Myricetin promotes peripheral nerve regeneration in rat model of sciatic nerve injury via regulation of BDNF- Akt/GSK-3β/mTOR signalling pathway

Purpose: To investigate the effects of myricetin on peripheral nerve regeneration in sciatic nerve crush injury model. Methods: Separate groups of rats were administered myricetin at 25, 50 or 100 mg/kg body weight/day for 2 weeks. Functional recovery following sciatic nerve injury was assessed by foot position and walking track analyses, measurement of mechanical hyperalgesia, and withdrawal reflex latency (WRL). Results: Myricetin treatment resulted in significantly enhanced recovery of sensorimotor functions as evidenced by increased scores in functional analysis tests. Myricetin treatment remarkably elevated brain derived neurotrophic factor (BDNF) expression, and also enhanced activation of Akt and mTORc1, reflecting up-regulation of PI3K/Akt/mTORC1 signalling involved in nerve regeneration. Conclusion: Myricetin enhances functional recovery and nerve regeneration in rats. These findings suggest that myricetin is a potent neuroprotective agent with potential for the management of peripheral nerve injury.


INTRODUCTION
In clinical practice, peripheral nerve injuries (PNIs) are encountered frequently due to tumours, accidental trauma, acute compression, surgeries or iatrogenic injuries.Traumatic injuries are on the increase, with estimated 500,000 new patients every year [1].The peripheral nerve damage results in total or partial function impairment affecting the motor, sensory and autonomic functions.Peripheral nerve injury (PNI) is characterized by disruption of axons and myelin sheaths [2].Nevertheless, Schwann cell tubes of the basal lamina remain intact and aid in regeneration of axons and re-innervation of tissues [3,4].However, the time taken for reinnervation of the target tissues is critical for improvement in sensory and motor functions, which makes accelerated regeneration vital for adequate functional recovery [5].Sciatic nerve crush injury is an adequate experimental model for studying the molecular events involved in peripheral nerve regeneration.The model is extensively employed in assessing the effectiveness of novel drugs in enhancing the speed of regeneration and effectively improving re-innervation [1].
The pivotal part of neurotrophins in neuroprotection and neuro-regeneration is well documented.The neurotrophic factors (NTFs) regulate neuronal survival, viability and differentiation [6].Following peripheral nerve injury, Schwann cells (SCs) aid in the removal of injured end of the axons.Several neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF), and neurotrophic factors-3, -4/5 and -6 are secreted by SCs [7,8].Studies have demonstrated that when neurotrophic factors are transported to the site of PNI, in vivo nerve regeneration is improved [7,9].
The mechanistic target-of-rapamycin (mTOR), a serine-threonine protein kinase, is critically involved in vital signaling pathways.It is the core constituent of mTOR complex 1 (mTORC1) and 2 (mTORC2).Studies have shown that mTORC1 is crucial for regulating various anabolic reactions [10], and is tightly associated with the major cell signaling pathway, viz, PI3K-Akt pathway.PI3K-Akt signaling, a major downstream target of growth factors, controls a wide range of functions including cell survival, proliferation, growth, and metabolism.Activation of mTORC1 regulates phosphatase and tensin homolog (PTEN), the chief inhibitor of PI3K/Akt [11].In addition, AKT phosphorylates and inhibits glycogen synthase kinase 3β (GSK-3β, another key downstream protein) which is critical for axon growth and branching [12].Studies have reported that the Akt-mTORC1 pathway plays a pivotal role in PNS and CNS myelination [13,14].Compounds that can regulate the PI3K-Akt-mTORC1 cascade would be of immense clinical value.Myricetin (3, 3', 4', 4, 5, 5', 7hexahydroxyflavone), a plant-derived flavonoid is present in various vegetable, fruits, nuts and red wine [15].Myricetin has been reported to possess anti-oxidant and antibacterial [16]; cardio-protective [17], and anti-proliferative properties [18].The lipid-lowering activities of myricetin [19], and its anti-diabetic properties [20] have been reported.However, not much work has been done on the effects of myricetin on nerve regeneration.In this study, the effect of myricetin on enhancement of nerve regeneration following PNI in sciatic nerve crush injury model was investigated.

