A self-developed MyD88 inhibitor across the blood-brain barrier fully reverses acute cerebral ischemia-reperfusion injury


 Background: The acute ischemia-reperfusion injury that occurs after an ischemic stroke is almost inevitable. In treating ischemic stroke, the prevention and treatment of cerebral ischemia-reperfusion injury (CIRI) is an undeniable difficulty clinically. Despite increasing studies being done on the subject, medicines showing effective neurological improvements after reperfusion is still minimal. This study aims to investigate the effects of a self-developed MyD88 inhibitor (TJ-M2010-5) on CIRI and its mechanism.Methods: The middle cerebral artery occlusion (MCAO) model was used to simulate CIRI in mice. BV-2 cells were cultured for the mechanism study by LPS stimulation in vitro. Neurological deficit score and cerebral infarction volume were studied. Immunofluorescence staining was used to measure neuronal damage and apoptosis in the brain. The anti-inflammation effect of TJ-M2010-5 was evaluated by analyzing the expression of inflammatory cytokines, activation of microglia and infiltration of peripheral myeloid cells. The expression of TLR4/Myd88/NF-κB signaling pathway protein was detected by Simple Western. The concentrations of TJ-M2010-5 in blood and brain were analyzed by LC-MS methods.Results: The cerebral infarction volume decreased in mice treated with TJ-M2010-5, with the most prominent decrease in approximately 80% of the original infarction volume. Moreover, the downregulation of apoptosis and NeuN protein expression in the brain of infarction area further indicated that neuronal damage was alleviated. TJ-M2010-5 treatment also reduced the infiltration ratio of peripheral myeloid cells in the cerebral infarction area and increased the proportion of inactive microglia. The inflammatory cytokines decreased after TJ-M2010-5 treatment in infarction area and supernatant. TJ-M2010-5 downregulated the expression of iNOS and inhibited TLR4/MyD88/NF-κB signaling pathway in vivo and in vitro. In addition, TJ-M2010-5 could freely pass through the blood-brain barrier.Conclusions: TJ-M2010-5 has shown to have an excellent therapeutic effect on CIRI by inhibiting TLR4/Myd88/NF-κB signaling pathway to decrease excessive inflammatory response, showing the potential to change the history that there is no effective medicine for the treatment of CIRI. and also has the potential to be utilized in brain neuroinflammatory diseases.


Introduction
Stroke is an acute cerebrovascular disease in which focal neurological loss suddenly occurs in relevant parts of the brain due to infarction or hemorrhage [1]. The most common type of stroke is ischemic stroke, accounting for about 80%. In recent years, vascular recanalization techniques such as thrombolysis and endovascular therapy have made great progress in the treatment of stroke, but the contradiction between the high rate of vascular recanalization and the low clinical bene t ratio of patients is still a prominent issue in clinical practice. There is some evidence that shows the acute immune-in ammatory reaction related to reperfusion can lead to secondary brain injury and expand the scope of brain injury [2,3]. Although increasing studies have been conducted regarding cerebral ischemia-reperfusion injury (CIRI) in the medical eld, targeted therapeutic drugs are still very scarce clinically.
MyD88 is a ligand-protein that plays a key role in the toll-like receptors (TLRs) signaling pathway [10]. After identifying pathogen-related and danger-related molecular patterns (DAMPs and PAMPs), TLRs activate the downstream pathway and lead to a series of in ammatory injuries [11]. Cerebral ischemiareperfusion (I/R) followed by in ltration of peripheral leukocytes and activation of endogenous microglia trigger a series of in ammatory responses [12]. The TLRs of these cells identify DAMPs and then activate downstream MyD88/NF-κB signaling, resulting in increased expression of pro-in ammatory factors [13,14]. Furthermore, the in ammatory responses induce more immune cells to activate and secrete increasing amounts of pro-in ammatory factors, leading to a cascade ampli cation of the in ammatory response, expanding the scope of damage and nally resulting in more neuron loss in CIRI. Recent studies have shown that blocking TLR/MyD88/NF-κB signaling pathway can downregulate in ammatory response and reduce CIRI [15][16][17]. Therefore, the activation of the TLR/MyD88/NF-κB pathway contributes to the neuroin ammatory response of CIRI, which causes an excessive in ammatory reaction.
However, many studies have only shown the effects of inhibiting the in ammatory pathway by gene knockout or biomacromolecules that are di cult to cross the BBB, which is not applicable in clinical settings. By analyzing the structural domain of MyD88, and using computer-aided systems such as drug design and virtual screening, small molecule aminothiazole derivative MyD88 inhibitor, TJ-M2010 series (WIPO Patent Application Number: PCT/CN2012/070811) has been innovatively developed, which can speci cally bind to the TIR domain of MyD88 and prevent homodimerization of MyD88 [18]. The chemical structure of MyD88 and its interaction with the MyD88 TIR domain was provided in the previous study( Fig. 1) [19]. The previous studies have shown that MyD88 inhibitor can inhibit the activation of peripheral innate immune cells such as macrophages and dendritic cells (DCs) [19][20][21], but it is unknown whether MyD88 inhibitor can directly inhibit the activation of microglia across the BBB, which is important for assessing the potential to treat diseases caused by neuroin ammation. Here, we focused on the in ammation inhibition effects of TJ-M2010-5 (TJ-5), a member of MyD88 inhibitor in mice of CIRI and BV-2 microglial cells. The neuroprotective potential of TJ-5 for treating CIRI was evaluated.

