Effect of lycopene on pain facilitation and the SIRT1/mTOR pathway in the dorsal horn of burn injury rats

Qin Yin a,b,1, Jin-Feng Wang c,1, Xiao-Hua Xu d, Hong Xie a,*

A B S T R A C T

To explore the effect of intrathecal injection of lycopene on pain facilitation, glial activation, and the SIRT1/ mTOR pathway in the dorsal horn of rats with burn injury pain (BIP).
Here we found that the mechanical pain threshold increased in the lycopene group compared with that of the control group, (P < 0.05). Compared with expression in the sham group, mTOR, pS6, p4EBP, GFAP, and Iba-1 decreased and SIRT1 increased in the lycopene group (P < 0.01). Glial activation in the spinal dorsal horn of BIP rats was alleviated by lycopene (P < 0.01). The SIRT1 and mTOR were mainly distributed in neurons in the spinal dorsal horn in the BIP model. Intrathecal injection of 3-MA (a mTOR agonist) or EX-527 (an inhibitor of Sirt1) partially antagonized lycopene-induced analgesia. Intrathecal injection of rapamycin (an mTOR inhibitor) or SRT1720 (an agonist of Sirt1) induced analgesia in BIP rats. 3-MA abrogated the SRT1720-induced analgesic effects.
The present data indicated that the SIRT1/mTOR pathway changed in the spinal dorsal horn of BIP rats; Lycopene alleviated the pain sensitization of BIP rats by regulating the SIRT1/mTOR pathway and glial activation in the spinal dorsal horn.

Keywords:
Burn injury pain
Lycopene SIRT1 mTOR
Glial activation

1. Introduction

More than 11 million patients suffer from burns of different degrees every year in the world, and most of them believe that the pain seriously affects daily life (Kim et al., 2019; Morgan et al., 2018).
Although burn injury pain (BIP) is very common, the choice of clinical treatment is still limited and ineffective. As the primary analgesics, opioids may produce serious adverse reactions and drug dependence for BIP (Kim et al., 2019). Therefore, it is urgent to study the mechanism of burn pain and find more effective treatment methods (Morgan et al., 2018).
Because of its high antioxidant activity, lycopene is of great value in the prevention and treatment of aging, chronic central nervous system diseases, chronic pain, tumor proliferation, and cardiovascular diseases related to oxidative damage (Caris-Veyrat et al., 2016; Chen et al., 2019; Hedayati et al., 2019; Senkus et al., 2019).
Lycopene increases the expression of SIRT1, FoxO1, and FAT/CD36 and decreases PPARγ in the adipose tissue of obese rats (Wang et al., 2015). ALA, a lycopene metabolite, prevents steatosis in ob/ob mice by upregulating the expression and activity of the SIRT1 gene (Chung et al., 2012). Furthermore, SIRT1 activation can deacetylate FOXO3a and increase the activity of superoxide dismutase and catalase in cells (Sun et al., 2018a,b; Wang et al., 2017). These enzymes can effectively reduce the accumulation of ROS (Sang et al., 2016; Wu et al., 2019; Yanagisawa et al., 2018) and inhibit the PI3K/Akt signaling pathway, thereby inhibiting activation of the mTOR pathway (H. Li et al., 2019; Sun et al., 2018a,b). The above studies suggest that lycopene may exert various pharmacological effects by mediating the SIRT1/mTOR pathway.
As an important signal transduction pathway in cells, the SIRT1/ mTOR pathway plays key roles in neuropathic pain and neurodegenerative disorders (Cetrullo et al., 2015; Maiese, 2018; Pani, 2015; Zhao et al., 2017). Previous findings suggest that lycopene induced the analgesic effect through the restoration of normal spinal Cx43 expression in partial sciatic nerve ligation (PSNL) (Zhang et al., 2016). In diabetic neuropathy and PSNL models, lycopene reduced the release of TNF-ɑ and NO, oxidative stress, reactive oxygen species and thermal hyperalgesia, suggesting that lycopene alleviates neuropathic pain through the anti-inflammatory mechanism (Goel and Tyagi, 2016; Kuhad and Chopra, 2008; Zhang et al., 2016).
However, there is no report that lycopene regulates the spinal SIRT1/ mTOR pathway and its downstream factors, thus participating in the development of chronic pain. Therefore, we hypothesize that lycopene mediates the SIRT1/mTOR pathway, thereby regulating pain sensitization in a BIP model. In the present study, we aimed to solve the following problems: (1) whether the SIRT1/mTOR pathway changes in the dorsal horn of the BIP model and (2) whether intrathecal injection of lycopene has analgesic effects on BIP by mediating the SIRT1/mTOR pathway.