EXPERIMENTAL Antibodies and chemicals
Myricetin was procured from Sigma-Aldrich (St. Louis, MO, USA).Antibodies against BDNF, TrkB, Akt, mTORc1, PTEN, GSK-3β, phospho-Akt, phospho-GSK-3β, and phospho-mTORC1 were also procured from Sigma-Aldrich.β-Actin and proliferating cell nuclear antigen (PCNA) used for expression analysis was purchased from Santa Cruz Biotechnology (CA, USA).All other chemicals used were of analytical grade and were obtained from Sigma-Aldrich unless otherwise stated.

Experimental animals
Adult Sprague-Dawley rats weighing 200 -220 g were procured from research laboratory animal facility of our University.The animals were housed in sterile polypropylene cages (2 per cage) under standard animal house settings (23 ± 2 ℃; 12-h dark/12-h light cycle, and 50 -55 % relative humidity), with unrestricted access to feed and clean drinking water.The animal experimental protocols were approved by the ethical committee of Southwest Medical University (approval number: TK345511), and were carried out in compliance with international animal welfare guidelines [21].

Surgical procedure for sciatic nerve injury
The rats were anesthetized by intraperitoneal (i.p.) injection of 0.7 mg/kg acepromazine, 10 mg/kg xylazine, and 95 mg/kg ketamine.The surgery sites were shaved neatly and wiped with alcohol (70 %).Using the gluteal muscle splitting method, the sciatic nerve was uncovered by incision made along the mid-thigh level with the help of a surgical microscope.At 10 mm from the sciatic notch, the sciatic nerve was crumpled for a total of 30 sec (10 sec at a time) with a pause of 10 s [22].The sciatic nerve was observed to make sure that the epineurial sheath was undamaged but translucent due to injury (axotomy).It was reimbursed back below the muscle [23], and the incision was sutured.
The rats exposed to sciatic nerve crush injury were randomly divided into 5 groups (groups II to V) with 12 rats/group.Treatment groups (groups III -V) received myricetin (25, 50 or 100 mg/kg body weight) via oral gavage every day for 14 days, starting 6 h after sciatic nerve crush damage.Group I rats (control) were not subjected to nerve injury.Groups I and II rats were treated with equivalent volumes of saline in place of myricetin.

Foot positioning
Following sciatic nerve injury, foot positioning was monitored.The animals were positioned on a plate at room temperature, and foot positioning was observed every 3 rd day following 24 h postinjury for 21 days.The foot positioning of the rats was scored based on altered locomotor scale ranging from 1 to 3 [24].

Measurement of withdrawal reflex latency
Withdrawal reflex latency (WRL) refers to the time taken for the withdrawal of the affected hind paw from a hotplate.For the measurement of WRL, animals with the injured hind paws were placed on a hotplate (at 56 ℃) after wrapping the rats with surgical cloth above the midriff.This test was conducted thrice with a break of 120 sec between repeated tests to avoid sensitization [25].The latency time was determined with a stopwatch, and was measured from the time the rats were placed on the plate to the time of withdrawal of the hind paw [26].

Mechanical hyperalgesia
To measure hyperalgesia following sciatic nerve crush damage, each rat was placed gently on a non-greasy surface, and onto the dorsal surface of the affected hind limbs, a piston was applied using computerized gauge.The display of pain by either making a noise or paw withdrawal was recorded.When animals exhibited pain, the pressure due to the device was released proximately and pain threshold was recorded by means of an Analgesic Meter (Model 21025, UGO Basile, Italy) [27].The mean of three successive values punctuated by an interval of 10 -20 min was determined.The pressure needed to induce withdrawal of paw in the animals is called the paw pressure threshold.Pain threshold levels were measured every 5 days and expressed in grams according to the method of Renno et al [24].

