Deletion of EGFL7 exacerbated chronic constriction-induced nerve damage and neuropathic pain by modulating the notch pathway

doi:10.21203/rs.3.rs-4727294/v1

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Deletion of EGFL7 exacerbated chronic constriction-induced nerve damage and neuropathic pain by modulating the notch pathway

https://doi.org/10.21203/rs.3.rs-4727294/v1

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The pathogenesis of neuropathic pain (NP) is complex, and there is an urgent need to study its underlying mechanisms. A CCI rat model was established and injected with shRNA plasmid lentivirus to knock down EGFL7. Behavioral experiments were used to analyze pain thresholds in rats. Histologic hematoxylin-eosin (H&E) and toluidine blue staining were used to investigate the changes in tissue structure. Apoptosis was analyzed by TUNEL staining, while VWF was assessed by IHC for nerve repair evaluation. Finally, Western blot (WB) experiments were performed to analyze the potential molecular mechanisms underlying the regulation of EGFL7 in a rat model of CCI. The results showed that he absence of EGFL7 exacerbated pain in rats with CCI, resulting in disorganization of nerve cells in the sciatic nerve. It also caused extensive necrosis of the sciatic nerve and promoted demyelination and degeneration of axonal myelin. Large amounts of apoptosis were also present in the groups where EGFL7 was knocked down. In addition, the nerve repair factor VWF was also reduced in the CCI rat model with EGFL7 knockdown. Finally, mechanistic studies revealed that the knockdown of EGFL7 led to the downregulation of proteins related to the Notch signaling pathway. Our results suggested that inhibiting EGFL7 expression might worsen CCI-induced neuropathic pain and nerve injury in rats by affecting the Notch signaling pathway. This indicated that EGFL7 played a crucial role in the process of nerve injury. EGFL7 could also be a potential target for the treatment of neuropathic pain.

EGFL7

neuropathic pain

sciatic nerve

Notch signaling pathway

apoptosis

Neuropathic pain (NP) is a chronic pain that occurs as a result of damage or lesions in the peripheral or central nervous system[1]. Injury or disease to the somatosensory nervous system, such as nerve compression, pathological changes, autoimmune diseases, and surgical incisions, can lead to the development of neuropathic pain[2]. Despite numerous studies targeting nerve injury regulation and downstream pathways over the past decades[3, 4], we are still unable to elucidate the underlying mechanisms of neuropathic pain. In the progression of neuropathic pain, small-diameter neurons play a crucial role in transmitting injurious and thermal information, while large-diameter neurons primarily function as non-injurious neurons, such as mechanoreceptors and proprioceptors. In the context of nerve injury, various neuronal responses are triggered, including cellular stress, apoptosis, regeneration, hyperexcitability, and functional maladaptation. These changes may contribute to the development of neuropathic pain[5].

Epidermal growth factor-like protein 7 (EGFL7) is a protein secreted by endothelial cells (EC) that binds to the extracellular matrix (ECM) and interacts with integrin αvβ3, which is expressed on ECs[6]. EGFL7 is, above all, an important tumor-promoting factor. One study found that targeting EGFL7 inhibited angiogenesis and increased the sensitivity of apatinib in colorectal cancer[7]. EGFL7 drove the evolution of resistance to EGFR inhibitors in lung cancer through the activation of the Notch signaling pathway[8]. In addition, it has been shown that EGFL7 promotes blood-brain barrier integrity and attenuates central nervous system (CNS) inflammation in experimental autoimmune encephalomyelitis (EAE) in mice. This result demonstrates the neuroprotective function of EGFL7[9]. Hao et al. [10] found that the expression of EGFL7 was reduced in a rat model of spinal cord injury (SCI). Conversely, increased expression of EGFL7 inhibited apoptosis and promoted angiogenesis, nerve regeneration, and repair of nerve defects. EGFL7 also positively regulates endothelial cell protection, and its reduced expression level may be associated with hyperoxia-induced endothelial cell death and lung injury[11]. The literature above demonstrates that EGFL7 has potential functions, such as neural repair and cryoprotection. Although the function of EGFL7 has been reported in a variety of disease models, its significance in the field of NP remains underreported.

In this study, we investigated the specific mechanism of EGFL7 in a rat model with chronic constriction injury (CCI). We found that knockdown of EGFL7 in the CCI model further aggravated neuronal apoptosis, promoted changes in neuronal axon morphology, such as myelin vacuolization and demyelination, and inhibited neural repair. These effects resulted in an increase in abnormal pain in CCI rats. This result may be related to the regulation of the Notch signaling pathway by EGFL7.