2. Materials and methods

2.1. Animals

Adult male SD rats (200–250g) were tested in a standard clean animal laboratory one week after adaptation. The animals were tested in a box (at 23 ± 1 ◦C, 50 ± 10% humidity, and a 12 h light/dark cycle with free access to food and drinking water). The Animal Ethics Committee of Xuzhou Medical University approved all procedures (SCXK (Jiangsu) 2015–0009).

2.2. Drugs and instruments

The PE-10 catheters were purchased from American Health Medical Instruments International Company and the von Frey filaments were obtained from the Institute of Bioengineering of the Chinese Academy of Medical Sciences. The anti-SIRT1, anti-mTOR and anti-GAPDH antibodies were purchased from Abcam. Lycopene, 3-MA (a mTOR agonist), EX-527 an (inhibitor of Sirt1), rapamycin (a mTOR inhibitor), and SRT1720 (an agonist of Sirt1) were prepared before behavior tests. According to the molecular weight of lycopene (536.87KD), the grams of each animal are approximately 20 nmol (10.8 μg), 40 nmol (21.6 μg) and 60 nmol (32.4 μg).

2.3. Intrathecal catheterization

The method of intrathecal catheterization has been previously described (Milligan et al., 1999). Briefly, the subarachnoid space was punctured with a 25 G needle at the L3-4 intervals in anesthetized rats, and then the PE-10 catheter was inserted approximately 2 cm. The other end of the catheter was transported subcutaneously to the back of the neck. The hind feet were paralyzed after injection of 2% lidocaine hydrochloride and recovered in approximately 30 min, which meant successful catheterization. Rats with spinal cord injury were excluded from the experiment.

2.4. BIP model

The BIP model method has been previously described (Chang et al., 2010). Briefly, after the animal was anesthetized, the right hind foot of the rat was fixed on a thermostatic metal plate (85.0 ± 0.5 ◦C, at 10g of pressure for 15.0 s). The area of thermal injury reached 1% of the total body area. Rats with motor impairment and no response to mechanical stimulation were excluded.

2.5. Behavioral tests

The behavioral tests have been previously described (Milligan et al., 1999). Briefly, after adapting for at least 30 min, von-Frey filaments (scales were 0.6, 1, 2, 4, 6, 8, 10 and 15g) were used to test the pain threshold at 30 s intervals. The PWTs, as the threshold, were calculated by the up-down method.

2.6. Immunoblotting

The L4-6 segments of the burn side were resected quickly and stored at − 70 ◦C immediately. After measuring and balancing the protein concentrations, 50 μg of each sample was separated by SDS-PAGE, and the proteins were transferred to a PVDF membrane. After incubation at 4 ◦C overnight with the primary antibodies (anti-SIRT1, 1:500 and anti- mTOR, 1:1000), anti-sheep HRP-IgG (1:2500, Sigma) was added after washing. After adding chemiluminescence reagent, X-ray exposure was performed. The relative protein expression level is expressed as the ratio of the integrated optical density (IOD) of each band to the corresponding IOD value of GAPDH.

