Artesunate Inhibits Apoptosis and Promotes Survival in Schwann Cells via the PI3K/AKT/mTOR Axis in Diabetic Peripheral Neuropathy

Abstract

Diabetic peripheral neuropathy (DPN) is an early developing complication of diabetes mellitus associated with nerve dysfunction. Artesunate (ART), a natural compound extracted from the herb Artemisia annua L., was reported to benefit neural injury. However, whether ART has a role in preventing DPN is still unknown. In this study, a rat model of DPN with a high fat diet feeding and streptozotocin (STZ) injection was established. The findings indicated that ART treatment significantly ameliorated hyperglycemia-induced hot plate reaction latency (HPRL) decline, cold sensitivity and mechanical allodynia, and nerve injury by inhibiting sciatic nerve apoptosis. Further, ART restored high glucose (HG)-induced elevated apoptosis and deficient survival in rat neuronal Schwann cells, RSC96 cells. We demonstrated that ART promoted protein kinase B (AKT) phosphorylation as well as its downstream factor mammalian target of rapamycin (mTOR) in vivo and in vitro. Of note, the protective effects of ART in RSC96 cells under HG condition could be counteracted by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Taken together, ART mitigated hyperglycemia-induced nerve injury by suppressing apoptosis and promoting the viability of Schwann cells via the PI3K/AKT/mTOR signaling pathway.

INTRODUCTION

Diabetic peripheral neuropathy (DPN), as one of the early developing complications of diabetes, is the second main cause of amputation worldwide and affects up to 50% of diabetic patients.^1,2) DPN is characterized by sensorimotor polyneuropathy with progressive distal to proximal peripheral nerve degeneration, including chronic pain, sensory loss, and recurrent ulcerations.³⁾ Up to now, there is no specific treatment to reverse the progression of DPN. The palliative drugs, such as anticonvulsants, opioids, and antidepressants were used to relieve pain temporarily but exhibited adverse side effects, including muscle weakness, dizziness, and confusion.^4,5)

The principal difficulty in DPN treatment attributes to the complicated etiology. Exacerbated glucose flux give rise to the hyperactivation of glucose metabolism, which results in abundant reactive oxygen species (ROS) production. Increased levels of oxygen free radical formation induce neuron apoptosis and nerve fiber lost in the peripheral nerves, subsequently posing the occurrence and development of DPN.⁶⁾ In streptozotocin (STZ)-induced diabetic animals, demyelination and axonal degeneration of the sciatic nerve were observed.^7,8) In diabetic patients, durations of the sensory block and the motor block in sciatic nerve were higher than that in the non-diabetes.⁹⁾ These reports indicated that sciatic nerve injury was an important manifestation in DPN. At the cellular level, one of the most predominant cells in the peripheral nervous system are Schwann cells, which maintain neuronal structure and promote nerve cell survival after injury.¹⁰⁾ Liu et al. reported that Schwann cells were sensitive to glucose concentration and closely correlated with the progression of DPN.¹¹⁾ High glucose (HG)-induced oxidative damage and apoptosis result in decreased viability of Schwann cells, and consequently posed peripheral nerve deficits.^12,13) Hence, capability promotion and apoptosis suppression in Schwann cells have been proposed to be effective protection from cellular insult under HG condition.

Multiple signaling pathways are involved in the recovery progression of DPN. Protein kinase B (AKT) pathway is one of the main intracellular signaling pathways for neurotrophic regeneration.¹⁴⁾ Strong evidence showed that elevation of phosphor-AKT might partly reduce retrograde axonal transport in the vagal nerve of diabetic rats.¹⁵⁾ Moreover, activation of the AKT signaling pathway mitigated neuronal apoptosis in the hippocampus of rats with STZ-induced diabetic encephalopathy.¹⁶⁾ Therefore, AKT activation might be a feasible strategy for alleviating nerve injury. Exploring the new drugs that activate the AKT signaling pathway may be useful for DPN therapy.