Animals
Male C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) weighing 22-28 g and aging 8-10 weeks were used. All the animal experiments were approved by the Institutional Animal Care and Use Committee at the Tongji Hospital, Wuhan, China. All breeding, management, and procedures with animals were in strict accordance with the SPF standard and protocols for the care and use of animals in research. Mice were randomly divided into the following seven experimental groups: sham group, I/R group, vehicle (I/R + saline) group, I/R + TJ-5 5 mg/kg group, I/R + TJ-5 10 mg/kg group, I/R + TJ-5 (I/R + TJ-5 15 mg/kg) group and I/R + Edaravone (I/R + Edaravone 3mg/kg) group.

CIRI model
The mice were anesthetized with 1% pentobarbital sodium solution by intraperitoneal injection, and the body temperature was maintained at 37.0-37.5 °C. The MCAO model was performed according to the Longa method as described previously [22]. Brie y, the right carotid artery was carefully separated and exposed. The distal end of the external carotid artery (ECA) was ligated with a silk ligature, and a siliconcoated embolic suture (Doccol Corporation, MA, USA) was inserted slowly from ECA into the internal carotid artery (ICA) and reached into the middle cerebral artery (MCA). After one hour of ischemia, the embolic suture was withdrawn, the ECA was ligated, and mice were injected with TJ-5 or edaravone intravenously immediately. Then, the mice were placed in a 32 °C incubator, and 1 ml of normal saline was injected intraperitoneally after they were awake. The surgical procedure in the sham group was the same as that of the ischemic model group, but the MCA was not obstructed.

Neurological de cit score
After one hour of ischemia and 24 hours of reperfusion, each group of mice was scored blindly according to the scoring system of the Longa method [22]. The score was 0 for no obvious neurological de cit; 1 for inability to fully extend the left forelimb; 2 for turning to the left; 3 for leaning to the left side while walking; and 4 for inability to walk spontaneously and impaired consciousness.

TTC Staining
The mice were sacri ced after 24 h I/R. The mice brains were harvested for the measurement of cerebral infarct volume. The brains were frozen at -80℃ for 5 min and cut into 5 slices of 2 mm thick brain slices.
The slices were then placed with the front side facing down in a small dish, 2% TTC solution (Sigma-Aldrich, USA) was added and then 4% paraformaldehyde was used for xation. After that, the slices were carefully taken out, the liquid was absorbed with lter paper, and pictures were taken with a digital camera. Image J, a professional image analysis software, was used to measure the infarct area of each brain tissue slice and the volume of brain infarction was calculated.