2.7. Immunofluorescence detection

After deep anesthesia and perfusion, the L4-5 segments were removed and fixed in 4% paraformaldehyde solution at 4 ◦C for 6 h, transferred to a 30% sucrose solution (0.1 mol/LPB, pH 7.4), incubated for 8 h at 4 ◦C and dehydrated completely. After frozen sections (25 μm) were washed and blocked, they were incubated for 48 h with primary antibody (Sirt1, 1:400; mTOR 1:1000) at 4 ◦C. After three rinses with PBS, FITC-labeled antibody (goat anti-rabbit IgG, 1:200) was added. After incubation at 37 ◦C for 2 h, the sections were rinsed with PBS three times. The coverslips were added in the dark and dried at 37 ◦C, and glycerin (containing anti-quenching agent) was used to seal the slides. Confocal microscopy photographs were then taken.

2.8. Statistical analysis

Data are expressed as the mean ± S.E.M. SPSS 22.0 statistical software was used to test the differences between groups. ANOVA, two-way ANOVA and repeated measures ANOVA were employed to test the data, and P < 0.05 indicates a statistical difference.

3. Results

3.1. The establishment of the BIP model

Changes in the mechanical pain threshold of the rats in each group are shown in Fig. 1. The ipsilateral PWTs of BIP rats decreased immediately and lasted until 21 days after thermal injury compared with those in the sham group (P < 0.01).

3.2. Intrathecal injection of lycopene affects the PWTs of BIP rats

The injection of the vehicle had no effect on the PWTs of BIP rats (P > 0.05) in Fig. 2. Compared with the PWTs in the BIP + vehicle group, the PWTs of burned limbs were significantly increased in the lycopene groups (40 and 60 nmol) (P < 0.01).

3.3. The effect of intrathecal injection of lycopene on SIRT1/mTOR pathway expression in the spinal dorsal horn of BIP rats (Fig. 3)

The expression of the SIRT1/mTOR pathway in the spinal dorsal horn of rats in the sham group was low. The SIRT1/mTOR pathway in the BIP group increased significantly after burn injury (P < 0.01). SIRT1 increased and the mTOR pathway decreased significantly in the BIP + lycopene group compared with those of the BIP + vehicle group (P < 0.01).

3.4. The distribution of SIRT1/mTOR in the spinal dorsal horn of BIP rats

Immunofluorescence results proved that SIRT1-and mTOR-positive cells were labeled with green fluorescence, while neurons, microglia and astrocyte markers were labeled with red fluorescence. SIRT1 and mTOR were not detected in almost all GFAP and Iba-1 IR cells. These results demonstrated that Sirt1 and mTOR were predominantly distributed in neurons in BIP rats (Fig. 4 A and 4 B). Fig. 4 C shows SIRT1 and mTOR co-expression in the spinal dorsal horn of BIP rats.

3.5. Lycopene reduced glial activation in the spines of BIP rats

Immunofluorescence results showed that the activation of glial cell markers (GFAP and Iba-1) was reduced in sham rats but increased in scalded pain rats; the activation of GFAP and Iba-1 decreased in the lycopene group compared with that of the vehicle group (Fig. 5).

3.6. Changes in lycopene-induced analgesia in BIP rats after regulation of SIRT1/mTOR

There was no significant difference in hindlimb PWTs between the groups prior to drug administration (P > 0.05). Compared with the pre- drug value, the PWTs in the BIP + lycopene 60 nmol group increased robustly (P < 0.05). The PWTs in the EX-527 or 3-MA group decreased significantly at each time point compared with those of the BIP + lycopene 60 nmol group (P < 0.05). These data indicated that the inhibition of Sirt1 or the activation of mTOR partially reduced lycopene- induced analgesia (Fig. 6).