Animals and models

A total of 24 male Sprague-Dawley rats (200–220 g, purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd.) were selected for the study. The rats were housed in standard plastic cages at 24 ± 1°C and 50–70% humidity. All rats were individually housed in cages according to a standard 12:12 h light/dark cycle, provided with unlimited water and food and acclimatized for 1 week in advance. All experimental manipulations followed the regulations of Ethics Committee of the Second Affiliated Hospital of Inner Mongolia Medical University. The study was conducted in accordance with the regulations published in the NIH Guide for the Care and Use of Laboratory Animals.

To investigate neuropathic pain following nerve injury, we created a unilateral sciatic nerve CCI rat model based on Bennett's method[12]. Briefly, a 10% chloral hydrate solution was injected intraperitoneally at a dosage of 30 mg/kg. A curved incision, approximately 4 cm in length, was made from the right buttock to the posterior lateral aspect of the rat's thigh. The skin was then incised, and the right sciatic nerve was exposed from the gluteal muscular space. A nerve defect was created by resecting approximately 10 mm of the nerve, starting 1 cm away from the beginning of the sciatic nerve. In the sham-operated group, the sciatic nerve was exposed without injury, and the incision was closed routinely.

Animal grouping

After establishing the CCI model and sham operation groups, all experimental rats were divided into three groups, each consisting of eight rats. These three groups were the sham operation group (sham), the model group (CCI), and the EGFL7 knockdown group (CCI + shEGFL7).

For the knockdown group, a recombinant shRNA-EGFL7 (shEGFL7) lentiviral plasmid was constructed by Genepharma (Shanghai, China). 1×10⁸ lentivirus particles were injected around the L5 dorsal root ganglion ipsilateral to the trauma 24 hours after CCI modeling. All rats were euthanized immediately after the behavioral tests, and sciatic nerve tissue from the surgical site was collected for the study.

Behavioral evaluation

For the determination of mechanical withdrawal threshold (MWT), we used an electronic tactile tester (38450, Ugo Basile, Varese, Italy) to examine the MWT values of each group of rats before modelling and 3, 7 and 14 days after modelling, respectively. Briefly, a glass box was positioned on a metal screen. After the rats had acclimated to the box for 15 minutes, the central area of the plantar foot on the hind limb of the rats was vertically stimulated using Von Frey filaments. The intensity of the pinprick was incrementally raised, and the occurrence of foot lifting or licking was considered a positive response and recorded as a value. Conversely, if there was no response, it was considered a negative response.

In addition, thermal withdrawal latency (TWL) was determined using the thermal radiation method. A plexiglass box was placed on a 3-mm-thick glass plate, and the soles of the rats were exposed to a thermal pain stimulator. The TWL was measured as the time from the beginning of the exposure to the point when the rats lifted their legs to avoid the stimulus. The automatic stopping time was 25 seconds to prevent tissue damage. The TWL was measured three times, with each measurement taken at a 3-minute interval. The average value of these measurements was considered as the TWL value of the rats. TWL was used to evaluate the nociceptive hypersensitivity of rats before modeling and at 3, 7, and 14 days after modeling, respectively.

Histologic hematoxylin-eosin (H&E) staining

At the conclusion of the behavioral experiments, the sciatic nerves of the rats in each group were collected and preserved in 10% neutral formaldehyde. Subsequently, they were dehydrated and embedded in paraffin. Subsequently, the samples were cut into 5 µm tissue sections and stained with hematoxylin and eosin (Sigma-Adrich, St. Louis, MO, USA). Three sections were randomly selected from each tissue sample. Each section was observed in five fields of view using a light microscope (Olympus, Tokyo, Japan).

Toluidine blue staining

Sciatic nerves from various rats were collected and post-fixed overnight in a solution containing 2% glutaraldehyde and 2% paraformaldehyde in a 0.1% cacodylate buffer (pH 7.4). These sciatic nerve samples were then dissected and washed three times with cacodylate buffer. The nerves were subsequently cut into 2-mm-long segments and postfixed in 1% osmium tetroxide (OsO4) for 2 hours. After washing several times with distilled water, the samples were dehydrated using ethanol at various concentrations and then embedded in Agar 100 resin. A semi-thin cross-section (0.5 µm) of the sciatic nerve, located 1 mm proximal to the injury site, was stained with 0.5% toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) and observed using an Olympus BX 40 microscope (Olympus, Tokyo, Japan).

