Skip to main content
NCBI home page
Springer logoLink to Springer
. 2025 Jan 8;41(1):24. doi: 10.1007/s10565-024-09970-6

A novel SIRT1 activator attenuates neuropathic pain by inhibiting spinal neuronal activation via the SIRT1-mGluR1/5 pathway

Xiaobao Ding 1,#, Guizhi Wang 1,#, Yuwen Lin 1,#, Wenli Hu 1, Chen Chen 2, Jian Gao 3,✉, Yuqing Wu 2,✉, Chenghua Zhou 1,✉
PMCID: PMC11711878  PMID: 39779529

Abstract

Neuropathic pain is a type of pain caused by an injury or disease of the somatosensory nervous system. Currently, there is still absence of effective therapeutic drugs for neuropathic pain, so developing new therapeutic drugs is urgently needed. In the present study, we observed the effect of Comp 6d, a novel silent information regulator 1 (SIRT1) activator synthesized in our laboratory, on neuropathic pain and investigated the mechanisms involved. We found that both intrathecal and intraperitoneal injections of Comp 6d effectively alleviated neuropathic pain induced by chronic constriction injury (CCI) or spared nerve injury (SNI). However, the effect of Comp 6d on neuropathic pain was abolished in SIRT1 knockout mice. These results demonstrated that Comp 6d alleviated neuropathic pain by specifically activating SIRT1 in the spinal cord. Moreover, long-term intraperitoneal injection of Comp 6d had no significant side effects on heart, liver and kidney in SNI mice. Further study showed that the improvement of neuropathic pain by Comp 6d was mediated by the downregulation of mGluR1/5 levels and the subsequent inhibition of spinal neuronal activation. Taken together, the present findings suggest that the novel SIRT1 activator Comp 6d inhibits the activation of spinal cord neurons via the SIRT1-mGluR1/5 pathway, thereby attenuating neuropathic pain. Comp 6d is expected to be an effective therapeutic agent for neuropathic pain.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10565-024-09970-6.

Keyword: SIRT1 activator, Neuropathic pain, MGluR1/5, Spinal cord

Introduction

Neuropathic pain is caused by primary lesions or dysfunctions of the nervous system and has a variety of causative factors, such as trauma, nerve injury or inflammation (Finnerup et al. 2021; Reichling and Levine 2009). It also has a long duration, lasting for months, years or even after the nerve injury has healed. Studies have shown that neuropathic pain affects approximately 8% of the world's population and is increasing every year, placing a heavy burden on society. However, the first-line analgesics commonly used in clinical practice are mainly tricyclic antidepressants and antiepileptics, such as amitriptyline, gabapentin or pregabalin, but the clinical efficacy of these drugs, either alone or in combination, has not been recognized (Onakpoya et al. 2019; Szok et al. 2019; Alcántara Montero et al. 2019; Curry et al. 2018). Therefore, the development of new drugs for neuropathic pain prevention and treatment is imminent.

Silent information regulator 1 (SIRT1), one of the most studied members of class III histone deacetylases (HDACs), plays an important role in the regulation of gene expression (Michán et al. 2010; Shao et al. 2014). Recently, SIRT1 has been demonstrated to play a significant part in the development and progression of neuropathic pain. For example, SIRT1 expression in the spinal cord is significantly reduced in neuropathic pain mice, while the SIRT1 agonist resveratrol can attenuate thermal nociceptive hyperalgesia and mechanical nociceptive hypersensitivity in CCI mice (Shao et al. 2014; Liu et al. 2018). In contrast, EX527, a potent inhibitor of SIRT1, blocked the analgesic effect of rutin extract in CCI model mice (Yin et al. 2013; Yang et al. 2019; Bennett and Xie 1988; Napper et al. 2005). Besides, the reduction of SIRT1 promotes SNI-induced neuropathic pain in rats (Zeng et al. 2020). In addition, our previous research shows that the level of SIRT1 is significantly downregulated in the spinal dorsal horn of diabetic neuropathic pain (DNP) rats and db/db mice, while intrathecal injection of the SRT1720 could obviously alleviate nociceptive hypersensitivity in DNP rats. However, knockdown of spinal SIRT1 could induce neuropathic pain (Zhang et al. 2019). Consequently, SIRT1 is an important regulatory factor for neuropathic pain, and the development of new drugs targeting SIRT1 will have important application prospects.

