Vespakinin-M, a natural peptide from Vespa magnifica, promotes functional recovery in stroke mice

Acute ischemic stroke triggers complex systemic pathological responses for which the exploration of drug resources remains a challenge. Wasp venom extracted from Vespa magnifica (Smith, 1852) is most commonly used to treat rheumatoid arthritis as well as neurological disorders. Vespakinin-M (VK), a natural peptide from wasp venom, has remained largely unexplored for stroke. Herein, we first confirmed the structure, stability, toxicity and distribution of VK as well as its penetration into the blood–brain barrier. VK (150 and 300 µg/kg, i.p.) was administered to improve stroke constructed by middle cerebral artery occlusion in mice. Our results indicate that VK promote functional recovery in mice after ischemia stroke, including an improvement of neurological impairment, reduction of infarct volume, maintenance of blood-brain barrier integrity, and an obstruction of the inflammatory response and oxidative stress. In addition, VK treatment led to reduced neuroinflammation and apoptosis associated with the activation of PI3K–AKT and inhibition of IκBα–NF-κB signaling pathways. Simultaneously, we confirmed that VK can combine with bradykinin receptor 2 (B2R) as detected by molecular docking, the B2R antagonist HOE140 could counteract the neuro-protective effects of VK on stroke in mice. Overall, targeting the VK–B2R interaction can be considered as a practical strategy for stroke therapy.

In the current manuscript Zhao H et al investigated the role of Vespakinin-M, a natural peptide from Vespa magnifica, on infarct volume and post-stroke functional recovery in preclinical stroke mouse model. The authors showed 1) the structure of VK by HPLC and stability/distribution test; 2) effect of VK on infarct volume, functional outcome post-stroke; oxidative stress; inflammation; BBB integrity and cell death and axonal injury; 3) for mechanism of VK on neuroprotection, the authors found VK treatment is associated with activated PI3K-AKT pathway and NF-kB pathway inhibition. This is a very well performed study. Overall, this reviewer was impressed with the body of work as presented, and it is certainly of broad significance across neuroscience fields. The experiments as presented are elegantly designed and straightforward in their interpretation towards defining a role for VK on stroke severity. This reviewer has a few minor concerns and clarifications that would benefit this manuscript prior to publication. 1. RIGOR guidelines should be followed for all effective translational stroke research. Specifically for this manuscript, female mice and a second stroke model should be used to investigate whether VK is only effective in male mice or MCAO model; longer period time of stroke severity should be demonstrated.
2. Systemic toxicity test for VK, such as liver/ kidney, and especial those related to coagulation, platelet function and bleeding should be performed.
3. The author do not demonstrated whether VK binds to B1R orB2R, the use of a single antagonist with one in vivo study does not reveal VK-B2R interaction, nor a practical strategy for stoke therapysuch a statement is not accepted. 4. In Fig. 7c lack of legend. Please check typos throughout the manuscript. For example, line 916 and 972, "1" for "i" , et al.

