VX‐765 enhances autophagy of human umbilical cord mesenchymal stem cells against stroke‐induced apoptosis and inflammatory responses via AMPK/mTOR signaling pathway

Abstract Introduction To investigate the protective effect of VX‐765 on human umbilical mesenchymal stem cells (HUMSCs) in stroke and its mechanism. Materials and methods Mouse models of ischemic stroke were established using the distal middle cerebral artery occlusion (dMCAO) method. The dMCAO mice were accordingly transplanted with HUMSCs, VX‐765‐treated HUMSCs, or VX‐765 + MHY185‐treated HUMSCs. The HUMSCs were inserted with green fluorescent protein (GFP) for measurement of transplantation efficiency which was determined by immunofluorescence assay. Oxygen‐glucose deprivation (OGD) was applied to mimic ischemic environment in vitro experiments, and the HUMSCs herein were transfected with AMPK inhibitor Compound C or autophagy inhibitor 3‐MA. MTT assay was used to test the toxicity of VX‐765. TUNEL staining and ELISA were applied to measure the levels of apoptosis and inflammatory cytokines (IL‐1β, IL‐6, and IL‐10), respectively. The expressions of autophagy‐associated proteins, AMPK, and mTOR were detected by Western blotting. TTC staining was applied to reveal the infarct lesions in the brain of dMCAO mice. Results The pro‐inflammatory cytokines, TUNEL‐positive cells, and p‐mTOR were decreased while the anti‐inflammatory cytokine, autophagy‐related proteins, and p‐AMPK were increased in HUMSCs treated with VX‐765 under OGD condition. Different expression patterns were found with the above factors after transfection of 3‐MA or Compound C. The pro‐inflammatory cytokines, TUNEL‐positive cells, and infarct sections were decreased while the anti‐inflammatory cytokine and autophagy‐related proteins were increased in dMCAO mice transplanted with VX‐765‐treated HUMSCs compared to those transplanted with HUMSCs only. The autophagy was inhibited while p‐mTOR was up‐regulated after transfection of MHY. Conclusion VX‐765 protects HUMSCs against stroke‐induced apoptosis and inflammatory responses by activating autophagy via the AMPK/mTOR signaling pathway in vivo and in vitro.


| INTRODUC TI ON
Stroke is one of the leading causes of death and disability worldwide. 1 Stroke brings serious neural damage to brain by triggering a series of pathophysiological responses including inflammatory changes. 2 Stem cell transplantation has emerged as a novel regenerative therapy for stroke. 3 Among the stem cell populations, human umbilical cord blood (HUCB) cells have been reported to exert anti-inflammatory effects and secrete growth factors to promote cell survival in treatment of neurodegenerative disorders. 4 Mannitol is a drug which can enhance the therapeutic efficacy of HUCB by facilitating the delivery of stem cells and their byproducts into ischemic brain. 5,6 However, inflammatory responses from the circulation may also permeate the brain when mannitol disrupts the blood-brain barrier (BBB). 7 Additionally, the central nervous system entry of HUCB cells is not necessarily required for neuroprotection in stroke if the specific cytokines they secreted could cross the BBB. 8 Therefore, a new drug is needed to strengthen the therapeutic outcomes of stem cell transplantation in stroke. Human umbilical mesenchymal stem cell (HUMSC) is another sort of stem cell which has already been evaluated for its therapeutic effects on ischemic stroke. 9 However, poor survival of donor cells impedes the efficacy of this promising method. 10 Improving stem cell survival in an ischemic environment has surfaced as the key challenge for HUMSC-mediated therapies in patients with ischemic stroke.
VX-765 is a newly developed, selective, small molecule caspase-1 inhibitor that can pass the BBB and reduce inflammation in vitro and in vivo. 11 The drug has been used clinically to treat epilepsy and can be administered orally. 12 Recent studies have shown that VX-765 exhibits a wide range of biological effects on central nervous system diseases such as experimental autoimmune encephalomyelitis and Alzheimer's disease. 13,14 Oxygen-glucose deprivation (OGD) is often used to mimic ischemic environment in vitro with the ability of inducing stem cell apoptosis. 15,16 To determine the potential of VX-765 for treatment of cerebral ischemic injury, we investigated the effect of VX-765 in OGD-treated HUMSCs and in a rat model of stroke.
Autophagy is a catabolic event that maintains cell homeostasis in reaction to various stimuli. 17 This self-catabolic process occurs when two membrane vacuoles, called autophagosomes, engulf components of cells and degrade them through lysosomal mechanisms. 18 Autophagy is critical in metabolic hunger and adaptive survival under stress for its potential in maintaining nutrient availability and energy levels in cells. 19,20 Apoptosis and autophagy are the main factors responsible for the poor survival of transplanted stem cells.
Collected evidence supported that the survival of bone marrow-derived mesenchymal stem cells could be enhanced when cell autophagy was activated by SDF-1/CXCR4 axis. 21 In addition, macrophage migration inhibitory factor was found to protect cells from apoptosis by regulating autophagy through AMPK/mTOR signaling pathway. 22 Under normal conditions, AMPK can sense changes in cellular energy and activate autophagy by reducing ATP/AMP ratio, thereby indirectly activating mTOR, one of its major downstream targets. 23 Activation of AMPK/mTOR signaling pathway may in turn stimulate autophagy and exert anti-apoptotic and anti-inflammatory effects. 24 The anti-inflammatory and anti-apoptosis role of VX-765 was documented in septic mice. 25 However, no available study has clarified either the effect of VX-765 on survival of transplanted cells or the potential relationship between VX-765 and autophagy. Therefore, the present article intends to elucidate the possible mechanism underlying the effect of VX-765 on HUMSC survival in OGD-treated cells and in rats with stroke.