In our previous study, using a structure-based drug design strategy, we have successfully identified several new SIRT1 activators, among which the activity of Comp 6d (Fig. 1B) is comparable to SRT2104, a known SIRT1 activator (Gao et al. 2021). However, the effect of Comp 6d on neuropathic pain is unknown. In addition, our previous study indicates that the expressions of mGluR1 and mGluR5, which belong to type I mGluRs, can be regulated by SIRT1 (Zhou et al. 2017). Moreover, mGluR1 and mGluR5 have been shown to be involved in the development of neuropathic pain (Chiechio and Nicoletti 2012). Therefore, in the present study, we first observed the ameliorative effect of the new SIRT1 activator Comp 6d on neuropathic pain, and then further investigated whether Comp 6d relieves neuropathic pain through the SIRT1-mGluR1/5 pathway.

Fig. 1.

Fig. 1

Open in a new tab

Intrathecal injection of Comp 6d reversed the decrease of SIRT1 level and activity and alleviated neuropathic pain in CCI rats. A Experimental timeline. B Chemical structure of Comp 6d. C, D Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of CCI rats after surgery. E, F Effects of intrathecal injection of Comp 6d on pain-like behaviors in CCI rats. G, H Effect of intrathecal injection of Comp 6d on pain-like behaviors in Sham rats. I Effect of intrathecal injection of Comp 6d on motor function in CCI rats. J-L Effect of intrathecal injection of Comp 6d on the protein (J), mRNA (K) and activity (L) of SIRT1 in the spinal cords of CCI rats. All data are expressed as mean ± SEM. n = 6–9, **P < 0.01 versus Sham; ##P < 0.01 versus CCI + DMSO

Materials and methods

Animals

The Animal Care and Use Committee of Xuzhou Medical University granted approval for all animal care practices and experimental procedures, and all surgical interventions were carried out following pertinent guidelines and standardized operating procedures. Adult male Sprague–Dawley rats and Kunming mice were purchased from the Experimental Animal Center of Xuzhou Medical University. B6;129-Sirt1 tm1Ygu/J (SIRT1flox/wt) mice and Wnt1-Cre (Tg(Wnt1-Cre)11Rth) mice were obtained from The Jackson Laboratory. SIRT1flox/flox mice were generated by intercrossing SIRT1flox/wt mice, and then SIRT1flox/flox, Cre mice were obtained by crossing SIRT1flox/flox mice with Wnt1-Cre mice. Cre-negative littermates were used as control.

Neuropathic pain models

The CCI-induced neuropathic pain model in rats and SNI-induced neuropathic pain model in mice were established according to the method we reported previously (Sun et al. 2021; Ding et al. 2023). Detailed method was shown in Supplemental Materials and Methods.

Behavioral tests

All behavioral tests were conducted by persons who were unaware of the experiment.

Von frey test

For the mechanical pain test, after acclimatization for 30 min, the hind paws of animals were then stimulated vertically for 5 s using von Frey fiber filaments (2.0–15.0 g for rats, 0.008–6 g for mice). The 50% mechanical withdrawal threshold (MWT) was calculated using the up-and-down method (Chaplan et al. 1994).

Hargreaves test

For the Hargreaves test, rats were irradiated with a thermal pain stimulator for 30 min after acclimation to the environment, and the period from the earliest irradiation until the rat lifted or licked its foot was the thermal withdrawal latency (TWL). The automatic cut-off time of the apparatus was set to 25 s to prevent tissue damage to the rat's foot.

Acetone test

To assess the cold pain threshold of SNI mice, the mice were placed in a quiet environment for 30 min. A small amount of pre-cooled acetone was sprayed onto the posterior part of the plantar foot with a syringe. The measurements were conducted three times at an interval of 3–5 min, and rated according to a 4-point scale (Flatters and Bennett 2004): 0, no response in the hind paw; 1, rapid foot retraction; 2, repeated foot retraction; 3, repeated foot retraction and licking of the plantar foot. The sum of the scores of the three measurements was calculated.

Rotarod test

Rats were placed on a rotating drum at an initial speed of 5 rpm and then gradually accelerated to a maximum speed of 20 rpm over a period of 5 min. The rats were tested for three trials separated by 15-min intervals. The time each rat remained on the rotarod (falling latency) was recorded for each trial, and the average falling latency was calculated.

Intrathecal injection

After anesthesia, the skin of the L5 to L6 segment of the rat's back was incised longitudinally and the attached muscles were bluntly separated with scissors to expose the vertebral plates. A PE-10 catheter was placed between the L5 and L6 vertebrae for drug delivery. Intubated rats were housed in solitary confinement. For intrathecal injection into mice, the Hamilton syringe was inserted into the L4 or L5 spinous process gap, and the sudden tail flap of the mouse indicated that the needle was in the correct position, then 5 μl of drug was injected into the subarachnoid space and maintained for 15 s to avoid drug runoff.