Cell culture of BV-2 cell line and Stimulation
The culture of BV-2 microglial cells was as previously described [23]. BV-2 cells were seeded on a 6-well plate at a density of 2 × 10 5 cells/well. After 2 days of culture, lipopolysaccharide (LPS #L2880, Sigma-Aldrich, USA) was added at 1 μg/ml to stimulate BV-2 cells. BV-2 cells were pretreated with different concentrations of TJ-5 for 2 h before LPS stimulation. Twenty-four hours later, BV-2 cells and the culture supernatant were harvested for subsequent experiments.

Fluorescence staining
As mentioned earlier, immuno uorescence staining was carried out on para n-embedded brain slices [24]. After standard histological procedures, the brain slices were treated with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction mixture to detect apoptosis according to the manufacturer's protocol (Roche, Germany). Furthermore, the brain slices were also used for immuno uorescence with rabbit anti-NeuN antibody (1:300, CST) and rabbit anti-Iba-1 antibody (1:500, CST). After incubating overnight at 4 ℃, the goat anti-rabbit IgG (1:1000, Alexa Fluor 594conjugated or Alexa Fluor 488-conjugated antibody) secondary antibodies were used and the brain slices were incubated for 2 h at room temperature. Finally, the slices were washed and labeled with 4′, 6diamidino-2-phenylindole(DAPI) for 10 min at room temperature. The images were captured using a uorescence microscope.

Isolation of immune cells
After the mice were anesthetized, 0.9% saline was used for transcardial perfusion. Right brain hemispheres were homogenized in a 6-well plate using 2 ml Hank's balanced salt solution (HBSS, Salarbio) per well. Collagenase IV (1 mg/ml) was added for myelin removal. Then, brain homogenate was ltered through a 70 µm cell strainer and centrifuged at 300 g for 5 min at 4 °C. Afterward, 2 ml of 30% Percoll (Sigma) were added to resuspend brain cells precipitate, The resuspended cells were slowly added to a 15ml centrifuge tube with 3 mL of 70% Percoll. Intermediate layer cells were taken for ow cytometry after density-gradient centrifugation.
2.11. ELISA assay ELISA kits were used to detect the levels of TNF-α, IL-1β, and IL-6 in the mice brain. The brain tissue was then homogenized in 1 mL of phosphate-buffered saline (PBS) by using a tissue homogenizer. Then, the homogenates were centrifuged at 14000 g for 10 min at 4 °C. The supernatant was immediately transferred for measuring, following the manufacturer's instructions. In vitro assay, BV-2 cells were incubated with different treatments. Then, the supernatant was collected and measured.

Western Blot analysis
The total proteins, nuclear proteins, and cytoplasmic proteins in BV-2 cells were extracted and quanti ed using the commercial bicinchoninic acid (BCA) protein quanti cation kit. The prepared proteins were loaded onto an SDS-PAGE to separate the proteins. Then, the proteins were transferred onto polyvinylidene uoride (PVDF) membranes and blocked with 5% non-fat milk for 2 h at room temperature. The membrane was immunoblotted with primary antibodies (GAPDH, TLR4, MyD88, iNOS, p-p38, p-JNK, p-ERK, NF-κB p65) at 4 ℃ overnight and detected with horseradish peroxidase-conjugated secondary antibodies by enhanced chemiluminescence (ECL) method. The band densities were analyzed with Image J.

Simple Western assay
The total proteins, nuclear proteins, and cytoplasmic proteins in brain were extracted and quanti ed using the BCA protein quanti cation kit. Here, Simple Western assay, a new method to detect proteins was used as previously described [25]. Brie y, the prepared proteins were diluted to a concentration of 0.5mg/ml by using a sample preparation kit. Then, according to the instructions of Protein Simple Kit, the prepared reagents were added to the detection plate in turn for an automated capillary western blot system, the WES System. The primary antibodies (iNOS, TLR4, MyD88, NeuN, Cleaved-cas-3, p-ERK, NF-κB p65) were diluted with the antibody diluter at a ratio of 1:50 and the dilution ratio of GAPDH was 1:1000. Compass software (Protein Simple, USA) was used to quantitatively analyze the signal intensity (area) of the protein.

