	− .103	.340	− .096	-1.024	.152	.169
Hunt-Hess grade (%)	− .162	.148	− .133	-1.380	.027	.814
Hypertension (%)	− .121	.262	.041	.437	− .190	.085
Treatment of diabetes (%)	.297	.006	.515	5.668	.277	.011
Smoking (%)	− .287	.136	− .311	-1.876	− .088	.652

3.2. The mortality rate, blood glucose and SAH severity evaluation in animal experiment

The mortality rate of SAH animals did not differ significantly from that of NS group (Table 3). And no significant changes in arterial blood glucose (Fig. 2a) and SAH severity evaluation (Fig. 2b) were observed in any of the SAH model groups.

Table 3

Mortality rates in SAH experiments
Group	Animal deaths (n)	Morality rate (%)
Sham	0/15	0
NS24	7/25	28
Met24	6/25	24
NS48	8/25	32
Met48	9/25	36
NS72	8/25	32
Met72	7/25	28

3.3. Met relieved EBI after SAH

To identify the effects of Met in neurological deficits after SAH, we tested neurological impairments before mice were sacrificed in each group. Neurological impairments were assessed by modified Garcia scale (Fig. 3a), brain water (Fig. 3d) content and BBB disruption (Fig. 3f). The cognitive and memory function (Fig. g-i) were performed by measuring the escape latency, speed and times of crossing the platform. Compared with the Sham group, the escape latency (Fig. 3b) of mice in the NS group was meaningfully prolonged (p < 0.05), and NS group can find the platform (Fig. 3c) slower (p < 0.05). Compared with the NS group, the times of crossing the platform (Fig. 3e) was significantly increased (p < 0.01).

Collectively, the results indicated that the Met treatment ameliorated the cognitive dysfunction in mice after SAH.

We performed modified Garcia scale before mice were sacrificed in each group. Significant neurological improvement was observed in Met group after SAH when compared with NS group as assessed by modified Garcia scale. All Met groups showed better neurological functions, compared with NS group. Especially, NS24 group showed impaired neurological functions worse, compared Met24 group (p < 0.05).

Brain water content was estimated to explore the properties of Met treatment on SAH-induced brain edema during 72h. It was illustrated that brain water content in the NS and Met group was significantly increased, compared with the Sham group. Significant differences were observed in the NS72 and Met72 group. We further ascertained the level of blood-brain barrier disruption. At 24 h after SAH induction, the amount of EB dye exuded decreased, compared with the NS group (p < 0.05). These data suggested that Met treatment could play a meaningful role in EBI of SAH.

3.4. Met upregulated SIRT1 and inhibits neuronal apoptosis following SAH

Western blot analysis revealed that total expression levels of SIRT1 and Bcl protein increased after SAH, especially at Met24 group (Fig. 4b-d). In addition, the protein levels of Bax, Cleaved-caspase 3 were down-regulated after SAH, which can be alleviated by the administration of Met (Fig. 4e-g). Western blot results suggested that Met treatment inhibits apoptosis after SAH. That is to say, the treatment of Met after SAH reduced the expression of Bax and Cleaved-caspase 3, and increased Bcl-2. To detect the effects of Met on neuronal death and degeneration after SAH, apoptosis detection by TUNEL was performed. In the Met and Sham group, rare injured neurons were detected (Fig. 4a). Similarly, TUNEL-positive cells were increased markedly in the NS group and this alteration was reversed by Met group.

3.5. Met reduced inflammatory cytokine production in SAH-induced model

NF-κB is a major protein involved in regulating inflammatory responses including IL-1β, TNF-a and IL-6. To elucidate the possible effect of the protective function of Met, inflammatory factors at different times after SAH were explored. In addition, the inflammatory cytokines IL-1β, IL-6, TNF-a and CRP were significantly increased in NS group, but the trend can be blocked by Met treatment (Fig. 5a-d). It was found that IL-1β, TNF-a and CRP in the NS48 and NS72 group was significantly increased compared with the sham group, which would be ameliorated by Met (p < 0.05). As for IL-6, significant differences were observed in the NS24 and Met24 group (Fig. 5b). These results suggest that treatment of Met contributes to the beneficial effects of reducing inflammation in vivo. This trend of CRP was consistent with the result of previous clinical retrospective analysis.