Determination of motor function recovery
For determination of recovery of locomotor activity, sciatic functional index (SFI) [28] and toe spread index (TSI) [29] assessments were conducted.The indices were constructed on measurements of walking footpaths which reflect functional sciatic nerve recovery.
For assessing locomotor activity, rats were trained to move on a white sheet of paper that covered the bottom of an 8.5-cm wide and 100cm long track in a dark box [30].Later, the hind feet of the animals were dipped in red dye to track the walk.To determine the following measurements, footprints of rats were used: Print length (PL) -the distance from the heel to the third toe; toe spread (TS) -the distance between the first and fifth toes, and intermediary toe spread (ITS) -the distance between the second and the fourth toes.The measurements were recorded for the affected limb (E) and normal (N) limb.For TSI assessment, TS was measured.Multiple prints from each foot were acquired, but only three prints were used to determine the mean extents in the E and N sides.The index of nerve dysfunction, SFI, ranges from 0 to 100, with 0 equivalent to regular function, and 100 equivalent to wide-range dysfunction.It was calculated using Eq 1.

Western blot analysis
The sciatic nerve tissues (6 per group) were normalized in cell lysis buffer and centrifuged for 30 min at 4 ℃ [32].Total protein content of the supernatant was measured using Bradford assay (Bio-Rad).Equivalent quantities of the protein samples (50 μg) were subjected to SDSpolyacrylamide gel electrophoresis SDS-PAGE).The protein bands were blotted and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore).The membranes were then blocked with non-fat milk (5 %) in TBST (20 mM Tris, 0.05 % Tween-20, and 150 mM NaCl) for 2 h at normal temperature.Thereafter, the PVDF membranes were washed with PBS and incubated at 4 ℃ with primary antibodies overnight followed by incubation with horseradish peroxidase-conjugated secondary antibody at room temperature for 60 min.The blots were washed further and the immune-reactive bands were quantified using enhanced chemiluminescence method (GE Healthcare, Piscataway, NJ).

RT-PCR analysis
In order to determine the gene expressions of TrkB and BDNF, RT-PCR analysis was performed.Total RNA was extracted from nerve tissues by means of TRIzol (Invitrogen, Carlsbad, CA, USA) as per manufacturer's guidelines.The RNA samples were then exposed to DNaseI digestion (Qiagen, Inc.Valencia, CA) to exclude any DNA residues, and the RNA density was assessed (Nanodrop spectrophotometer -ND-1000, Bio-Rad, USA).The initial strand of cDNA was created by reverse transcription using Fermentas Company (USA) Revert Aid First Strand cDNA Synthesis Kit.The PCR procedures were carried out as directed in the kit protocol (Fermentas Company, USA) using the following primer sequences for BDNF and TrkB: GAPDH expression was used as the internal control.The PCR mixture obtained was run on agarose gel (1 %) and the products were visualised with ethidium bromide (0.05 %).Band intensities were scanned and analysed with Bio-Gel imagery apparatus (Bio-Rad, USA).

Statistical analysis
The results of the study are presented as mean ± standard deviation (SD) obtained from six independent trials.Statistical differences between different groups were analysed using SPSS package (version 22.0).The results were subjected to one-way analysis of variance (ANOVA) with subsequent post-hoc analysis using Duncan's multiple range test.Values of p < 0.05 were assumed to be statistically significant.

Myricetin treatment enhanced functional recovery after nerve crush injury
The effects of myricetin action on functional recovery following sciatic nerve crush injury were assessed.The foot location analysis revealed a progressive retrieval in the animals treated with myricetin (25, 50 or 100 mg/kg).Myricetin-treated group exhibited marked recovery on day 21 postcrush injury, relative to the saline-treated injured rats.Interestingly, rats treated with higher dose of myricetin (100 mg/kg) presented negligible foot location movement or any irregular foot placing from day 14 post-surgery through day 21 (Figure 1).