3.7. Regulation of SIRT1/mTOR affected the PWTs of BIP rats (Fig. 7)

There was no significant difference in basal hindlimb PWTs among the groups (P > 0.05). Compared with the basal PWTs, the PWTs of BIP rats decreased significantly at each time point (P < 0.01). PWTs in the SRT1720 or rapamycin group increased after injection compared with those of the BIP + vehicle group (P < 0.05), suggesting that activating Sirt1 or inhibiting mTOR decreases hyperalgesia in BIP rats. PWTs decreased in the BIP + SRT1720+3-MA group compared with those of the BIP + SRT1720 group (P < 0.05), suggesting that mTOR activation partially antagonizes SRT1720-induced analgesia.

4. Discussion

In this study, the BIP animal model was established by using the temperature of 85.0 ± 0.5 ◦C and maintaining the pressure at 10g for 15 s. In the pre-experiment, the skin of the injured foot of the rat appeared red and swollen, with local blister formation 3–5 days after the heat injury. The skin on the surface of the toe healed and formed scars approximately 7 days after the heat injury. The entire healing period lasted approximately 4 weeks after the heat injury, and during the healing period, rats showed mechanical pain facilitation on the affected side. There was no significant change in the threshold of thermal pain, which is similar to that of patients with chronic pain after heat injury in clinical practice. Previous studies also confirmed that chronic pain after a burn is mainly affected by tactile pain stimuli. The temperature pain threshold was less affected (Malenfant et al., 1998; Summer et al., 2008). Therefore, in our study, we only measured the change in the mechanical pain threshold in rats after burn injury. This method caused deep second degree burns on the affected side of the rats, which accounted for approximately 1% of the total skin area. On the first day after the burn, mechanical pain appeared and lasted for 21 days.
Previous studies have shown that resveratrol and other antioxidant drugs have analgesic effects in a certain concentration range, and the analgesic effect is dose-dependent; when the drug concentration is too high, there are some adverse reactions, such as in vitro experiments producing cytotoxic effects and in vivo experiments showing a stress state (Gupta et al., 2004; Tillu et al., 2012; Tsai et al., 2012).
In the preliminary test, the low dose of lycopene (20 nmol) did not show analgesic effects in BIP rats, while other dosage (lycopene 40 nmol and 60 nmol) can obviously alleviate the pain sensitization in BIP rats. In our experiment, rats in the groups did not show stress states, and so it is safe to administer lycopene (60 nmol) intrathecally. In western blotting and morphological experiments, we selected a high dose of lycopene (60 nmol) to detect changes in the SIRT1/mTOR signaling pathway in the spinal cord.
It has been proved that lycopene can produce obvious analgesic effects through spinal Cx43 expression and the anti-inflammatory mechanism. (Goel and Tyagi, 2016; Kuhad and Chopra, 2008; Zhang et al., 2016). In this paper, we firstly report that the analgesic effects of lycopene are also achieved by activating SIRT1. Another interesting finding of our research is that lycopene not only regulates SIRT1 expression, but also induces mTOR level in the spinal dorsal horn.
The studies have also shown that the upregulation of SIRT1 may protect tissues from noxious stimulation (Bazzo et al., 2013; Sorkin et al., 2009). Activation of SIRT1 can improve insulin sensitivity, inhibit tumor growth, inhibit inflammation, improve neuropathic pain, and prevent neurodegenerative diseases (Baur and Sinclair, 2006; Gui et al., 2018; Richard et al., 2011). Bai et al. found that SIRT1 inhibits apoptosis and proinflammatory factors, avoiding pathological damage in burn tissues and helping tissue repair (Bai et al., 2015). As a response to scald stress, the SIRT1/mTOR pathway was changed in the spinal cords of BIP rats, corresponding to an increase in hyperalgesia after burn injury in our experiment. After intrathecal administration of lycopene, SIRT1 and the pain threshold increased and the mTOR pathway decreased in the spinal cords of BIP rats. The increase in the pain threshold in the BIP + lycopene group may be due to changes in the SIRT1/mTOR pathway in the spinal cord.
