Neuroprotective effect of Indobufen against pyroptosis following cerebral ischemia-reperfusion injury both in vivo and in vitro

Background: Indobufen is a new generation of antiplatelet agents and has been shown to have antithrombotic effects in animal models. However, the ecacy of Indobufen on cerebral ischemia/reperfusion (I/R) injury and its mechanisms remain to be investigated. Methods: In this study, the ecacy of Indobufen with both pre- and post-treatment on rats suffering middle cerebral artery occlusion/reperfusion (MCAO/R) was investigated. Furthermore, human umbilical vein endothelial cells (HUVECs) were cultured and underwent oxygen glucose deprivation/reoxygenation (OGD/R) injury for in vitro studies. Relationship between Indobufen and pyroptosis associated NF-κB/Caspase-1/GSDMD pathway was preliminarily discussed. Results: The pharmacodynamic tests revealed that Indobufen ameliorated I/R injury by decreasing the platelet aggregation, infarct size, brain edema and neurologic impairment in rats and rescuing cell apoptosis/pyroptosis in HUVECs. The underlying mechanisms were probably related to pyroptosis suppression by regulating the NF-κB/Caspase-1/GSDMD pathway. Conclusion: Overall, these studies indicates that Indobufen exerts protective and therapeutic effects against I/R injury by pyroptosis suppression via downregulating NF-κB/Caspase-1/GSDMD pathway.


Background
Stroke is characterized by high morbidity and disability rate, and is also one of the deadliest diseases globally [1]. However, only a limited number of strategies are now available for ischemic stroke [2,3]. Therefore, the prevention and treatment of stroke has important clinical value. Indobufen (lab no. K3920, Figure 1), with a chemical structure of 2-[p-(1-oxo-2-isoindoline) phenyl] butyrate, is a new generation of antiplatelet aggregation drugs that reversely inhibits platelets, cycoperxygenase and reduces the production of thromboxane A2 (TXA2) [4]. We have previously reported that Indobufen could inhibit the coagulation process and reduce thrombosis [5], so we speculated that Indobufen may also have therapeutic and preventive effects against ischemia/reperfusion (I/R) injury.
Ischemic stroke is attributed to the blockage of brain blood circulation, which leads to the accumulation of hypoxia and toxic substances such as in ammatory cytokines, as well as the death of brain cells, and may induce subsequent neurobehavioral de cits in stroke survivors. There are three classical forms of brain cell death: necrosis, apoptosis and pyroptosis, among which pyroptosis, also known as Caspase-1 dependent cell death, is recently a hot topic [6]. Caspase-1 is activated by the in ammasome NLRP3 after receiving the signal of NF-κB nuclear translocation, followed by the activation of in ammatory cytokines IL-1β and IL-18 into active forms, which play important roles in the maintenance and development of in ammatory responses [7]. In addition, Caspase-1 also speci cally cleaves Gasdermin D protein (GSDMD) into carboxy-terminal Gasdermin-c (GSDMD-C) and amino-terminal Gasdermin-n (GSDMD-NT), where GSDMD-NT is associated with cell membrane rupture [8]. In this study, we investigated by prophylactic and therapeutic administration the effects of Indobufen on I/R injury as well as its anti-apoptosis/apoptosis e cacy in rat brain and HUVECs cells, conjecturing that the molecular mechanisms involved signal regulation of NF-κB/GSDMD/NLRP3 both in vivo and in vitro. This study may provide us new insights into the effects of Indobufen on I/R injury and suggest Indobufen as a potential strategy for ischemic stroke.

Animals and Treatments
Male SPF Sprague-Dawley (SD) rats (250-300g) were purchased from Qinglongshan Animal Farm of Nanjing, China (Production License No. SCXX (su) 2018-0019; SCXK (zhe) 2019-0002). All animals were housed under a 12 hour light-dark cycle at temperature of 22-26℃ in a room of 40-70% humidity. Rats were fasted for 12 hours before the surgery. Rats were randomly divided into 4 groups: (1) sham operation group; (2) MCAO/R model group; (3) Indobufen (20 mg/kg) group; (4) Aspirin (10 mg/kg) group. The in vivo trial was divided into 5 day prophylactic administration (5-day tests) and 15 day therapeutic administration (15-day tests). In the 5-day prophylactic administration experiment, oral administration was given for 5 days, and surgery was performed 30min after the 5th-day administration.In the 15-day treatment administration experiment, the drugs were administered 3 hours after the operation, once a day for 15 days. All rats were kept at a relative humidity of 55 ± 5% (at 25 ± 2 ℃) in compliance with institutional guidelines of China Pharmaceutical University and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). All experiments were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (SYXK (Su) 2016-0011).