Artesunate (ART), which is extracted from the leaf of the Chinese herbal medicine Artemisia annua L., is a water-soluble compound with molecular C₁₉H₂₈O₈ and molecular weight 384.42.¹⁷⁾ The previous study demonstrated that ART played a positive role in peripheral nerve repair by increasing myelinated axon regeneration after injury.¹⁸⁾ In addition, ART was reported to attenuate diabetic symptoms, such as weight loss and hyperglycemia, and simultaneously inhibit apoptosis of salivary glands in type 2 diabetic rats via the phosphatidylinositol 3-kinase (PI3K)/AKT pathway.¹⁹⁾ Nevertheless, the effects of ART on DPN and the mechanistic details remain to be further investigated.

Here, we suggested that ART might have a functional role in anti-apoptosis and pro-survival after peripheral nerve injury. Using rat neuronal Schwann cells under HG condition and a rat model of DPN, this current research evaluated the protective effects of ART on DPN. This study suggests that ART could be a potential therapeutic agent for DPN treatment.

MATERIALS AND METHODS

Animals and Induction of Diabetes

Male Sprague–Dawley (SD) rats weighing between 180 and 200 g were purchased from Beijing Huafukang Bioscience Co., Inc. (Beijing, China) and housed in humidity (45–55%) and temperature (22 ± 1 °C) controlled rooms with a 12 h light/12 h dark cycle. All experiments were carried out under an Institutional Animal Care and Use Committee (IACUC)-approved protocol of the Guangxi Medical University. Diabetes was induced in SD rats given high fat diet feeding (common feed 57.5%, sucrose 25%, lard 15%, cholesterol 1.5%, sodium cholate 1%) for 4 weeks followed by a single intraperitoneal injection of streptozotocin (STZ, 25 mg/kg, Aladdin, Shanghai, China). The sham rats were injected with an equal volume of citrate buffer. Three days after STZ injection, rats with blood glucose concentrations of 16.7 mmol/L or more were screened for diabetic models. For ART administration, rats were treated with ART (0.7 mg/mL, Aladdin) in the drinking water and maintained on the high-fat diet for 8 weeks. The blood glucose levels were monitored at 5, 6, 8, and 12 weeks. The chemical structure of ART and the detailed experimental protocol were shown in Fig. 1.

Fig. 1. The Chemical Structure of ART and the Detailed Experimental Protocol

(A) Chemical structure of ART. (B) Detailed experimental protocol representing the DPN induction and ART treatment.

Hot Plate Test

The rats were placed on the hot plate apparatus at a temperature of 52 ± 0.3 °C. The hot plate reaction latency (HPRL) was recorded when rats jumped or licked the hind paws or abnormally flicked the hind paws to avoid pain.

Acetone Test

Cold allodynia was performed by applying a drop of 100% acetone (Sinopharm, Shanghai, China) to the plantar surface of rats. A foot withdrawal response evoked by the cooling of acetone was considered a sign of cold allodynia. Acetone was applied 3 times to the hind paw with an interval of 3 min between tests. The pain-related behavior was graded as follows: 0, no response; 1, stamp, flick, or quick withdrawal of the hind paw; 2, repeated flicking or prolonged withdrawal of the hind paw; 3, repeated licking and flicking of the hind paw.

Von Frey Test

Mechanical allodynia was detected in rats using the up and down method with von Frey filaments (RWD Life Science, Shenzhen, China). The filaments weighing between 0.4 and 15 g were applied 4 times in each hind paw of rats. The 50% paw-withdrawal threshold was recorded.

Histology Examination

All harvested sciatic nerve samples fixed in 4% paraformaldehyde were dehydrated, embedded in paraffin, and cut into 5 µm-thick slices. After deparaffinized with xylene (Aladdin), sections were rehydrated in descending ethanol gradients. Subsequently, sciatic nerve segments were stained with hematoxylin (Solarbio, Beijing, China) for 5 min and eosin (Sangon, Shanghai, China) for 3 min, and ultimately examined by light microscope (Olympus, Tokyo, Japan).