Detection of drug concentration by LC-MS
The mice were injected 15mg/kg TJ-5 intravenously at the time of reperfusion after 1 h of MCAO. Then, mice were anesthetized at 5 min, 10 min, 30 min, 1 h, 2 h, or 6 h post-dose. The blood was collected to prepare serum samples and then right ipsilateral brain (IL brain), left contralateral brain(CL brain), heart, and liver samples were obtained after transcardial perfusion using 0.9% saline. All samples were frozen at -80 °C for later analysis. The liquid chromatography-mass spectrometry (LC-MS) method was performed to detect the concentration of TJ-5 in the samples. Time-concentration curve, area under the curve (AUC), and pharmacokinetic (PK) parameters of TJ-5 were analyzed using PKslover 2.0 PK software [26]. The BBB permeability was evaluated as the brain-to-serum partition coe cient (K p ) which was calculated as AUC brain /AUC serum .

Statistical analysis
All experimental data were statistically analyzed using the professional analysis software GraphPad 8.0. The data obtained were expressed as Mean ± standard error of the mean (SEM) values. The paired comparison was conducted using a t-test and a one-way analysis of variance (ANOVA) was used for comparison among multiple groups. P values < 0.05 were considered statistically signi cant.

TJ-5 improves neurological function, reduces the infarct volume and neuronal loss in CIRI mice
To investigate the therapeutic effects of TJ-5 in cerebral I/R induced acute injury, the neurological de cit test of mice was evaluated after 24 h reperfusion. The results showed that the neurological de cit score of the TJ-5 15 mg/kg group was signi cantly reduced compared with the I/R group (Fig. 2a). The infarct volume was evaluated by TTC staining. We found that TJ-5 signi cantly reduced the infarct volume, showing more effective neuroprotection, especially on the cortex with the increase in dosage amounts (Fig. 2b). The percentage of infarct volume was 33.24% in the I/R group, while the most effective TJ-5 15 mg/kg group was only 6.02%, reducing by approximately 80%, Furthermore, compared with 18.59% in the Edaravone group, TJ-5 at 15 mg/kg achieved a better effect (Fig. 2c). We regarded TJ-5 at 15 mg/kg as the I/R + TJ-5 group in subsequent trials because of its best effect. Immuno uorescence staining of the neuron-speci c nuclear protein (NeuN) in the brain showed that the number of neurons on the injury hemisphere in the I/R + TJ-5 group signi cantly increased when compared to that in the Vehicle group (Fig. 2d). Furthermore, the expression of NeuN protein in the I/R group was signi cantly lower than that in the Sham group, and the results demonstrated that TJ-5 could inhibit this decrease (Fig. 2e and 2f). Together, these data indicate that TJ-5 has powerful neuroprotective effects and reduces neuron loss caused by CIRI.

TJ-5 inhibits apoptosis and alleviates the excessive in ammatory response in CIRI mice
To investigate the protective effects of TJ-5 on CIRI-induced in ammatory responses, the mRNA expression and protein content of TNF-α, IL-1β and IL-6 in brain were detected by RT-qPCR and ELISA, respectively. The results showed that I/R injury signi cantly increased the expression and production of in ammatory factors in the brain tissue, while TJ-5 reduced the expression of TNF-α, IL-1β and IL-6 ( Fig.  3a and 3b). The expression of iNOS in brain was detected via western blotting and RT-qPCR, and TJ-5 was found to inhibit the upregulation of iNOS expression caused by CIRI (Fig. 3c and 3e). TUNEL uorescence staining was used to detect apoptosis. As shown in Fig. 3d, the scope of apoptosis in brain tissue and the apoptotic cells in the ischemic penumbra were signi cantly reduced with the treatment of TJ-5. And the expression level of cleaved caspase-3 was signi cantly upregulated in the I/R group, while the treatment of TJ-5 signi cantly downregulated the activation of caspase-3 (Fig. 3f). These results suggest that TJ-5 reduces the scope of damage by inhibiting the excessive in ammatory response and apoptosis.