TUNEL staining

After collecting the rat sciatic nerve, the tissue will be fixed, dehydrated, embedded in paraffin, and sectioned. Sections were incubated at 37°C for 60 minutes under light protection using the TUNEL kit, following the manufacturer's instructions (Beyotime, C1088, Shanghai, China). Finally, the tissue sections were washed three times with PBS and observed using a light microscope (Olympus, Tokyo, Japan).

Immunohistochemistry (IHC)

Sciatic nerve tissues were routinely paraffin-embedded, sectioned, deparaffinized, and hydrated. They were then subjected to antigen retrieval using proteinase K (0.2 mg/mL) and a 10 mM sodium citrate solution (pH 6.0). It was closed with normal goat serum and then incubated with EGFL7 primary antibody (1:200, ab256451, Abcam, Cambridge, UK) and VWF (1:200, ab287962, Abcam, Cambridge, UK) overnight at 4°C. Next, they were incubated with HRP-labelled secondary antibodies (1:200, S0001, Affinity, Changzhou, China) for 2 hours and observed under a light microscope (Olympus, Tokyo, Japan).

Western blotting (WB)

Total protein samples were obtained from sciatic nerves after being processed in RIPA lysis buffer (Beyotime, Shanghai, China) and quantified using a BCA Protein Assay Kit (Solarbio, Beijing, China). 20 µg of protein extracts were separated using SDS-10% polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Burlington, MA, USA). 5% non-fat milk was used to block these membranes, which were then incubated with specific antibodies overnight at 4°C. The specific antibodies used in this study were obtained from Abcam (Cambridge, UK). These included anti-EGFL7 (1:2000, ab256451), anti-Notch1 (1:2000, ab167441), anti-Delta4 (1:2000, ab183532), and anti-VEGF (1:2000, ab214424). The internal reference antibody used was anti-GAPDH (1:3000, AF7021, Affinity, Changzhou, China). Protein levels were detected using an Immobilon Western Chemilum HRP Substrate (Millipore, Burlington, MA, USA).

Statistical Analysis

Statistical analysis of data and image generation were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Normality testing was conducted using a Shapiro-Wilk test, and statistically significant differences were determined by a one-way ANOVA with post-hoc Bonferroni corrections for multiple comparisons. All data are expressed as mean ± standard deviation (SD). Additionally, all experiments were repeated three times. A value of p < 0.05 was considered to indicate statistical significance.

Effect of knockdown of EGFL7 on MWT and TWL values in rats

To investigate the effect of the EGFL7 gene on the progression of neuropathic pain, we constructed a CCI model to induce NP in rats. Subsequently, behavioral experiments were conducted to measure the neuropathic pain threshold using the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests. The results showed that the MWT and TWL values of the CCI group were higher than those of the sham group. And knocking down EGFL7 further increased MWT and TWL values (Fig. 1A-B). This result indicated that CCI and knockdown of EGFL7 increased pain ability in rats and may have affected the mobility of the animals.

Knockdown of EGFL7 exacerbated CCI-induced neuropathological damage

Next, H&E staining was used to analyze whether knockdown of EGFL7 influenced the extent of sciatic nerve damage. Compared to the H&E staining results of the sham group, the nerve cells in the model sections were disarranged, and extensive areas of sciatic nerve necrosis were observed, accompanied by a significant infiltration of inflammatory cells. And this phenomenon was further aggravated after treatment with shEGFL7 (Fig. 1C). The above results suggested that knockdown of shEGFL7 exacerbated sciatic nerve injury.

Effect of knockdown EGFL7 on the morphology and apoptosis of CCI-Induced sciatic nerves

Toluidine blue staining has been used to assess morphological changes to the sciatic nerve. Under normal conditions, myelinated axons of various sizes are arranged in an orderly, parallel fashion in the sciatic nerve with minimal inter-axonal space. However, in the case of CCI-induced injury, significant morphological changes including myelin vacuolization and demyelination were clearly observed and the ordering of these neurons was markedly disturbed. Knockdown of EGFL7 further promotes myelin vacuolization and demyelination (Fig. 2A).

Next, TUNEL staining was used to assess apoptosis in sciatic nerve sections. The results are presented in Fig. 2B and revealed that CCI induced a significant amount of apoptosis, which was exacerbated by knockdown EGFL7.