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate (dUTP) Nick End Labeling (TUNEL) Assay

DNA fragmentation in sciatic nerve sections was tested using the In Situ Cell Death Detection Kit (Roche, Nutley, NJ, U.S.A.). Briefly, sections were immersed in 0.1% Triton X-100 (Beyotime, Shanghai, China) at room temperature for 8 min, and then incubated with enzyme and label solution at 37 °C for 60 min. Later the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Aladdin) and observed under a fluorescent microscope (Olympus).

Apoptosis Observation

Cysteine-requiring aspartate protease 3 (Caspase-3) plays an important role in cell apoptosis. Apoptosis in sciatic nerve tissues was determined using the Caspase-3 Activity Assay Kit (Beyotime) according to the manufacturer’s instruments.

Immunofluorescence Analysis

To evaluate the fibers in the sciatic nerve of rats, immunofluorescence was performed with NF-200 antibody. Sciatic nerve sections were blocked using 1% bovine serum albumin (BSA, Sangon) for 15 min and then incubated with primary antibody against NF-200 (1 : 50, Abclonal, Shanghai, China) at 4 °C overnight. A goat anti-rabbit antibody labeled with cy3 (1 : 200, Invitrogen, Carlsbad, CA, U.S.A.) was used as a secondary antibody. After nuclei were stained with DAPI (Aladdin), sections were examined on a fluorescent microscope (Olympus).

Electron Microscopy Study

Sciatic nerve samples were fixed in 1% osmium tetroxide at room temperature for 2 h. After washing with 0.1 mol/L phosphate buffer, the samples were dehydrated with an ascending series of ethanol and then dehydrated in 100% acetone (Sinopharm). Subsequently, the samples were blocked in resin and sectioned at 60–80 nm thickness. Ultrathin sections were stained with 2% uranyl acetate in ethanol and 2.6% lead citrate for 8 min, respectively. Photos were obtained with an electron microscope (Hitachi, Tokyo, Japan).

Cell Culture

Rat neuronal Schwann cell line RSC96 (iCell Bioscience lnc, Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO₂ at 37 °C. RSC96 cells were incubated with 5.5 mmol/L glucose (normal glucose, NG), 25 mmol/L glucose (high glucose, HG), or 5.5 mmol/L glucose plus 19.5 mmol/L mannitol (M), respectively. ART administration was performed by combining treatment with 25 mmol/L glucose and different concentrations of ART (0.15 µg/mL, HG + ART-L and 0.3 µg/mL, HG + ART-H) for 48 h. For rescue experiments, cells were treated with ART (0.3 µg/mL) combined with LY294002 (20 µmol/L, the PI3K inhibitor) for 48 h.

Cell Viability Detection

Cell Counting Kit-8 (CCK-8, Beyotime) was used to measure the viability of RSC96 cells. RSC cells were seeded at 3 × 10³ cells per well in 96-well plates. After treatment with glucose (5.5, 25 mmol/L), ART (0.15, 0.3 µg/mL), and LY294002, 20 µmol/L) for 48 h, 10 µL of CCK-8 kit reagents were added to cells and incubated at 37 °C for 2 h. The optical density of live cells was monitored using an absorbance reader (Biotek, Winooski, VT, U.S.A.) at 450 nm.

Cell Apoptosis Evaluation

All harvested RSC96 cells were washed with phosphate buffered saline (PBS), incubated with 5 µL Annexin V-fluorescein isothiocyanate (FITC) (AV) and 10 µL propidium Iodide (PI) (AV/PI Cell Apoptosis Detection Kit, Beyotime) at room temperature in dark for 15 min. A flow cytometer (ACEA, San Diego, CA, U.S.A.) was used to determine the percent of apoptotic cells.

Western Blotting Analysis

Total protein was extracted from sciatic nerve tissues or RSC96 cells using Whole Cell Lysis Assay (Solarbio) and quantified by a BCA Protein Assay Kit (Solarbio). Equal protein was mixed with loading buffer, electrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (8, 10, and 14%), and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, U.S.A.). Membranes were immunoblotted with primary antibodies against Bcl-xl (1 : 500, Abcam, Cambridge, MA, U.S.A.), p-AKT, mammalian target of rapamycin (mTOR) (1 : 500, CST, Danvers, MA, U.S.A.), Bax, AKT (1 : 1000, CST), and p-mTOR (1 : 400, CST) at 4 °C overnight. Subsequently, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1 : 5000, Solarbio) followed by ECL Western blotting detection system (Solarbio).