Myricetin improved withdrawal reflex latency
Withdrawal reflex latency values reflect the nociceptive and motor functions of peripheral nerves.The latency values were higher in the saline-treated injured group than in the normal control group.Myricetin administration significantly (p < 0.05) reduced the latency values, when compared to group II animals.Furthermore, the latency values of rats treated with 100 mg myricetin on days 8, 12 post-injury were similar to latency values of normal control rats (Figure 2).These observations suggest that myricetin treatment enhances recovery of motor and nociceptive functions of injured sciatic nerve.

Effect of myricetin on withdrawal reflex thresholds
The withdrawal reflex thresholds (WRT) of affected hind limb in response to external pressure stimulus was measured and is termed powered hyperalgesia.It was measured every week following injury, for 3 weeks.On the 7 th day following SNC injury, the rats displayed higher WRT values, when compared to normal control rats.On the 2 nd and 3 rd weeks, there was a slight decrease in mechanical hyperalgesia in the saline-treated injured rats, when compared to the 1 st week.Nevertheless, the myricetin-treated rats presented a marked decrease in paw WRT to powered stimulus, relative to saline-treated crush group from 14 th to 21 st day post-injury (Figure 3).There was no considerable difference in WRT values between rats treated with 100 mg myricetin and rats in the normal control group.

Myricetin enhanced motor function recovery
In the normal control-saline treated animals, SFI and TSI values were normal, with the SFI value around -2, and TSI value zero.However, the TSI and SFI values in injured-saline treated rats were significantly reduced (Table 1 and Table 2), indicating functional impairment following nerve crush injury.Interestingly, treatment with myricetin produced recovery of sensorimotor function as evidenced by significant (p < 0.05) increases in SFI and TSI values.On day 21, the SFI scores increased from -40.21 ± 1.91 to -13.16 ± 0.81; while TSI increased to -0.09 ± 0.001 from -0.29 ± 0.011 on treatment with myricetin at 100 mg.These observations demonstrate that myricetin enhanced recovery of sensorimotor functions following SNC injury.

Myricetin raised BDNF-TrkB expression
The mRNA levels of BDNF and TrkB in the crushed nerves were determined using RT-PCR.Following SNC injury, the BDNF and TrkB mRNA levels were significantly reduced, when compared to normal control rats (p < 0.05).However, the BDNF expression and TrkB mRNA increased significantly in myricetin-treated SNC injury rats (Figures 4 A and B), relative to the saline-treated SNC injure rats.The raised levels of BDNF expression and TrkB mRNA reflect increased nerve regeneration following myricetin treatment, since BDNF is pivotal in regulating and controlling neuronal cell growth, survival and proliferation.

Myricetin up-regulated Akt/mTORC1 signalling
The effects of myricetin on Akt/mTORC1 signalling following SNC injury was assessed.Following SNC injury, noticeably raised levels of phosphorylated forms of Akt, mTORc1 and GSK-3β was observed (Figure 5 A -C).

