BMP7 attenuates propofol induced neuron apoptosis via modulating JNK and AKT signaling

doi:10.21203/rs.3.rs-4620682/v1

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BMP7 attenuates propofol induced neuron apoptosis via modulating JNK and AKT signaling

https://doi.org/10.21203/rs.3.rs-4620682/v1

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Background

The aim of this study is to elucidate the protective role of bone morphogenetic protein 7 (BMP7) in mitigating the propofol-induced apoptosis on the HT-22 cells.

Method

Firstly, mouse hippocampal HT22 cells were treated with propofol at the concentrations of 0, 10, 50, 100uM for 12- and 24-hours to detect the propofol induced cell viability change and apoptotic effect. Secondly, BMP7 at the concentrations of 0, 20, 40, 60 ng/ml was co-incubated for 12-hrs to test whether BMP7 application could mitigate the propofol induced pro-apoptotic effect on the H22 cells. Finally, a specific BMP7 antagonist (TWSG-1) and an AKT inhibitor Akti-1/2 were applied to further investigate the downstream signals responsible for the BMP7’s protective property. In all sections, CCK-8 and LDH assay were employed to determine the cell viability and cytotoxicity; TUNEL staining was performed to investigate the apoptotic effect, and western blotting was conducted to examine the changes of the ratios of p-AKT/AKT and p-JNK/JNK.

Results

Firstly, propofol at the concentrations of 10, 50 and 100uM robustly induced cell viability decline, LDH release and morphological changes after 12- and 24-hours incubation. Secondly, BMP7 treatment at the concentration of 40 ng/ml statistically relieved propofol-induced cell cytotoxicity and apoptosis after 12-hours co-incubation. Thirdly, western blotting results showed that BMP7 initiated the upregulations of both p-AKT/AKT and p-JNK/JNK signals, which was antagonized by the treatment of TWSG-1 at 1ug/ml; In the meantime, Akti-1/2 prominently downregulated the ratio of p-AKT/AKT while upregulated the ratio of p-JNK/JNK. Finally, applications of both TWSG-1 and Akti-1/2 abolished BMP7 induced neuroprotective and anti-apoptotic effect.

Conclusion

BMP7 plays a protective role in reversing the propofol-induced apoptotic effect on HT22 cells possibly through the modulation of the downstream signals including JNK and AKT.

Bone morphogenetic protein-7

propofol

neural cells

apoptosis

AKT

JNK

As one of the most common intravenous anesthetics, propofol has been widely used for sedation in the critical care unit and general anesthesia [1–2]. However, a growing body of in-vivo [3–7] and in-vitro [8–9] evidence has documented that subjects treated with high dose of propofol over long duration shall develop abnormalities in learning, memory and cognitive functions, which is correlated with the neuronal apoptosis. Thus, therapeutic targeting the propofol-induced neuron injury should be addressed clinically.

Bone morphogenetic protein-7 (BMP-7) belongs to the family of transforming growth factor-β (TGF-β), and plays a critical role in the homeostasis in the central nervous system (CNS). Importantly, BMP7 and its specific receptors are reported to be expressed in the brain [10–11]. Previous researches have demonstrated that exogenous BMP7 administration profoundly enhances the neuronal anti-oxidant and anti-apoptotic ability through preventing cytochrome C release and inhibiting the activation of the caspase-3/8/9, thus exert neuroprotective property in the process of cerebral ischemia and reperfusion injury [12] and spinal cord injury [13]. In light of the evidence above, we further hypothesize that BMP7 administration could attenuate the propofol-induced neuronal injury and apoptosis.

In the present study, we designed an in-vitro study to explore the neuro-protective role of the BMP7 against the propofol-induced apoptosis in the neuronal HT22 cells, and further investigate the underlying mechanisms by testing the possible downstream signals.

Cell Culture and Modeling

Mouse hippocampal neural HT22 cells were purchased from the Procell (PNS-MC-83, Wuhan, CHN), and were grown to full confluences at 37℃, 5% CO₂ and saturated humidity. The culture medium contained high-glucose DMEM (GNM12800-5, Genobio, Beijing, CHN), 10% fetal bovine serum (FBS, FSP500, ExCell Bio, Shanghai, CHN), 100U/ml penicillin and streptomycin (P1400, Solarbio, Beijing, CHN). The medium was renewed every 48 h, and the cells were passaged when grew to 80% ~ 90%.