Statistics

Results are reported as mean ± standard deviation (S.D.). Statistical analyses were performed with GraphPad Prism using ANOVA followed by Tukey’s multiple comparisons test as indicated. A p-value of less than 0.05 was considered significant.

RESULTS

ART Preserved the Sciatic Nerve Structure and Function in DPN

Hyperglycemia caused a significant reduction in body weight of rats (Fig. 2A, p < 0.01). Blood glucose levels in the DPN rats were remarkably elevated when compared to the sham rats (Fig. 2B, p < 0.01). Neural injury was primarily evaluated by the histomorphological detection following hematoxylin and hematoxylin–eosin (H&E) staining of the sciatic nerve tissues. Figure 2C illustrated histopathological changes in the sciatic nerves of DPN rats compared with the sham-operated control rats, such as lower myelin width, less numbers of axon (the red arrow), and wider neuron gaps (the black arrow). In line with the findings of light microscopy, the sciatic nerves of DPN rats exhibited myelin destruction, miniaturization, and shrinkage of axons under electron microscopy examination (the blue arrow, Fig. 2D). Immunofluorescence analysis in sciatic nerves of DPN rats revealed a similar decrease in NF-200, which is a regeneration-associated protein of injured peripheral nerves²⁰⁾ (Fig. 2E). Functionally, the HPRL sharply reduced to 32.41, 23.54, 18.02, and 12.47% of the sham group at 5, 6, 8 and 12 weeks, respectively (Fig. 3A, p < 0.01). Furthermore, increased cold sensitivity and mechanical allodynia of DPN rats were observed in Figs. 3B and C (p < 0.05). Treatment with ART displayed a better outcome in ameliorating the pain-related behavior of DPN rats via decreasing the blood glucose levels and maintaining the histopathological structure. These findings indicated that therapeutic intervention with ART alleviated adverse pathological changes and preserved sciatic nerve function.

Fig. 2. Detection of the Sciatic Nerve Function and Morphology in Rats with DPN

Effects of ART on body weight (A) and blood glucose (B) in DPN rats. HE stained images (C) and electron micrographs (D) of sciatic nerve tissues. (E) Immunofluorescence analysis of sciatic nerve sections for NF-200. The arrows pointed at axon (red), neuron gap (black), and damaged nerve fibers and myelin sheath (blue). ^ns p > 0.05, ** p < 0.01.

Fig. 3. ART Partially Alleviated Tactile Allodynia

Effects of ART on HPRL (A), cold allodynia (B), and mechanical hyperalgesia (C) in DPN rats. ^ns p > 0.05, * p < 0.05, ** p < 0.01.

ART Attenuated Apoptosis in the Sciatic Nerves of the DPN Rats

TUNEL assay was performed to detect cell apoptosis in DPN rats (Fig. 4A). Multiple red nuclei of apoptotic cells were observed in the sciatic nerves of the rats from the DPN group. However, TUNEL-positive cells with ART treatment were markedly lower than that in the DNP rats. Consistent with these observations, the Western blotting analysis showed that hyperglycemia-induced upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic protein Bcl-xl were restored by ART treatment (Figs. 4B, C, p < 0.01). Furthermore, the activation of Caspase-3 increased by 3-fold in the damaged sciatic nerves, whereas ART notably decreased caspase-3 activity (Fig. 4D, p < 0.05). Here, we showed that ART inhibited hyperglycemia-induced apoptosis in the sciatic nerves of the DPN rats.

Fig. 4. ART Inhibited Apoptosis in the Sciatic Nerves of the DPN Rats

(A) TUNEL assay was used to measure the apoptotic cells in the sciatic nerve tissues. (B) The levels of Bcl-xl and Bax were tested by Western blot. (C) The intensity of bands was quantified. (D) Detection of the Caspase-3 activity in the sciatic nerve tissues. * p < 0.05, ** p < 0.01.