The SAH is prone to cause brain tissue and nerve cell damage in patients, so perioperative brain protection has become a research concern [15]. In the retrospective case study, we identified Met to be associated with inflammation factors in patients with SAH, after adjusting for potential confounding the multivariate linear regression analysis. Met treatment can decrease the change of leukocyte, neutrophil and CRP in patients with SAH and alleviate inflammatory reaction. Blood neutrophil count has been shown significantly increased after SAH and is associated with poor clinical outcome in patients after SAH [16]. And preclinical data have implicated the immunologic response as a significant exponent of the neuroinflammatory cascade following SAH with neutrophils being one of several crucial factors of this process [17]. Regardless of its precise etiopathogenesis, the relationship among these factors presents clinically significant information for the treatment of SAH.

In the animal experiment, Met treatment also depressed inflammation, illustrating that the protective effects of Met in SAH-induced brain injury are mediated in part by decreasing IL-1β, IL-6, TNF-a and CRP. A growing body of evidence has shown that EBI after SAH is closely associated with inflammatory response [3,18]. In addition to inflammation, numerous factors after SAH can induce neuronal death, such as oxidative damage, neuroexcitotoxicity, and apoptosis. We know that the initiation of neuronal apoptosis is caused by the up-regulation of Cleaved-Caspase 3 and Bax. Pro-apoptotic Bcl-2 family members take part in and activate caspase-9 to trigger further the downstream effector caspases, including caspase-3 [19].

In our study, we found that Met could reverse the situation, decrease the number of apoptotic neurons in the cortex during SAH, suggesting that Met exerted its anti-apoptotic effects on neuronal apoptosis induced by SAH. The expression levels of Bax and cleaved caspase-3 were significantly elevated while Bcl-2 was suppressed in mice after SAH, especially in the model of 48h. Our study clarified the mechanism of Met’s anti- inflammation and anti-apoptotic effects on EBI following SAH. In addition, administration of Met can improve cognitive and memory function. The previous studies [20] demonstrated that Met alleviated EBI following SAH by the up-regulation of Sirt1, and Met pre-treatment can diminish memory impairment by inhibiting apoptosis [21].

According to the previous study [22], the EBI was much worse at 24h when compared with other time points post-SAH. In our study, we found that SIRT1 expression in brain was up-regulated and reach the highest level at 48 h after SAH. Integrating all of our experimental data, Met can alleviate the EBI of the SAH-induced mouse model better, when compared with others.

We also realize there were some limitations in our experiment. Our method and retrospective approach are susceptible to selection bias: Met and Non-Met patients are hospitalized patients, admittedly in part due their risk profile. In addition, other inflammation factors (including IL-1, IL-6 and TNF-a) of patients are missing. Because of the complexity of a common problem in clinical and experimental pharmacology studies, medicine rarely acts at only one molecular target. Consequently, the effect and mechanism of Met on SAH remain unclear, which deserve to distinguish the key molecular targets of drug function. Secondly, in our animal experiment, we ignored Met’s anti-oxidative. After all, there is an interaction between antioxidant stress, anti-inflammatory and anti-apoptosis following SAH.

In summary, we provide the first evidence that Met treatment up-regulate SIRT1 mediated inflammatory injury and apoptosis after SAH. At the same time, in patients with SAH, Met oral administration can decrease the changes of neutrophils, neutrophils and CRP. The general mechanism of Met was illustrated in Fig. 6. Although more research is required, these findings suggest that Met may be a promising beneficial mediator for the treatment of SAH.

early brain injury (EBI), subarachnoid hemorrhage (SAH), metformin (Met), c reaction protein (CPR), American society of Anesthesiologists (ASA), standard deviation (SD), Morris Water Maze（MWM）, neurological severity score (NSS), Enzyme-linked immunosorbent assay (Elisa), blood brain barrier (BBB), Terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL), one-way analysis of variance (ANOVA), Evans blue (EB)

Author contribution Zhong-hua Zhang designed the experiments, analyzed the data and prepared the manuscript. Wei-zhen He and Xiao-ming Zhou selected the materials. Xin Zhang helped with manuscript writing. All authors read and approved the final manuscript.

Funding This work was supported by grants from the National Natural Science Foundation of China

(No. 82071328).

Data availability statement Data sharing is not applicable to this article.

Declarations Conflict Interest The authors declare that they have no conflicts of interest with the contents of this article.

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No competing interests reported.

Metformin mitigates early brain injury after subarachnoid hemorrhage primarily by increasing SIRT1 and inhibiting inflammation factors

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

3. Result

3.2. The mortality rate, blood glucose and SAH severity evaluation in animal experiment

3.3. Met relieved EBI after SAH

3.4. Met upregulated SIRT1 and inhibits neuronal apoptosis following SAH

3.5. Met reduced inflammatory cytokine production in SAH-induced model

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1