TJ-5 inhibits activation of microglia and in ltration of peripheral myeloid cells in CIRI mice
This study further explored how TJ-5 inhibits the excessive in ammatory response at the level of cellular mechanism. Microglia in brain were labeled with Iba-1 staining and the results indicated that the number of Iba-1 positive cells in the I/R group increased, while that in the I/R + TJ-5 group signi cantly decreased (Fig. 4a). Flow cytometry results showed that the proportions of CD11b + CD45hiLy6G + neutrophils (PMNs), CD11b+CD45hiLy6G − mononuclear macrophages (Mo/MΦ) increased and the proportion of CD11b+CD45int inactive microglia decreased in CIRI mice. However, TJ-5 reversed this proportional change and inhibited the activation and in ltration of these in ammatory cells (Fig. 4b). Injecting of TJ-5 at the time of reperfusion reduced brain-in ltrating CD11b+CD45hi myeloid cells and increased the proportion of inactive microglia (Fig. 4c and 4d). Thus, we clarify the level of cellular mechanism at which TJ-5 inhibits both in ltration of myeloid cells and activation of microglia to alleviate neuroin ammation.

TJ-5 inhibits the activation of BV-2 cells after LPS stimulation
To evaluate the anti-in ammatory effect of TJ-5 in vitro, neuroin ammation was induced by LPS in BV-2 cells. The CCK8 experiment con rmed that TJ-5 did not affect the viability of BV2 cells below a concentration of 20 μM (Fig. 5a). Observation of cell morphology under the microscope after 24 h of LPS stimulation showed that after LPS stimulation, the activation of BV-2 cells became amoebic-like and had shown more protrusions, while the cells in the control group were spherical with a small number of protrusions and the activation of BV2 cells were inhibited with TJ-5 intervention (Fig. 5b). The expression of iNOS in LPS-stimulated BV-2 cells was reduced with TJ-5 intervention (Fig. 5c). The levels of TNF-α and IL-6 in the supernatant were detected by ELISA, and treatment with TJ-5 markedly reduced the levels of these pro-in ammatory cytokines in LPS-stimulated BV-2 (Fig. 5d). These results demonstrate that TJ-5 inhibits the activation of BV-2 cells and attenuates the in ammatory reaction induced by LPS.

TJ-5 down-regulates the TLR4/MyD88/NF-κB pathway signaling in CIRI mice and LPS-induced BV-2 cells
TLR4/MyD88/NF-κB signaling pathway played a key role in neuroin ammation induced by CIRI, we investigated this pathway in CIRI mice and LPS-induced BV-2 cells with the treatment of TJ-5. In vivo, the results showed that TJ-5 down-regulated the expression levels of TLR4 and MyD88 and inhibited the phosphorylation of ERK (Fig. 6a-f). Nuclear translocation of NF-κB p65 activated the excessive expression of pro-in ammation factors, and the results indicated that nuclear translocation of NF-κB p65 was obstructed with TJ-5 treatment in CIRI mice (Fig. 6g-i). In vitro, under the stimulation of LPS, the expressions of TLR4 and MyD88, the phosphorylation level of the MAPK pathways and the nuclear translocation of NF-κB p65 increased, but these signals were down-regulated with TJ-5 treatment (Fig. 6j). Therefore, in molecular mechanism, TJ-5 inhibits the TLR/MyD88 signaling pathway to reduce neuroin ammation injury.
3.6. The blood-brain barrier permeability and pharmacokinetics of TJ-5 To investigate whether TJ-5 could directly inhibit microglia activation across the blood-brain barrier, the concentration of TJ-5 in brain was measured and compared with those in serum, liver, and heart. The concentrations of TJ-5 were summarized in Table 1 and the calculated PK parameters were summarized in Table 2. The time-concentration curves of TJ-5 in different tissues indicated that TJ-5 was eliminated according to rst-order kinetics. Surprisingly, there was no signi cant difference between the injury hemisphere and normal hemisphere of brain in BBB permeability of TJ-5. Besides, the drug was quickly distributed from blood to organ after intravenous injection (Fig. 7a and 7b). The concentration ratio of brain to serum at each time point was displayed (Fig. 7c). Thus, we found that TJ-5 may freely pass through the BBB and directly inhibit microglial activation in CIRI mice.