Effect of EGFL7 knockdown on the morphology and apoptosis of CCI-induced sciatic nerves

Toluidine blue staining has been used to assess morphological changes in the sciatic nerve. Under normal conditions, myelinated axons of various sizes are arranged in an orderly, parallel fashion in the sciatic nerve with minimal inter-axonal space. However, in the case of CCI-induced injury, significant morphological changes, including myelin vacuolization and demyelination, were clearly observed, and the arrangement of these neurons was markedly disturbed. Knockdown of EGFL7 further promoted myelin vacuolization and demyelination (Fig. 2A).

TUNEL staining was used to assess apoptosis in sciatic nerve sections. The results were presented in Fig. 2B and reveal that CCI induced a significant amount of apoptosis, which was exacerbated by EGFL7 knockdown.

Knockdown of EGFL7 inhibited nerve regeneration in the sciatic nerve induced by CCI

IHC was first used to analyze the expression of EGFL7. It was found that CCI-induced EGFL7 expression was reduced at the axonal site of the tissue medulla. Additionally, knockdown of EGFL7 resulted in a more significant reduction in EGFL7 expression (Fig. 3A). In addition, the potential for nerve regeneration was assessed by evaluating the expression of the growth factor VWF. As shown in Fig. 3B, VWF expression was significantly reduced at the myelinated axonal sites in the CCI model. Furthermore, knockdown of EGFL7 resulted in an even greater reduction in VWF expression. The above results suggested that EGFL7 may have a positive regulatory role in nerve regeneration after injury.

EGFL7 modulated the Notch signaling pathway

Finally, we investigated the specific molecular mechanisms of EGFL7 in regulating sciatic nerve injury. The results of WB were shown in Fig. 4, and it was found that CCI initially down-regulated EGFL7 expression, and knockdown of EGFL7 further decreased its expression. This result was similar to previous immunohistochemistry (IHC) results. Secondly, the protein expression of Notch signaling pathway-related proteins, Notch1 and Delta4, was down-regulated in the sciatic nerve of the CCI-treated model. Additionally, knockdown of EGFL7 further suppressed the protein expression of Notch1 and Delta4. The protein VEGF, which is an endothelial growth factor that promotes nerve regeneration, was also evaluated. The results were consistent with the IHC findings, indicating that VEGF expression was downregulated by CCI. Furthermore, knockdown of EGFL7 resulted in an even greater downregulation of VEGF expression. These results demonstrated that knockdown of EGFL7 downregulated the Notch pathway and inhibited the expression of nerve regeneration factors to exacerbate nerve injury.

The diagnosis and treatment of neuropathic pain (NP) remain challenging due to its diverse etiology and clinical presentation[13]. It is therefore urgent that we explore the pathogenesis of NP. The present study aimed to investigate the mechanism of EGFL7 in neuropathic pain. The results suggest that a deficiency of EGFL7 worsens neuropathic pain and nerve damage in a CCI rat model by reducing the expression of proteins in the Notch1 pathway.

Firstly, appropriate animal models are important for preclinical studies on pain. We established a CCI model by ligating the sciatic nerve, following the method described by Bennett's method[12], in order to replicate the characteristics of clinical neuropathic pain. In this study, we found that the sciatic nerve pathology sections of the CCI model group showed disrupted nerve cell arrangement, extensive sciatic nerve necrosis, and significant infiltration of inflammatory cells. Consistent with the results of the current study, Muthuraman[14] and Ye et al[15]. also found nerve fiber disarray and neuronal cell loss, as well as inflammatory cell infiltration, in sciatic nerve tissues induced by CCI. These findings are indicative of typical tissue features associated with nerve injury.

In addition, the results of Toluidine blue staining of sciatic nerve sections showed that CCI promotes myelin vacuolization. Furthermore, in the case of myelinated axons, it revealed complete degeneration of both the axon and the myelin sheath. This result is consistent with the findings of Sibel Türedi et al.[16], who observed changes in the status of the sciatic nerve in an injury model. In their study, they found that sciatic nerve injury led to myelin disorders and degeneration of both myelin and axons. Myelin is a multilayered fatty tissue that surrounds the axon of a neuron. It serves to insulate and protect the neuron, as well as increase the rate of information transmission along the axon[17]. In the case of nerve injury, selective A afferent fibers undergo a process of demyelination that can result in electrical instability, ectopic insertion of ion channels, and ultimately increased mechanical pain[18–21]. Thus, the normal function of myelin sheaths plays an important role in nociceptive transmission.