| Distal middle cerebral artery occlusion (dMCAO) model
All procedural and ethical considerations acquired approval of the Wenzhou Medical University Experimental Animal Ethics Committee.
Adult Sprague Dawley rats (250-300 g) were purchased from the Animal Center of the Shanghai Branch of the Chinese Academy of Sciences and maintained at the Experimental Animal Center of Wenzhou Medical University. Permanent dMCAO method and ipsilateral common carotid artery (CCA) occlusion were used to establish the focal cerebral ischemic stroke models. Firstly, the rats were subjected to anesthesia using 10% chloral hydrate (3.5 mL/kg, intraperitoneal injection). A vertical skin incision was made at the midline of the neck to separate the two lateral CCAs. Then, a 2-cm incision was made between the right eyelid and the tragus, allowing the diaphragm to be removed from the exposed skull. After that, a microdrill was used to punch a 3-mm-diameter hole in the skull to reveal the brain. The dura mater and arachnoid were carefully peeled off using forceps under a surgical microscope. The bilateral CCA was ligated, and then, the right MCA was occluded using electrocoagulation. After 60-minute ligation, the two lateral CCAs were released and the incision was sutured.
Sham-operated rats underwent the same procedure without occlusion Conclusion: VX-765 protects HUMSCs against stroke-induced apoptosis and inflammatory responses by activating autophagy via the AMPK/mTOR signaling pathway in vivo and in vitro.

K E Y W O R D S
autophagy, human umbilical mesenchymal stem cells, stroke, VX-765 of the distal MCA. Rectal temperature was controlled at 37.0 ± 0.5°C during surgery using a temperature-regulated heating pad.

| Cell preparation and transplantation
HUMSCs were incubated in a proprietary complete medium with 5% CO 2 at 37°C (both the cells and the medium were purchased from Cyagen Biosciences Inc). Cells exposed to OGD alone were

| Enzyme-linked immunosorbent assay (ELISA)
Levels of interleukin (IL)-6, IL-10, and IL-1β in grinded brain tissues and HUMSCs were measured using ELISA kits (Beyotime) based on the manufacturer's instructions. HUMSC culture supernatant was collected and centrifuged at 500 g for 5 minutes, after which the supernatant was obtained and homogenized before ELISA. The absorbance value was measured at 450 nm using a microplate reader.

| MTT assay
The HUMSCs were incubated in a 96-well plate with 5 × 10 3 cells per well. After primary incubation, 10 μL of MTT labeling reagent (5 mg/mL) was added to each well for another 4 hours of incubation at 37°C. DMSO (100 μL) was added to terminate the reaction at 37C° overnight. Finally, the optical density (OD) of the samples was measured at 590 nm using a microplate reader (Sectramax 190, Molecular Devices Corp.).

| Western blotting
Cells were lysed in lysis buffer containing protease inhibitor cocktail

| TUNEL staining
Apoptosis of HUMSCs or cerebral tissues from rats was measured by TUNEL staining using a TUNEL Apoptosis Assay kit (Beyotime Institute of Biotechnology, Inc). TUNEL stained apoptotic nuclei; DAPI and fluorescein-dUTP stained all nuclei. The apoptotic index (AI) = the number of TUNEL-positive cells/ the total number of cells.
AI was evaluated in 15 randomly selected fields.