DISCUSSION
Peripheral nerve injuries following accidental trauma and sport injuries are frequently seen in clinical practice worldwide.Post-traumatic peripheral nerve regeneration and functional recovery pose a huge challenge since inadequate restoration of function and innervation leads to subsequent loss of function of the target organ [33].
Thus, accelerated repair of the damaged nerves, enhanced regeneration of nerves and sensorimotor functional recovery are crucial in restorative medicine.Sciatic nerve crush injury prototype is a widely employed animal model in studies of regeneration of nerves [34].In this study, the effects of myricetin on nerve regeneration and functional recovery following sciatic nerve crush injury were investigated.
It was observed that myricetin treatment effectively improved the foot positioning of the rats.The WRL and WRT values reflect the beneficial effects of myricetin on enhancement of functional recovery of the sciatic nerve, while SFI and TSI values obtained following rat walking track analysis reflect the motor function of the injured hind limb.The SFI values are regarded as a standard index for evaluating motor function of nerves following nerve crush injury and repair [35].In this study, all the experimental groups exhibited gradual recovery of sciatic nerve function.Interestingly, the myricetin-treated rats showed significantly higher SFI and TSI values than the saline treated-injured group.The recovery of sensorimotor functions was enhanced and myricetin-treated injured rats presented SFI very close to that of normal control rats on the 3 rd week post-injury.The SFI and TSI values reflect gain in function following SNC injury.
It is well documented that the neurotrophic factors are crucial in neuronal cell proliferation and growth following nerve injuries [36].The BDNF is a major neurotrophic factor that is involved in axonal regeneration and myelinogenesis of injured nerve cells.Studies have demonstrated that BDNF increased axonal regeneration and myelination in nerve fibres following sciatic nerve crush [37].In this study, myricetin treatment clearly enhanced BDNF and TrkB mRNA expressions.The raised levels of BDNF were in line with increased axonal regeneration and myelination as observed.The increase in BDNF levels could have, in part enhanced nerve regeneration observed following myricetin treatment.
In injured nerve cells, multiple factors are associated with the process of repair and regeneration.The PI3K/AKT/mTORC1 pathway is the major pathway involved in nerve cell growth, proliferation and survival [38].Studies have shown that activation of the mTORC1 pathway and deletion of inhibitor PTEN leads to enhanced axonal regeneration, and AKT activation triggers regeneration of axon fibres at the site of crush injury [38].A marked upregulation in the levels of phosphorylated AKT and mTORC1 along with reduced PTEN expressions was observed in this study.Furthermore, elevated expression of phosphorylated forms of GSK3β were observed on myricetin treatment.The GSK3 family comprises GSK3α and GSK3β.It is known that AKT phosphorylates and inactivates the kinase activity of GSK3α at Ser21 or GSK3β at Ser9 [39].Interestingly, GSK3β inactivation has been shown to mediate AKT signalling-induced axon regeneration [40].Thus, the results obtained in this study suggest activation of Akt leading to raised levels of p-mTORC1 and p-GSK3β, and supressed PTEN levels.The up-regulation of the pathway could have subsequently led to raised nerve regeneration.

CONCLUSION
The results obtained in this study reveal the effectiveness of myricetin in enhancing functional recovery and nerve regeneration following peripheral nerve injury.Myricetin activates the BDNF and PI3K/AKT/mTORC1/GSK3β signalling pathways which are crucial in nerve cell growth.
The findings reveal the effectiveness of myricetin in enhancing functional recovery and nerve regeneration following peripheral nerve injury via activation of the BDNF and PI3K/AKT/mTORC1/GSK3β signalling pathways involved in nerve cell growth.

Figure 4 :
Figure 4: Myricetin regulated the expressions of BDNF and TrkB.A: Representative RT-PCR Gel; B: Relative expression of proteins.Data are represented as mean ± SD (n = 6).*p < 0.05, compared with control; #p < 0.05, SNC compared with SNC + 100 mg myricetin (a, b, c & d represent mean values from different experimental groups that differ significantly) However, myricetin treatment significantly (p < 0.05) enhanced phosphorylated forms of Akt, mTORc1 and GSK-3β.Markedly reduced PTEN expressions were seen in myricetin treated rats (p < 0.05), indicating activation of the pathway.The levels of total Akt, mTORc1 and GSK-3β were not altered, although slightly raised expressions were noticed on myricetin treatment.These results suggest possible activation of the PI3K/Akt/mTORC1 signalling by myricetin.

Figure 5 :
Figure 5: Myricetin up-regulated Akt activation following SNC injury.A: Representative immunoblot; B: Relative expression of proteins following SNC injury.Data are presented as mean ± SD (n = 6).*p < 0.05, compared with control; #p < 0.05 (SNC compared with SNC + 100 mg myricetin; a-c represent mean values from different experimental groups that differ significantly)

Table 1 :
Sciatic functional index

Table 2 :
Toe spread index