Drug administration
Indobufen (Huadong medicine Co., LTD) and Aspirin (Shanghai yuanye Bio-Technology Co., LTD) were both dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na). Drugs were administered intragastrically with a volume of 0.5mL/100g body weight. Sham and model group were given the same volume of 0.5% CMC-Na.

In vivo MCAO/R establishment
MCAO/R injury was established as previously described after anesthetization with 3% iso urane [9] with 2h of ischemia followed by reperfusion. After the operation, the rats with successful surgury according to the behavioral scoring were retained. All efforts were made to minimize animal suffering and reduce the number of animals used. Five rats died from anesthesia and ten died from intracranial hemorrhage, which were excluded in the analysis.
2.4 Neurological defect scoring, cylinder test and postural re ex test One days following MCAO/R, the neurological function of the rats was scored from 0 to 4 according to Bederson's method [10]. For the limb-use asymmetry test and postural re ex test, the rats were tested as previous reported [2]. In 5-day tests: neurological scoring was conducted at 24 h after reperfusion. In 15day tests: cylinder test and posture re ex test were carried out 30 min after drug administration as shown in Figure 2B.

Measurement of infarct size
In the 5-day tests, rats were sacri ced by cervical dislocation 24h after the MCAO surgury and in the 15day tests, rats were sacri ced by cervical dislocation 30min after the last drug-administration. 2,3,5-Triphenyltetrazolium chloride (TTC, Sangon Biotech CO., LTD) stainng were used to measure brain infact size [11]. The percentage of infarction was calculated as following: Infarct rate (%) = (left hemisphere area -right white brain infarction area)/left hemisphere area × 100%.

Evaluation of brain edema
Brain sections were weighed right after TTC staining to obtain wet weight. Then the brain was placed in an oven at 110 ℃ for 24 h to dry to constant weight to obtain dry weight, and the brain water content was calculated: Brain water content (%) = (1 -dry weight/wet weight) × 100% [12].

Morris water maze (MWM)
In the 15-day tests, Morris Water Maze evaluation was conducted as mentioned before [13,14]. The escape latency and swimming path were recorded along the rst ve days and percentage of time spent in the quadrant IV and number of crossing platform was measured in probe trial of the fth day of MWM.

Primary cell culture of HUVECs
Primary cultures of Human umbilical vein endothelial (HUVEC) were purchased from ScienCell Research Laboratories, Inc (Cat. #8000). Endothelial cell medium (ECM, ScienCell Research Laboratories, Inc, Cat. #1001) containing 5% heat-inactivated fetal bovine serum (FBS) with 100 U/mL penicillin and 100 μg/mL streptomycin was used and cells was placed in a humidi ed atmosphere with 5% CO 2 at 37 ℃. The medium was changed every 2 days.

ELISA assay
The concentrations of 6-keto-PGF 1α and TXB 2 in the brain tissue homogenates and culture cell supernatants were assessed by ELISA (Nanjing Jiancheng Bioengineering Institute) according to manufacturer's instructions. The levels were normalized to cell protein concentrations.

Real-time PCR
Changes in mRNA levels of IL-18, IL-1β and NLRP3 were detected in brain tissues in the 5-day pretreatment groups, and real-time PCR was performed as described previously [16]. Quantitative PCR At 24 h after MCAO/R in the 5-day pretreatment groups, the rats were anesthetized, 4% paraformaldehyde was perfused through the heart, and brain tissue was quickly removed and xed in 4% paraformaldehyde. Brain samples were performed on xed frozen ultrathin sections (Leica CM3050s, Germany) as previously described [17]. As for in vitro experiments, HUVEC cells were xed with 4% paraformaldehyde for 20 min after OGD/R treatment. Thereafter, brain sections or culture cells were incubated with rabbit antibody NF-κB p65 (1: min. Images were captured with a uorescence microscope (Nikon Ts2R, Japan).