ART Inhibited HG-Induced Apoptosis in RSC96 Cells

To verify the effects of ART on apoptosis in DPN, the apoptotic phenotype of RSC96 cells under HG condition was assessed by AV/PI staining in the experiments in vitro. Forty-eight hours after treatment with HG, more than 7% of RSC96 cells exhibited apoptosis, which was reduced in RSC96 cells treated with ART (Figs. 5A, B, p < 0.05). The altered expressions of the crucial apoptotic players were consistent with the apoptotic phenotype of RSC96 cells after ART treatment (Figs. 5C, D, p < 0.01). In addition, cell viability was decreased in HG-treated RSC96 cells and was increased with ART treatment (Fig. 5E, p < 0.01). These results further demonstrated that ART treatment diminished apoptosis in HG-treated RSC96 cells.

Fig. 5. ART Restrained Apoptosis in RSC96 Cells Induced by HG

(A) AV/PI staining followed by flow cytometry analysis and (B) quantitation for cell apoptosis. (C) Western blot analysis and (D) quantitation of Bcl-xl and Bax. (E) CCK-8 was performed to examine the cell viability of RSC96 cells. ^ns p > 0.05, * p < 0.05, ** p < 0.01.

ART Activated the PI3K/AKT/mTOR Signaling Pathway

Compared with the sham group, the phosphorylation levels of AKT and mTOR were reduced under hyperglycemia administration, whereas ART notably elevated their phosphorylation (Figs. 6A–D, p < 0.05). Furthermore, ART treatment restored HG-induced decreased viability and elevated apoptosis in RSC96 cells (Figs. 6E–I, p < 0.01). Notably, the above effects were impeded by LY294002, the PI3K inhibitor. Overall, ART inhibited apoptosis and promoted survival in RSC96 cells via activating the PI3K/AKT/mTOR signaling pathway under HG condition.

Fig. 6. ART Reduced Apoptosis and Accelerated Survival in Sciatic Nerves of Rats and RSC96 Cells via Activating the PI3K/AKT/mTOR Signaling Pathway

The protein levels of p-AKT, AKT, p-mTOR, and mTOR were detected by Western blot analysis in sciatic nerve tissues of rats (A) and RSC96 cells (B). (C, D) Bar graphs summarized quantitative analysis by the intensity of bands. (E) The cell viability of RSC96 cells was analyzed by CCK-8 assay. Determination of cell apoptosis (F, G) and detection of key apoptotic proteins (H, I) in RSC96 cells. * p < 0.05, ** p < 0.01.

DISCUSSION

This present study showed the role of ART in inhibiting nerve injury in DPN through a variety of experiments in vivo and in vitro. ART maintained nerve structure and function by alleviating the apoptosis induced by hyperglycemia in the sciatic nerves of the DPN rats. In addition, our results indicated that ART promoted survival and decrease apoptosis in Schwann cells exposure to HG by the activation of the PI3K/AKT/mTOR signaling pathway.

The neurotrophic protection effects of ART were revealed by a previous study demonstrated in sciatic nerve regeneration.¹⁸⁾ Apart from alleviating pathological damage and increasing the latency values, ART exerted anti-apoptotic effects in DPN in vivo and in vitro. One of the most predominant anti-apoptotic factors in nerve cell apoptosis was Bcl-xl, which controlled the mitochondrial membrane potential balance, increased neural plasticity, and promoted nerve survival.^21,22) As a neuroprotective molecule, Bcl-xl protected Schwann cells from apoptosis.^23,24) Noteworthily, ART suppressed cell apoptosis by increasing mitochondrial respiratory enzyme activity and subsequently improving mitochondrial function.²⁵⁾ Moreover, during the process of nerve cell apoptosis, the level of Bax was upregulated. Bax, as a pro-apoptotic factor, was reported to facilitate mitochondrial permeabilization.²⁶⁾ Interestingly, ART was shown to suppress apoptosis by inhibiting Bax protein expression.^27,28) Furthermore, Severe mitochondria dysfunction induced activation of caspase-3, the executioner of apoptosis, and consequential cell apoptosis.²⁹⁾ A previous study showed that the levels of cleaved caspase 3 were increased in the spinal tissues of DPN rats.³⁰⁾ In this present study, administration of ART led to increased Bcl-xl expression and decreased Bax expression as well as caspase-3 activity in the sciatic nerve tissues of DPN rats. Thus, ART might restrain apoptosis by maintain mitochondrial function after nerve injury in DPN. Methylcobalamin, a clinical drug for DPN therapy, was shown to prevent mitochondria dysfunction and inhibit apoptosis.³¹⁾ Our results indicating ART as an anti-apoptotic factor might provide preliminary evidence for DPN treatment.