Cell Counting Kit-8 (CCK-8) assay

Cell viability was determined using a CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (CA1210, Solarbio). Cells were seeded in the 96-well plate (1× 10⁵ per well in 100ul medium) and incubated with CCK-8 solution (10ul per well) for 4 h at 37°C. The absorbance was measured at 450 nm with a microplate reader (Biochromatic, Helsinki, Finland). The calculation of the cell viability was based on the following equation: Cell viability (%) = [(experimental absorbance - blank absorbance)/ (control absorbance - blank absorbance)]×100%

lactate dehydrogenase (LDH) assay

Cytotoxicity was measured by the LDH released from cells with compromised membranes using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (G7890, Promega, WI, US). Cells (3 ×10⁴ per well) were seeded in a 96-well plate at 37 ℃ and 5% CO₂. Then, the plate was removed from the 37 ℃ incubator and equilibrated to 22 ℃ for 30 min. Next, each well was added with 100ul CytoTox-ONE™ Reagent at 22 ℃ for 10 min, followed by 50ul of stop solution. Afterwards, the resorufin fluorescence was measured at 560 nm of excitation and 590 nm of emission using a microplate reader (Biochromatic). The percentage of LDH release was calculated by the equation: Percent Cytotoxicity of LDH release = 100 × (Experimental - Culture Medium Background)/ (Maximum LDH Release - Culture Medium Background).

TdT-Mediated dUTP Nick-End Labeling (TUNEL) staining

Cell apoptosis analysis was performed using a TUNEL staining kit (FXP142-050, 4abio, CHN). In detail, cells of each group were fixed with 4% paraformaldehyde for 15 min, then the membrane was punched with 0.3%Triton X-100 for 10 min. Next, in a light-shielding wet box, each sample was incubated with 100ul TUNEL equilibrated recoil solution for 5min, followed with 50ul fresh labeled reaction solution at 37 ℃ for 60 min. After that, each section was stained with DAPI (S2110, Solarbio) for 10 min, sealed and stored at 4 ℃ away from light. All fluorescent images were captured (5 fields per section) using the fluorescence microscopy (Nikon Eclipse E600, Japan) and were calculated for the intensity using Image-Pro Plus 6.0 (Rockville, MD, US).

Western Blotting

Cells were washed with PBS 3 times, homogenized in RIPA lysis buffer (C5029, Bioss, Beijing, CHN) containing proteinase inhibitor (04693159001, Roche, Mannheim, GER) and phosphatase inhibitor (4906845001, Roche), followed by the centrifugation at 4 ℃ at 12000 rpm for 15 minutes to collect the supernatants. The protein concentrations then were tested by the bicinchoninic acid method (BCA protein assay kit, CW0014S, CWBIO, JS, CHN). Each sample then was mixed and boiled with the loading buffer, and 20 ug protein was loaded in each blot in the 10% SDS-polyacrylamide gels for electrophoresis, followed by transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, US). Next, the membrane was blocked with 5% non-fat dried milk for 1 hour at room temperature, and incubated overnight with the following primary antibodies: rabbit anti-BMP7 (1:2000, ab129156, Abcam, Cambridge, UK) rabbit anti-AKT (1:1000, 9272, Cell Signaling, MA, US), rabbit anti-p-AKT (1:2000, 4060, Cell Signaling), rabbit anti-JNK (1:1000, 9252, Cell Signaling), rabbit anti-p-JNK (1:1000, 4668, Cell Signaling) and rabbit anti-β-actin (1:6000, Proteintech, IL, US), which was followed by the incubation of the second antibody HRP-conjugated goat anti-rabbit IgG (1:6000, Proteintech) for 1 hour at room temperature. After washed three times with TBST, the membranes were processed with Luminol Reagent (Millipore, MA, US) and exposed to the MP-ECL films in a dark room. Finally, the films were scanned and analyzed with Image-Pro Plus 6.0 software (Rockville)

Statistical analysis

All data were presented as the mean and standard deviation. Comparisons among groups were performed using one-way analysis of variance (ANOVA), followed by the Sidak’s multiple comparison tests. All data were analyzed and charted with SPSS (version 19.0, IBM, NY, US) and Prism software (version 8.0.1 GraphPad Inc. CA, US), and P < 0.05 was considered to be statistically significant.