Discussion
This study is the rst to prove that TJ-5 can alleviate cerebral ischemia-reperfusion injury and inhibit the excessive immune-in ammatory reaction in CIRI mice. In addition, current data and pharmacologic evidence demonstrate that TJ-5, a small molecule Myd88 inhibitor, can pass through the BBB and directly inhibit the activation of microglia in CIRI mice. These results may indicate that TJ-5 has the potential for treating ischemic stroke and brain diseases caused by neuroin ammation in clinical settings, opening a novel eld for the clinical treatment of CIRI.
In recent decades, ischemic stroke has become one of the most common causes of disability and death worldwide [27]. Recanalization as soon as possible is the primary treatment after ischemic stroke, but the ensuing reperfusion injury will aggravate the brain injury and expand the infarct size. Unfortunately, there is a lack of effective treatment use in clinical settings currently [2]. At present, there has been a breakthrough in many aspects of the research on the mechanism of CIRI, involving multiple complex pathological processes such as oxidative stress, amino acid toxicity, the release of endogenous substances, in ammation and apoptosis [28]. The interaction between these factors forms a complex regulatory network, which leads to a series of pathological cascade reactions [29]. Although the pathogenesis of CIRI is multifactorial, many experimental studies suggest that the in ammatory response of immune system is essential to the occurrence and progression of CIRI [30]. Although the relationship is still unclear, recent studies continue to elucidate the role of immune-in ammatory response in CIRI. TLR4 has been found to be involved in the development of neuroin ammation in CIRI, which initiates a cascade of in ammatory responses through the TLR4/MyD88/NF-κB signaling pathway [31][32][33]. Furthermore, MyD88 is the core transduction protein in the toll-like receptor signaling pathway [34], and the immune-in ammatory response induced by CIRI in the MyD88 knockout mice is alleviated [35]. Considering that Myd88 activation may enhance neuroin ammation caused by ischemic stroke, we hypothesized that TJ-5 can potently inhibit neuroin ammation to protect against CIRI (Fig. 8).
BBB is the interface that controls the exchange of substances between the central nervous system (CNS) and blood, which brings di culty in developing drugs that can cross such barrier [36]. Currently, not many drugs can enter the CNS e ciently through the BBB, which limits the development of therapies for brain diseases [37,38]. Microglia are resident macrophages in the brain. Studies have shown that the activation of microglia in CNS is an important factor that contributes to the occurrence and development of CIRI [39][40][41]. In CIRI mice, activation of endogenous microglia and in ltration of exogenous immune cells promote the cascade of in ammation in the focal brain and enlarge the scope of injury [42]. Inhibiting the activation of microglia and its transformation to M1 type is the main focus in the research of drugs for treating ischemic stroke [23,40]. However, many studies lack evidence for the BBB permeability of drugs, which is an essential factor as it determines whether drugs can directly affect microglia in the brain. Therefore, the inhibitory effect of TJ-5 on microglia activation and its BBB permeability is examined in the present study.
In this research, we investigated the protective effects of edaravone and different concentrations of TJ-5 in CIRI mice and found that TJ-5 at 15mg/kg was the most effective as the infarction volume was reduced by approximately 80%, which achieved better neuroprotection effects compared with that of edaravone, a medicine currently in use clinically for treating ischemic stroke. This indicates that TJ-5 has signi cant clinical application value for treating CIRI. The ndings also prove that TJ-5 reduces the in ltration ratio of peripheral myeloid cells in the cerebral infarction area, increases the proportion of inactive microglia, and decreases the expression levels of iNOS, TNF-α, IL-1β and IL-6 in infarction areas after 24 h of reperfusion, which suggests that TJ-5 could interrupt the in ammatory cascade to inhibit excessive in ammation. Furthermore, we found that TJ-5 was able to cross the BBB and the activation of LPS-induced BV-2 cells were effectively inhibited with the intervention of TJ-5, demonstrating that TJ-5 can directly inhibit the activation of microglia in brain to alleviate neuroin ammation. In addition, the results showed that TJ-5 inhibited the TLR4/MyD88/NF-κB signaling pathway in brain of CIRI mice and BV-2 cells, which were consistent with previous studies that focused on peripheral innate immune cells [19,43].
This study a rms the potency of TJ-5 in treating CIRI as it demonstrates a better neuroprotective effect in the early stage of cerebral I/R and the ability to cross the blood-brain barrier, as compared with other treatments. Moreover, we verify that TJ-5 not only can act on peripheral innate immune cells but also directly on brain tissue cells, which may be an in uential factor contributing to its exceptional antiin ammatory and neuroprotective effects. However, this study is only limited to microglia alone, so further complementary studies are needed to demonstrate the effects of TJ-5 on the cell interaction and molecular mechanism of CIRI. The transformation mechanism from the acute in ammation induced by CIRI to the chronic in ammation in the entity of the brain and the mechanisms of the adaptive immune system in CIRI both need to be further explored [44]. On the other hand, the in ammatory response in CIRI is a "double-edged sword". The excessive in ammatory response causes the injury to expand, but the in ammatory response also promotes the immune cells to devour the necrotic tissue, which can promote tissue repair [45]. MyD88 inhibitor regulates the immune response in CIRI from its origin and has e cacy in the acute phase, but the e cacy in the chronic phase still requires further investigation. Taken together, for aseptic in ammatory reactions like CIRI, it can be concluded that the key to the treatment of CIRI is to balance the regulation of the immune system and minimize neuron loss.