It has been shown that neuronal apoptosis is a mechanism that contributes to the maintenance of neuropathic pain. Inhibition of apoptosis has been found to alleviate nociceptive hypersensitivity and mechanically abnormal pain[22–24]. Therefore, TUNEL staining was used to assess apoptosis in the present study, and a significant increase in apoptosis was found in the sciatic nerve induced by CCI. This finding is consistent with the necrosis observed in the H&E results, indicating that these cells may have undergone necrosis through the apoptotic pathway. The potential for nerve regeneration using outer conduits was assessed by evaluating the expression of growth factors such as VWF and VEGF[25]. IHC and WB results showed that CCI inhibited the expression of VWF and VEGF, suggesting that CCI inhibits nerve regeneration. Nerve regeneration is particularly important in the later stages of nerve injury. Therefore, enhancing nerve regeneration is considered a key strategy for alleviating nerve damage.

However, deficiency in EGFL7 further exacerbated the damage to neuronal cell morphology and outcome. It also promoted neuronal apoptosis and inhibited neural regeneration. These results highlight the indispensable role of EGFL7 in nerve injury. Previous reports have found that EGFL7 has neuroprotective properties. On one hand, EGFL7 increased angiogenesis and nerve regeneration, promoting nerve defect repair, and reducing neuronal apoptosis in rats with spinal cord injury[10]. On the other hand, EGFL7 overexpression reduced T lymphocyte infiltration of the spinal cord parenchyma, strengthened the blood-brain barrier, and decreased inflammation in the central nervous system during spinal cord inflammation (EAE)[9].

Finally, mechanistic analyses revealed that CCI inhibited the expression of Notch1 and Delta4. Additionally, knockdown of EGFL7 further suppressed the expression of Notch-related proteins. It was found that activation of the Notch pathway improved nerve function[26, 27], promoted myelination, and facilitated myelin regeneration[28–30]. Consistent with the results of this study.

However, there are limitations in this study. This study explored EGFL7’s research on the pathological changes in nerve tissue structure and morphology induced by CCI. Therefore, the study of other physiological processes of EGFL7, such as autophagy, inflammation, and oxidative stress, is lacking. On the other hand, this study lacks clinical samples to validate the role of EGFL7 in neuralgia and nerve injury disorders. Therefore, future studies could focus on these two directions.

To summarize, the present study found that the absence of EGFL7 worsened the progression of neuropathic pain by reducing the activity of the Notch pathway, intensifying CCI-induced nerve injury and structural changes, promoting neuronal apoptosis, and blocking nerve modification processes.

Authors' contributions

ZQ. H, ZT. H and T. W contributed to the study's conception, design, and data collection. QC. AXJL, BH. W, YH. C, JL. Z, SJ. Y and MM. C contributed to data collection and statistical analysis. ZQ. H and ZT. H write the draft of the manuscript. All authors commented on previous versions. ZT. H and T. W finished the critical revision and approved the final manuscript.

Funding

This work was supported by the Inner Mongolia Natural Science Foundation (joint project) (2021LHMS08051) and Surface Program of Inner Mongolia Medical University (YKD2022MS004).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

Not applicable

Ethics approval and consent to participate

All animal procedures performed in this study were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Inner Mongolia Medical University and were conducted in accordance with the guidelines of the International Association for the Study of Pain. The study is reported in accordance with ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Deletion of EGFL7 exacerbated chronic constriction-induced nerve damage and neuropathic pain by modulating the notch pathway

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Version 1

Abstract

Figures

Background

Materials and methods

Animals and models

Animal grouping

Behavioral evaluation

Histologic hematoxylin-eosin (H&E) staining

Toluidine blue staining

TUNEL staining

Immunohistochemistry (IHC)

Western blotting (WB)

Statistical Analysis

Result

Effect of knockdown of EGFL7 on MWT and TWL values in rats

Knockdown of EGFL7 exacerbated CCI-induced neuropathological damage

Effect of knockdown EGFL7 on the morphology and apoptosis of CCI-Induced sciatic nerves

Effect of EGFL7 knockdown on the morphology and apoptosis of CCI-induced sciatic nerves

Knockdown of EGFL7 inhibited nerve regeneration in the sciatic nerve induced by CCI

EGFL7 modulated the Notch signaling pathway

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

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