Propofol administration induced the cytotoxicity, morphological change and downregulated BMP7 expression of the HT22 cells

In order to test the effect of propofol on the HT22 cells, propofol at the concentrations of 10, 50, 100uM was chosen for 12-hours (h) and 24-hrs incubation respectively. Compared with the Sham (non-treated) group, the CCK-8 assay showed that the cell viability sharply decreased upon the propofol incubation at each concentration for both 12h (Fig. 1A) and 24h (Fig. 1B) (both P < 0.001); Besides, the LDH test similarly indicated that the LDH release was also robustly elevated after propofol treatment at each concentration after 12h (Fig. 1C) and 24h (Fig. 1D) incubation (both P < 0.01). Next, cell morphology was displayed in Fig. 1E and F. Compared with the Sham group (Fig. 1E), there was prominent increase in the morphological changes in propofol treated group at the concentration of 100uM for 12h, which was characterized with blurry outlines and shrinkages of the cell body (Fig. 1F). Finally, compared with the Sham group, 100 uM-propofol treatment for 12h induced a significant decrease of the BMP7 expressions (P < 0.05, Fig. 1G and H). All results above suggested that propofol could initiate the decline of cell viability, aggravate the neurotoxicity, induce the morphological changes and downregulation of the BMP7 levels in the HT22 cells.

BMP7 treatment attenuated propofol induced cell cytotoxicity and apoptosis

Considering that endogenous BMP7 is downregulated after the propofol treatment, we next investigated that if exogenous BMP7 supplement has the protective role against the propofol-induced cell injury. Here, exogenous BMP7 at the concentration of 20, 40 and 60 ng/ml were co-incubated with propofol at 100uM for 12-hrs. As shown in Fig. 2A, the CCK8 results showed that BMP7 at 20 and 40 ng/ml significantly restored the cell viability compared with the propofol treated group (P < 0.05). However, no statistical difference was found when the concentration of BMP7 reached to 60 ng/ml (P > 0.05). Besides, the LDH test elucidated that BMP7 at 40 ng/ml but not 20 or 60 ng/ml statistically relieved the LDH levels compared with the propofol-treated group (P < 0.05, Fig. 2B). Finally, the TUNEL results further revealed that after the treatment of propofol at 100uM for 12-hrs, the number of TUNEL positive nucleus (green) sharply increased compared with the Sham group (P < 0.001, Fig. 2C, D and E), which was further decreased after the BMP7 co-incubation at the concentration of 40 ng/ml (P < 0.001, Fig. 2C, E and F).

The above findings indicated that BMP7 at 40 ng/ml could effectively attenuated propofol-induced cell viability decline, LDH release and cell apoptosis.

AKT and JNK signaling mediated the protective role of BMP7 against the propofol-induced cell injury

Upon the above findings that BMP7 could protect the HT22 cells against propofol induced cytotoxicity and apoptosis, we further seek out the possible downstream signaling responsible for the BMP7’s protective role. In this scenario, the BMP7 specific antagonist TWSG-1 [14–15] and the serine/threonine kinase (AKT) signaling inhibitor Akti-1/2 were applied to block the BMP7’s effect.

Firstly, western blotting was performed to investigate the degree of activation in the downstream signals including c-Jun N-terminal kinase (JNK) and AKT (protein kinase B). As shown in Fig. 3A to C, compared with the propofol-treated group, BMP7 treatment at 40 ng/ml statistically upregulated the ratios of both p-AKT/AKT (P < 0.001) and p-JNK/JNK (P < 0.01), indicating that BMP7 treatment initiated both AKT and JNK activation. Then, co-incubation of the antagonist TWSG-1 at 1ug/ml could markedly decrease BMP7 induced-upregulation of both p-AKT/AKT and p-JNK/JNK (P < 0.01, compared with the BMP7-treated group). In the meantime, co-incubation of Akti-1/2 at the concentration of 400nM sharply decreased the ratio of p-AKT/AKT (P < 0.01, compared with the BMP7-treated group), but meanwhile elevated the ratio of p-JNK/JNK, which was statistically higher than both propofol + BMP7-treated and propofol + BMP7 + TWSG-1-treated group (P < 0.001).