Conclusion
In summary, we con rm that the self-developed MyD88 inhibitor TJ-5 presented in this study has an impressive therapeutic effect on CIRI after acute ischemic stroke and the possible mechanisms behind its potency are that TJ-5 reduces neuroin ammation by inhibiting TLR4/MyD88/NF-κB signaling pathway and crosses the BBB to directly inhibit the activation of microglia, providing new insight for the potential of TJ-5 treating acute ischemic stroke and other neuroin ammatory diseases in clinical settings.  Tables   Table 1. Distribution of TJ-5 in tissues during the different time points (ng/ml or ng/g, mean ± SD) Table 2. PK parameters of TJ-5 in tissues of mice. a The tissue density was assumed to be 1 g/ml. K p : Brain-serum ratio was calculated by the mean of AUC 0-6h ratios. t 1/2 : elimination half-life.

Figure 2
Neuroprotective effect of TJ-5 treatment in CIRI model. a Effects of TJ-5 on neurological de cit score (n = 6/group; *P < 0.05, **P < 0.01 vs I/R group). b c Representative TTC-stained slices at 24 h after reperfusion and statistical analyses of infarct volume (n = 6/group; **P < 0.01, ****P < 0.0001 vs I/R group; ## P < 0.01 vs I/R + TJ-5 15 mg/kg group). d Representative NeuN immuno uorescence staining 1β were reduced with TJ-5 treatment in CIRI mice. c The mRNA level of iNOS in brain was detected. d Representative TUNEL immuno uorescence staining image of brain slices (bar = 500 μm). e TJ-5 downregulated the expression of iNOS in CIRI mice. f The expression of cleaved caspase-3 was analyzed.

Figure 7
Pharmacokinetic pro le of TJ-5. The concentrations of TJ-5 were analyzed in serum, IL brain, CL brain, liver and heart at speci ed time points after intravenous injection of TJ-5 at 15 mg/kg. a The concentration-time pro les of TJ-5 in serum, IL brain and CL brain. TJ-5 was quickly distributed into the brain after intravenous injection. b The concentration-time pro les of TJ-5 with expressed in logarithmic ordinate. Elimination of TJ-5 followed the rst-order elimination kinetics. c Concentration ratios of IL brain or CL brain to serum at 5 min, 10min, 30 min, 1 h, 2 h and 6 h.