As previously shown in animal models and human patients, remarkable neuron injury, as characterized by a reduction of nerve fiber density, was observed in the sciatic nerve tissues in DPN.^32,33) Decreased HPRL level and increased tail-flick response observed in diabetic rats could be associated with sciatic nerve injury and therefore a decrease in the number of motor units.^34,35) Nerve is formed from a cluster of assembled nerve fibers, which include many neurons. Axons are the elongated portion of the neuron that functioned for signal transmission. Myelin is produced by Schwann cells, which wrap around the axons to establish a thick myelin sheath.³⁶⁾ Schwann cell degeneration and apoptosis are marked features in the occurrence and development of DPN.³⁷⁾ As the crucial supporting glial cells of the peripheral nervous system, Schwann cells maintain axonal survival via secreting a variety of neuroprotective factors.³⁸⁾ Acetylcholinesterase (AChE) and brain derived neurotrophic factor (BDNF) are two well-known factors for neural development and regeneration.^39,40) Of note, an earlier study demonstrated that ART treatment significantly promoted AchE and BDNF expression.¹⁹⁾ Moreover, ART was observed to alleviate hippocampal neuronal injury and maintain neuronal vitality.⁴¹⁾ By evaluating the viability of RSC96 cells under HG condition, we showed that ART treatment facilitated RSC96 cell survival, which was vital for neuroprotective factors secretion. Based on the above evidence, we were aware of the probability that ART might promote axonal regeneration in DPN via increasing secretion of neuroprotective factors, for instance, AChE and BDNF. Whether these two factors were mediated by ART for neurotrophic regeneration in DPN needs further exploration.

Axon injury and myelin loss in the sciatic nerve are the main physiological features of DPN.⁴²⁾ As an insulator, myelin boosts the neural signal transmissibility among neurons and preserved the connectivity of a healthy nervous system.⁴³⁾ The regulation program is extremely complex and associated with multiple signaling pathways. It is generally believed that Schwann cells maintain proper myelination via the PI3K/AKT/mTOR signaling pathway.⁴⁴⁾ High mTOR activity accelerates Schwann cell survival and myelin growth during nerve development.⁴⁵⁾ By detecting the viability of HG-treated RSC96 cells, we found a crucial phenomenon that ART induced survival was partly counteracted in RSC96 cells treated with PI3K inhibitor LY294002. These results showed the involvement of ART in PI3K/AKT/mTOR-mediated myelination. Furthermore, Hackett et al. reported that insulin-like growth factor 1 (IGF-1) induced PI3K activation in various cells types, including Schwann cells.⁴⁶⁾ Interestingly, ART was known to elevate IGF-1.⁴⁷⁾ Therefore, ART might promote myelination in peripheral nerves by facilitating IGF-1 expression, which induced phosphorylation of AKT, and subsequent phosphorylation of mTOR in DPN. The underlying mechanisms involved in myelination resulting from ART await further elucidation.

In conclusion, this current study indicated that ART effectively decreased neuropathological injury and HPRL in DPN rats. In addition, ART suppressed HG-induced apoptosis as well as neurologic deficits in vivo and in vitro. Furthermore, our results showed that ART exerted its neuroprotective effects by inhibiting apoptosis and promoting viability in neurons via activating the PI3K/AKT/mTOR signaling pathway. Hence, ART might be a novel therapeutic drug for the treatment of DPN.

Acknowledgments

This research was funded by the High-level Talents Scientific Research Developmental Fund of Liuzhou People’s Hospital (Grant No. LRYGCC202109).

Conflict of Interest

The authors declare no conflict of interest.

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