Secondly, the CCK-8 analysis showed that co-incubations of both TWSG-1 (1 ug/ml) and Akti-1/2 (400nM) both significantly decreased the cell viability compared with the propofol + BMP7 group (P < 0.001). Moreover, compared with TWSG-1, Akti-1/2 treatment exacerbated the decline of the cell viability (P < 0.01). (Fig. 3D). In the meantime, both TWSG-1 and Akti-1/2 co-incubation aggravated the LDH release compared with the BMP7 + propofol-treated group (P < 0.01). (Fig. 3E)

Finally, as shown in Fig. 3F to J, the TUNEL assay revealed that the TUNEL positive nucleus were significantly increased after the co-incubations of both TWSG-1 (P < 0.01) and Akti-1/2 (P < 0.001). Additionally, compared with the TWSG-1, Akti-1/2 induced heavier elevation in the percentage of TUNEL positive cells (P < 0.001).

In ensemble, all results above indicated that BMP7 may exert the protective role though modulating both AKT and JNK signaling, Antagonizing BMP7 downstream signals especially AKT could completely abolish the protective property of BMP7.

The present study showed that administration of exogenous BMP7 could antagonize propofol-induced cytotoxicity and apoptosis in the HT22 cells. Besides, antagonizing BMP7 signaling using TWSG-1 and Akti-1/2 could abolish BMP7 induced anti-apoptotic effect, which may serve as a new treating candidate in reversing general anesthetic agents (GAs) induced neuro-toxicity.

Propofol has been world-widely used in general anesthesia for patients in all age groups due to its superior rapid-acting, amnesia and antiemetic effect [16–17]. Recently, its regulatory effect on the CNS has been drawn attentions, and studies from the last two decades remain controversial on whether propofol application exerts protective or harmful property on the neuron. On the one hand, some studies have elucidated that in the process of brain ischemic injury, propofol displays the neuroprotective effect and decreases the infarct volume via its metabolism-reducing and anti-oxidant actions [18–20]. On the other hand, substantial evidence has revealed that upon the high doses and long duration, propofol possesses the neurotoxicity instead of neuroprotection effect especially for the developing and childhood CNS. For instance, it has been proven that propofol could dose-dependently induce the apoptosis in HT22 cells via downregulating the brain derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B receptor (TrkB)-AKT signaling, which is known as a critical component of endogenous defense mechanism [8]. Besides, Creeley et al further demonstrated that 6-day aged rhesus macaques exposed to 5hrs-propofol infusion developed more serious cell death of both neurons and oligodendrocytes. Similarly, in the present in-vitro study, we also found after 12hrs- and 24hrs-treatment, propofol at 10, 50 and 100uM prominently induced the viability decline and cyto-toxicity on the HT22 cells. Meanwhile, propofol at the concentration of 100uM for 12hrs-incubation induced significant morphogenetical changes and increased number of TUNEL + cells, further indicating that propofol has the ability to induce the neuron apoptosis.

BMP7 is a secreted protein that belongs to the TGF-β super-family. Accumulating studies have collaborated the fact that BMP7 plays an essential role in the regulation of the proliferation, differentiation, and apoptosis in series of cell-lines and tissues. Notably, BMP7 is reported to be expressed at the early stage of brain development and critical for the neuron-glia crosstalk and homeostasis. For instance, in the cortical and hippocampal cultures, BMP7 is capable to promote the neurite growth and display neuroprotective effect via its antioxidant and antiapoptotic properties [21–23]. Besides, after the cerebral ischemic injury takes place, the central levels of BMP7 and its specific receptors are upregulated [24] and a growing body of evidence has elucidated that over-expression of BMP7 protects the neuron against the traumatic or ischemic injury, which is mainly through inhibiting oxidative stress and apoptotic pathways, therefore restoring the neurological deficit [12–13, 25]. Based on our limited knowledge, till now, whether BMP7 could reverse the anesthetic induced neuron toxicity still remains unclear. Here, we firstly proved that propofol treatment would decrease the endogenous BMP7 expressions in the HT22 cells; Besides, exogenous BMP7 at the concentration of 40 ng/ml after 12h co-incubation could effectively attenuated propofol induced cell viability decline, LDH release and cell apoptosis, prompting BMP7 might be capable to protect against the propofol-induced neuron injury.