Reviewer #1 (Remarks to the Author):
The manuscript entitled "Vespakinin-M, a natural peptide from Vespa magnifica, promotes functional recovery in stroke mice" by Dr. Zhao et al. describes that vespakinin-M (VK), a natural peptide from wasp venom, promotes functional recovery in mice after ischemia stroke, including an improvement of neurological impairment, a reduction of infarct volume, protection of the BBB, and the inhibition of inflammatory responses and oxidative stress. Mechanistically, they found that reduced neuroinflammation and apoptosis by treatment with VK were associated with PI3K-AKT-mediated NF-κB inhibition. The results are potentially interesting and the techniques used in this study is appropriate to conclude the results. However, there are some major issues that should be addressed.
Thank you very much . We appreciate your constructive comments and suggestions on our manuscript entitled "Vespakinin-M, a natural peptide from Vespa magnifica, promotes functional recovery in stroke mice".
1. How was the dose (150 and 300 μg/kg) determined? any references? Will increasing the dose of VK yield better protection effect in vivo? Determined the dose (150 and 300 μg/kg) consistent with effective dose of bradykinin1. We have added reference Line 335-338 (discussion).Ischemic stroke were treated VK (75, 150, 300 μg/kg) in preliminary experiment. Infarct size and behavior were evaluated. Compared with MCAO/R group, decreased area of cerebral infarction and mortality were seen in VK group. However, there were no significant differences between the three groups (75, 150, 300 μg/kg). The effect of increased the dose of VK was not illustrated in this paper. But, we are working on the long-term pharmacodynamics of VK on ischemic stroke, such as angiogenesis and synaptic remodeling.
2. Oxidative stress is caused by elevated production of reactive oxygen species (ROS). It can cause neuron apoptosis. The level of ROS should be detected in figure 3. Thank you. ROS level have be demonstrated using a DCFHDA kit. We have added to Figure 3 .Lines 184-188.
3. In this study, microglia and neuron were used to do some experiments in vitro. From my point of view, the experiments of OGD induced co-culture of these two kinds of cells should be done. Because VK has effect on both microglia and neuron, and in vitro co-culture experiment can really reflect the situation in vivo.
Thanks. Our experimental design is lacking in this part. For co-culture experiment, primary microglia and neuron extracted from newborn or embryonic mice. We confirmed VK protects neurons, and avoids persecution of proinflammatory secreted by microglia. Figure 6 and lines 276-282.
4. Please note that Iba1 is not a specific marker for microglia; it can also be used for macrophages. Therefore, the results from figure 5 did not necessarily mean microglia activation. Thank you very much. Iba1 is a marker for microglia, activated microglia can be defined as larger with shorter and thicker axons from morphology. As you say, it's not objective. Therefore, TNF-α co-localization with IBA-1 was performed by immunofluorescence ( Supplementary Fig. 4d). We also isolated these microglia for phenotype study by flow cytometry to analyse the expression of CD45+F4/80+CD11b+MHCⅡ+(M1 polarization). Lines 239-244.
5. The authors did not provide exclusion criteria for the MCAO model. Are all the mice included in this study? And the authors did not show the mortality rate of the animals. Did all the mice survive? Please provide the data.
6. There is no information about mice in the method part, and why did the authors use 3-month year old mice? Many studies used 2-month year old mice. Thank you. In fact, 8-10 weeks male mice were used to build MCAO/R, we have revised and added reference. Line 496 and line 503. 7. Please explain that "brain tissues" in this study stand for which part of the brain? For the MCAO model, the infarct size implicated caudate-putamen, striatum, and cortex. This study focus on the cortex of ischemic hemisphere. Thank you for your suggestion, we have revised, Such as Line 226-229. For rozen section, brain (interaural 1.7-4.30 mm, bregma -2.06-1.94 mm) was collected Lines 594.
8. I would like to suggest the authors to conduct the autodock experiment to evaluate the combination of VK and B2R or show us some other direct evidence to prove your conclusion. According to your suggestion, To reveal VK-B1R or B2R interaction, the HDOCK server for integrated protein-protein docking was performed. we predicted VK-B1R or B2R interaction sites (Fig.8 a-d) , and the peptide docking model of VK and B1R with the highest score. The docking summary of the top 10 models were also displayed (Supplementary Fig.7 and Fig.8). Lines 310-315. in preclinical stroke mouse model. The authors showed 1) the structure of VK by HPLC and stability/distribution test; 2) effect of VK on infarct volume, functional outcome post-stroke; oxidative stress; inflammation; BBB integrity and cell death and axonal injury; 3) for mechanism of VK on neuroprotection, the authors found VK treatment is associated with activated PI3K-AKT pathway and NF-kB pathway inhibition. This is a very well performed study. Overall, this reviewer was impressed with the body of work as presented, and it is certainly of broad significance across neuroscience fields. The experiments as presented are elegantly designed and straightforward in their interpretation towards defining a role for VK on stroke severity. This reviewer has a few minor concerns and clarifications that would benefit this manuscript prior to publication.
Thank you very much for your valuable comments and suggestions from the overall structure of the article and the future application of VK. We take your suggestions.
1. RIGOR guidelines should be followed for all effective translational stroke research.
Specifically for this manuscript, female mice and a second stroke model should be used to investigate whether VK is only effective in male mice or MCAO model; longer period time of stroke severity should be demonstrated.
Thank you. We only evaluated the neuroprotective effect of VK (150 and 300 μg/kg) during the period of acute cerebral ischemia in male mice. However, protective effect of longer period time (such as 14d, 21d, 28d) of VK on stroke severity was not demonstrated in this paper. We are working on the long-term pharmacodynamics of VK on ischemic stroke, such as angiogenesis and synaptic remodeling for effective translational stroke research. We have put your suggestion into the discussion section. The deficiencies of this study. Lines 462-472.
Female mice were not selected because estrogen has a protective effect on cerebral ischemia. In future, We will carry out all these experiments.
2. Systemic toxicity test for VK, such as liver/ kidney, and especial those related to coagulation, platelet function and bleeding should be performed.
Thank you very much. We sought to understand whether VK (0.0091-0.294 µM) inhibit platelet aggregation platelet aggregation ( Supplementary Fig. 2a). Platelet-activating factor (PAF),arachidonic acid (AA), adenosine diphosphate (ADP), thrombin, and collagen (COL) or saline as inducing agents was employed. ADPinduced platelet aggregation was decreased in VK groups as compare to control group, detailed values are shown in (Supplementary Fig. 2a) Fig. 2b). The index of brain, liver and kindey had no difference in all treatment dose groups, whereas increased liver index was found (VK, 384 mg/kg) (Supplementary Fig. 2c-f). No significant change was observed for four coagulation during the study (Supplementary Fig. 2g-j). In the histopathological study, it was observed that in all treated groups after 14 days ( Supplementary Fig. 3a-d) the organs (brain, liver and kindey) showed no changes at the cellular level in comparison to the control. Karyopyknosisa and the cellular swelling of the hepatocyte was moderate in liver of mice treat with VK (96 and 384 mg/kg).Taken together, this finding reveal these dosages of VK are safe to mice.
3. The author do not demonstrated whether VK binds to B1R or B2R, the use of a single antagonist with one in vivo study does not reveal VK-B2R interaction, nor a practical strategy for stoke therapy-such a statement is not accepted.
Thanks. BIAcore experiment were selected to reveal VK-B2R interaction. However, B1R or B2R were seven-span transmembrane protein. We have tried many methods to express and purify it, and very difficult. Finally, to reveal VK-B1R or B2R interaction, the HDOCK server for integrated protein-protein docking was performed as described 47. Known three-dimensional structure of VK and threedimensional structure of B1R or B2R ; we predicted VK-B1R or B2R interaction sites (Fig.8 a-d) , and the peptide docking model of VK and B1R with the highest score. The docking summary of the top 10 models were also displayed (Supplementary Fig.7 and Fig.8). Lines 311-316.