| TTC staining
Infarct size was assessed using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. After the rats were deeply anesthetized and decapitated, the brains were quickly removed and manually cut into coronal sections from the head to the forehead with a scalpel. These sections were then cultured in 2.0% (wt/vol) TTC (Sigma) at 37°C for 20 minutes. Then, the brain sections were fixed in 4% paraformaldehyde (PFA) for 30 minutes at 4°C and finally photographed with a digital camera. The infarct area in each section was evaluated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

| Neurobehavioral testing
To assess overall neurological deficits, modified neurological severity scores (mNSS) were assessed on rats 1 and 3 days after dMCAO.
The test included a task portfolio that assessed the rats' ability to move, feel, reflect, and balance. The mNSS test scores ranged from 0 (normal performance) to 18 (maximum defect), with higher scores indicating severe neurological dysfunction. Rats with abnormal preoperative scores (>0) were excluded.

| Immunofluorescence assay (IFA)
Rats were anesthetized with a lethal dose of chloral hydrate and perfused with 100 mL of saline followed by 100 mL of 4% PFA in 0.1 mol/L PBS (pH 7.6). Tissues were fixed in 4% PFA in 0.1 mol/L PBS at 4°C overnight and cryoprotected in 30% sucrose for 36 hours.
Frozen sections of 10-μm thickness were prepared and fixed in 4% PFA for 20 minutes. After being washed for 3 × 5 minutes with PBS, these sections were then permeated with 0.3% Triton X-100 for 15 minutes and finally subjected to washing (3 × 5 minutes). After GFP transfection, transplanted HUMSCs were directly detected at 488 nm; nuclei were counterstained with DAPI. All samples were analyzed with a fluorescence microscope (BX51, Olympus).

| Statistical analysis
Data are presented as mean ± standard deviation (SD). All values were analyzed in Prism software (GraphPad Software Inc). Unpaired Student's t tests were used to compare differences between two groups. One-way analysis of variance was performed to compare differences involving three or more groups. The Kolmogorov-Smirnov (K-S) method was applied to check the normality of data distribution. P < .05 was considered statistically significant.

| VX-765 protects HUMSCs from OGD-induced apoptosis and inflammatory responses
The molecular structure of VX-765 is shown in Figure 1A.
Furthermore, the expression of mTOR was measured by Western blotting ( Figure 4C). p-mTOR was up-regulated in dMCAO group compared to sham group; p-mTOR decreased from dMCAO group, dMCAO + HUMSCs group to dMCAO + VX-765 + HUMSCs group while increased in dMCAO + VX-765 + HUMSCs+MHY group compared to dMCAO + VX-765 + HUMSCs group. The above results F I G U R E 2 VX-765 promotes autophagy through AMPK/mTOR signaling in HUMSCs exposed to OGD. Notes: A, B, Western blot tested the expressions of autophagy-associated proteins. C, D, Western blot tested expressions of proteins involved in AMPK/mTOR signaling pathway. E, TUNEL staining after transfection of 3-MA or compound C. *P < .05, **P < .01. Scale bars: 50 μm; HUMSC, human umbilical mesenchymal stem cell; AMPK/mTOR, AMP-activated rapamycin protein kinase/mammalian target protein; OGD, oxygen-glucose deprivation indicate that VX-765 regulates mTOR to enhance autophagy so as to protect HUMSCs from OGD-induced injury.

| D ISCUSS I ON
Stroke, a highly fatal cerebrovascular disease, ranks the second leading cause of global deaths. 27 Ischemic stroke, closely related to inflammatory responses, is the most common stroke type which results in severe neurologic disability. 28,29 Cell-based neurovascular regeneration is a novel direction of therapies for stroke, among which autologous MSCs enjoy the most popularity. 30  signaling thus protecting rat brain in MCAO models. 38 Abundant research has claimed that autophagy is activated in cerebral ischemic injury. 39 For instance, AMPK-mediated autophagy induced by ischemic preconditioning (IPC) reduced infarct volume and cell apoptosis in pMCAO rat. 40 Recently, many stem cells have been discovered to have therapeutic potential for ischemic stroke. 41 MSCs are one of those promising stems cells which suppress ischemic damage by modulating inflammatory response and promoting endogenous repair in stroke. 42 Autophagy is verified to play a pivotal role in modulating proliferation and differentiation of MSCs. 43 The present study found that VX-765-treated HUMSCs exerted superior protective effect on rat brain than HUMSCs alone by enhancing cell autophagy.