It has been well characterized that BMP7 transduces signals via binding its specific membrane receptor BMPR2 (BMP receptor II), which then complexes with the BMPRI and induce the activation of SMAD-1/5/8 (phosphorylation) [26]. Both p-SMAD1/5/8 and BMPR2 then initiate the phosphatidylinositol3-kinase (PI3K)/AKT signaling. Meanwhile, BMP7 also augments JNK signal transduction via initiating TGF-β activated kinase 1 (TAK1) with the non-canonical manner [27–29]. Notably, recent studies have demonstrated that BMP7 potentiating PI3K-AKT signaling could drive the monocyte polarized towards the M2 anti-inflammatory subtype [30–31], restoring the insulin sensitivity in the process of type II diabetes mellitus [32], and protect the tissue against the pro-inflammatory, pro-apoptotic and pro-fibrotic injury [33–36]. Otherwise, Substantial evidence has proven that JNK activation mediates cytotoxicity and apoptosis [37–40]. In light of the evidence above, one can assume that AKT and JNK might play a different even opposite role in the process of inflammation and apoptosis regulation. In the present study, we found that after BMP7 treatment, the ratio of both p-JNK/JNK and p-AKT/AKT were significantly elevated, and the BMP7 antagonist TWSG-1 co-incubation effectively abolished BMP7-induced JNK and AKT activation, indicating that both JNK and AKT should be the possible downstream signals initiated by BMP7. Furthermore, we also found that antagonizing AKT signals using Akti-1/2 aggravated the cell viability decline and apoptosis compared with propofol + BMP7 + TWSG-1 group, which might be partly due to the fact that Akti-1/2 relieved the AKT activation but reinforced the JNK initiation. Additionally, we also found that BMP7 at the concentration of 40 ng/ml mainly displayed neuro-protective effect, which was downregulated when the concentration reached up to 60 ng/ml. We assumed that high dose of BMP7 might augment the JNK signal transduction, thus antagonizing BMP7-AKT-induced cyto-protection and anti-apoptosis property.

The major limitation of the present study is that all the results were based on the in-vitro research instead of in-vivo validation, which rule out the crosstalk between neurons and other cell types. Previous researches [41–43] have elucidated that although propofol shows pro-apoptotic effect on neonatal neurons but astrocytes remain refractory to propofol toxicity, which further attenuates propofol induced neurotoxicity. Thus, the actual role of propofol on the CNS needs further elucidation.

Overall, our research showed that administration of exogenous BMP7 could antagonize propofol-induced cytotoxicity and apoptosis in the HT22 cells possibly due to the modulation of both JNK and AKT signals. Antagonizing AKT signaling could completely abolished BMP7-induced protective property.

Ethics approval and consent to participate

Not applicable.

Availability of data and materials

All individual identified participant data and statistical analysis code that underlie the results reported in this article will be available after publication. Data can be requested for academic use by contacting the corresponding author (Xin Wang, Email: [email protected])

Competing interests

All authors declare that they have no competing interests.

Funding

This research is funded by the Natural Science Foundation of Hunan Province (2024JJ5504 to XW) and Foundation of Hunan Provincial Health Commission (HNA202101002 to LY).

Authors’ Contributions:

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Acknowledgments:

None.

Author information:

QS: [email protected]

RS: [email protected]

LY: [email protected]

QW: [email protected]

YS: [email protected]

XW: [email protected]

JX: [email protected]

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BMP7 attenuates propofol induced neuron apoptosis via modulating JNK and AKT signaling

Status:

Version 1

Abstract

Background

Method

Results

Conclusion

Figures

Introduction

Materials and Methods

Cell Culture and Modeling

Cell Counting Kit-8 (CCK-8) assay

lactate dehydrogenase (LDH) assay

TdT-Mediated dUTP Nick-End Labeling (TUNEL) staining

Western Blotting

Statistical analysis

Results

Propofol administration induced the cytotoxicity, morphological change and downregulated BMP7 expression of the HT22 cells

BMP7 treatment attenuated propofol induced cell cytotoxicity and apoptosis

AKT and JNK signaling mediated the protective role of BMP7 against the propofol-induced cell injury

Discussion

Declarations

References

Additional Declarations

Status:

Version 1