Statistical analysis
The data in this study were expressed as mean ± SD. Kruskal-Wallis test was used for behavioral tests, the number of crosses in the probe test in quadrant IV of MWM. The remaining data were analyzed by one-way ANOVA followed by Bonferroni test post hoc test for equal variance data and Dunnett 3 post hoc test for unequal variance by IBM SPSS 25.0 software. Differences were considered signi cant when pvalues were smaller than 0.05. Photoshop 2020, Image J and GraphPad Prism Version 8.0 were used for statistical analysis.
Representative swimming tracks in MWM of rats on the 2nd day on visible platform were presented in Fig. 3C. Other results of MWM were in Fig. 3D-F. The MCAO/R group had an apparently longer escape latency than the sham group (Day10, Day11, Day13: P < 0.01; Day12, Day14: P < 0.01, Fig. 3D). Compared with model group, escape latency of Indobufen and Aspirin groups were signi cantly reduced in the visible platform trial and escape latency of Indobufen group was signi cantly reduced in the invisible trial (Fig. 3C). In the probe trial, model group had fewer numbers of platform crossings (P < 0.01) compared with sham group, while Indobufen group increased the crossing numbers (P < 0.01, Fig. 3E). Time spent in the target quadrant also showed an increase in Indobufen group (P < 0.01, Fig. 3F).
In both the cylinder test and posture re ex test, Indobufen groups showed a signi cantly difference (Cylinder test: P < 0.01; Posture re ex test: Day 2, 10,15: P <0.01, Day5: P <0.05) compared with model group, and particulary showed better effects than Aspirin in cylinder test (Day10 and 15, P <0.01), which suggested that Indobufen (20 mg/kg) has an obvious effect on behavioral imporvement (Fig. 3G-H).

Indobufen reduces cerebral pyroptosis and regulates NF-κB/Caspase-1/GSDMD expressions in vivo
To evaluate the protective effects of Indobufen on cerebral cell survival especially pyroptosis against I/R injury, 24 h after MCAO/R, pyroptosis related components Caspase-1, NLRP3 and NF-κB-p65 was measured through IF in the brains of rats in 5-day tests. GSDMD, a hallmark of pyroptosis, was stained along with TUNEL assay to distinguish cerebral pyroptosis cells (Fig. 4-5). Fig. 4A-B showed the ipsilateral cortex with Caspase-1 or NLRP3 costainging with DAPI. It was obvious that MCAO/R group showed more apparent Caspase-1/NLRP3 expression compared with sham group, while Aspirin and Indobufen groups exhibited similar expression as sham group. In Fig. 5A, B showed the ipsilateral cortex with NF-κB costainging with DAPI or GSDMD costaining with TUNEL and DAPI. It was also apparent to see that MCAO/R group showed more nuclear import of NF-κB compared with sham group, while Aspirin and Iindobufen groups exhibited similar level of nuclear import as sham group. Results from Fig. 5B showed that GSDMD-positive TUNEL cells were markedly reduced in both the pretreated groups, indicating the protective effect of Indobufen against cerebral cell pyroptosis (Fig. 5C).

Indobufen protects OGD/R induced cell injury in HUVEC cells
HUVEC cells were used to determine the effect of Indobufen on cell viability using the MTT assay. As shown in (Fig. 6A-B), compared with the control group, the HUVEC cells subjected to OGD for 4h and reperfusion for 2h exhibited a decrease in cell viability, and this effect was potently reversed by pretreatment of Indobufen (200μM) and Aspirin (100μM). Moreover, consistent with the in vivo results, Indobufen showed similar effects on TXA2/PGI2 balance. (Fig. 6C).