Previous studies have proved that lycopene can upregulate SIRT1 mRNA levels in adipose tissue of HD-fed rats (Luvizotto et al., 2015). Moreover, the lycopene metabolite apo-100-lycopene acid can induce SIRT1 expression in the liver of ob/ob mice. SIRT1 participates in a variety of biological processes (Alcain and Villalba, 2009; Chung et al., 2012). We are not sure that the analgesia of intrathecal injection of lycopene is by lycopene itself or by its possible metabolites. The specific mechanism needs further study.
Furthermore, we investigated the effects of the SIRT1 inhibitor or mTOR agonist on lycopene-induced analgesia. Referring to the preliminary data, we chose the concentrations of EX527 (8 μg)and 3-MA (10 μg) to investigate the involvement of SIRT1/mTOR in lycopene- induced analgesia. The dosages of EX527 or 3-MA used did not produce pain sensitization. However, they alleviated the analgesic effect of lycopene in BIP rats. Moreover, it was found that EX527 or 3-MA could not completely antagonize the analgesic effects of lycopene. It was further confirmed that lycopene partially participated in the analgesia of BIP rats through the SIRT1/mTOR pathway.
Intrathecal injection of SRT1720 (a SIRT1 agonist) can also produce analgesic effects (M.Y. Li et al., 2019; Yang et al., 2019; Zhang et al., 2019; Zhou et al., 2017). In our study, we further studied the relationship between the analgesic effect of SIRT1 agonist and mTOR. As shown in the results, intrathecal injection of SRT1720 or rapamycin increased the pain threshold in BIP rats. Compared with PWTs in the BIP + SRT1720 group, PWTs decreased at each time point in the BIP + SRT1720+3-MA group, suggesting that intrathecal injection of the mTOR agonist partially antagonized the analgesic effect of SRT1720, which indicated that the analgesic effects of activating Sirt1 are partially mediated by mTOR.
As mentioned in the introduction, lycopene can upregulate Sirt1 and inhibit the mTOR signaling pathway in different cells (Chung et al., 2012; Sun et al., 2018a,b; Wang et al., 2015). Previous studies have found that Sirt1 may regulate lipid metabolism and cell proliferation through the mTOR signaling pathway (Cetrullo et al., 2015; Maiese, 2018; Pani, 2015). (Erdogan et al., 2017; Park et al., 2016; Shiratsuki et al., 2016; Suzuki and Bartlett, 2014). In the studies of different pain models, the spinal mTOR signaling pathway (mTOR and its downstream effectors) is crucial to the plasticity of spinal cord neurons and the high sensitivity of pain behaviors (Fang et al., 2019; Guo et al., 2017; Xie et al., 2019) (Liu et al., 2019; Shih et al., 2012).
The immunofluorescence results demonstrated that the expression of SIRT1/mTOR was not detected in GFAP or Iba-1 IR cells. However, the positive expression of SIRT1/mTOR was detected in NeuN IR cells, and both proteins colocalize in the posterior horn of poliomyelitis. Our finding is the first to demonstrate that Sirt1 and mTOR are dominantly co-distributed in spinal neurons, which indicates that the regulation of neuronal function by Sirt1/mTOR may be involved in the development of BIP. Several studies have confirmed that the downstream molecules pS6 and p4EBP of SIRT1/mTOR were also found in activated glial cells (Eady et al., 2012; Hayashi et al., 2015; Liu et al., 2014; Xu et al., 2011). Combined with the results of this experiment, we hypothesize that SIRT1/mTOR signals could regulate the downstream pS6 and p4EBP pathways and further activate glial cell-mediated pain sensitization of BIP.
In conclusion, the upregulation of SIRT1/mTOR and glial activation in the spines of rats may mediate pain sensitization of the BIP model. Lycopene can alleviate pain sensitization in scalded rats by mediating the SIRT1/mTOR pathway and glial activation.

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