Discussion
In this research, we rst examined the neuroprotective effects of Indobufen against cerebral I/R injury and proved its possible mechanism of reducing pyroptosis by inhibiting NF-κB/Caspase-1/GSDMD signal pathway.
Our previous studies showed that Indobufen had antithrombotic and anticoagulant effects on rats [5]. Therefore, we speculated that Indobufen may be able to treat cerebral ischemia reperfusion injury. In MCAO/R induced I/R injury, exacerbate damage from numerous in ammatory responses occurs, leading to cell injury and eventually programed cell death, in which pyroptosis is dependent on an in ammatory response and a hot topic recently [20]. In vivo pharmacodynamic tests with pretreatment or posttreatment were managed to assess the e cacy of Indobufen on I/R injury. Aspirin was used as the positive drug for its common use in the clinic [21,22]. In the pre-treatment test, Indobufen signi cantly alleviated I/R injury by reducing infarct area and water edema 24 hours after MCAO/R treatment, and Indobufen could adjust the balance of TXA2 and PGI2 expression after I/R injury both in vivo and in vitro, preliminarily proving its anti-thrombotic ability. In post-treatment tests, the assessment of motor function, learning and memory ability 24 hours to 15 days after reperfusion showed that the I/R injury was signi cantly reduced in rats treated with Indobufen, and Indobufen signi cantly improved spatial cognition and motor function in rats over a longer period of time. In vitro, we simulated I/R injury with HUVEC cells by OGD/R, and Indobufen pretreatment reduced OGD/R-induced cytotoxicity and cellular morphological changes, and increased cell viability. The reduction of I/R injury and the improvement of motor, memory and learning abilities are all closely related to the reduction of brain cell injury and death by Indobufen. Recently, more and more studies have shown that cell pyroptosis, also known as in ammatory death, can be caused by cerebral ischemia-reperfusion injury [23]. A hallmark of pyroptosis is GSDMD-NT-dependent membrane rupture due to Caspase-1 activation, which, unlike apoptosis, allows the release of intracellular pathogens such as pro-in ammatory mediators (e.g., IL-1β and IL-18) [24].
Caspase-1 is stimulated by the typical signal of NF-κB-p65 entry into the nucleus and is activated by canonical in ammasomes such as NLRP3, which promotes brain tissue damage [25]. Therefore, in order to determine the role of Indobufen against cerebral ischemia and cell pyroptosis, IF, PCR, western blot method were conducted 24 hours after MCAO/R to detect the Indobufen in vivo and in vitro NF-κB/Caspase-1/GSDMD gene and protein expression in composition of pyroptosis signaling pathways, and using TUNEL co-staining with GSDMD-NT to measure the apoptosis/pyroptosis levels in the 5-day pretreatment rats. The results showed that Indobufen signi cantly reduced the mRNA expression levels of IL-1β, IL-18 and NLRP3 in the rat brain, reducing the entry of NF-κB into the nucleus, and apparently inhibited the expression levels of Caspase-1 and NLRP3, thus lowering the number of pyroptosis cells exhibiting certain anti-apoptosis/pyroptosis effects. Consistent with the results in vivo, Indobufen had a strong anti-pyroptosis effect on OGD/R-induced cell death by potently decreasing the pyroptosis signal of NF-κB/Caspase-1/GSDMD in HUVEC cell culture. In addition, TUNEL+ GSDMD-NT co-staining also con rmed this anti-pyroptosis effect in vitro. However, since this experiment was only a preliminary study of the relationship between Indobufen and pyroptosis related pathway, further studies are needed on whether Indobufen directly inhibits pyroptosis signals and whether it further activates the downstream signals.

Conclusion
Collectively, we observed that pre-treatment or post-treatment of Indobufen exerts anti-pyroptosis effects against cerebral I/R injury and is bene cial to long term memory, learning and cognitive recovery. The underlying mechanisms are probably related to its potential of down-regulating pyroptosis related NF-κB/Caspase-1/GSDMD signaling pathway. This study suggests that aside from anti-thrombotic and anticoagulant effects, Indobufen may have neuroprotective e cacy in cerebral ischemic injury and is ideal as a new candidate drug for ischemic stroke in clinical trials.     Data are shown as mean ± SD, n=3. *P < 0.05, **P < 0.01 vs. MCAO/R; #P < 0.05, ##P < 0.01 vs. Sham. shown as mean ± SD, n=8. *P < 0.05, **P < 0.01 vs. OGD/R; #P < 0.05, ##P < 0.01 vs. Control Figure 7