Drugs and chemicals

The drugs used in this study are as follows: Dimethyl sulfoxide (DMSO, Sigma-Aldrich, St Louis, USA); Resveratrol (Res, Meilun Biotechnology, Dalian, CN); Pregabalin (Meilun Biotechnology, Dalian, CN); SRT1720 (Selleck Chemicals, Houston, USA). To observe the effect of Comp 6d on neuropathic pain, CCI models of rats were established, the drugs were administered by intrathecal injection for 5 days, starting from 3 to 4 days after intrathecal cannulation, which was day 9 after CCI surgery. The final concentration of solvent DMSO was 1%, and the concentrations of SRT1720 and Comp 6d were 2 µg /µl, and the volume of administration was 10 µl. To evaluate the long-term effect and toxicity of Comp 6d, a longer duration SNI mouse model of neuropathic pain was established. Mice in SNI + Comp 6d (L), SNI + Comp 6d (M), and SNI + Comp 6d (H) groups were administered intraperitoneally 2.5 mg/kg, 5 mg/kg, and 10 mg/kg of Comp 6d, respectively, SNI + Res group was given 10 mg/kg of resveratrol (Res), and SNI + Pregabalin was given 15 mg/kg of pregabalin. The above drugs were injected on day 1 after SNI surgery and administered continuously for 28 days. All control groups were given equal amounts of lysate. In addition, SIRT1flox/flox, Cre mice were used to confirm the specific effect of Comp 6d on SIRT1 by intrathecal injection of Comp 6d 5 µl/d for 5 consecutive days.

Western blotting analysis, real-time quantitative PCR (RT-qPCR), SIRT1 activity assay and double immunofluorescent staining

Western blotting analysis, RT-qPCR, SIRT1 activity assay and double immunofluorescent staining were performed as we reported previously (Zhou et al. 2017). Detailed methods were shown in Supplemental Materials and Methods. Information for the primers used was included in the Supplemental Table 1 and Table 2.

Hematoxylin–eosin (HE) staining

The heart, liver and kidney tissues were embedded in paraffin, and sliced into sections of 4 µm thickness. Paraffin sections were regularly dewaxed and rehydrated, stained with hematoxylin solution. After differentiation for 30 s, the sections were soaked, then restained with eosin solution. The sections were observed under a light microscope.

Biochemical analysis

The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), creatinine (CRE), and urea nitrogen (BUN) in serum were detected according to the corresponding detection kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

Statistical analysis

Data are expressed as mean ± SEM and analyzed with GraphPad Prism v8.0. Two-way (ANOVA) was applied for pain behavioral analysis, and one-way ANOVA with LSD or Dunnett's T3 post-hoc tests was used for multiple comparisons. P < 0.05 was considered to be statistically significant.

Results

Intrathecal injection of Comp 6d reversed the decrease of SIRT1 level and activity and alleviated neuropathic pain in CCI rats

A rat CCI model was established, and the effect of intrathecal injection of Comp 6d on neuropathic pain was observed (Fig. 1A). The mechanical and thermal pain tests were applied to assess the pain hypersensitivity in rats. As shown in Fig. 1C and D, compared with Sham rats, there was no significant difference in pain thresholds in CCI rats at baseline, but significant mechanical hyperalgesia and thermal hyperalgesia were shown from postoperative day 3 to day 14. After intrathecal injection of the SIRT1 activator Comp 6d for 5 consecutive days, the MWT and TWL in the CCI + Comp 6d rats were significantly increased compared with the CCI + DMSO group, and the effect of Comp 6d was similar to the well-known SIRT1 activator SRT1720 (Fig. 1E, F). However, Comp 6d did not influence the pain behavior in the Sham rats (Fig. 1G, H). In addition, we found that compared with the CCI + DMSO group, intrathecal injection of Comp 6d or SRT1720 had no influence on the motor function in CCI rats (Fig. 1I).

To assess the influence of the novel SIRT1 activator Comp 6d on spinal SIRT1 levels in CCI rats, we determined its effect on SIRT1 expression and activity. We found that the spinal SIRT1 level and activity were reduced in CCI rats compared with the Sham group. Intrathecal injection of Comp 6d could reverse the decrease of SIRT1 expression and activity in CCI rats, and the effect of Comp 6d was comparable to SRT1720 (Fig. 1J-L). These results suggest that intrathecal injection of Comp 6d could alleviate CCI-induced neuropathic pain by upregulating SIRT1 expression and activity.

Intraperitoneal injection of Comp 6d reversed the decrease of SIRT1 level and activity and alleviated neuropathic pain in SNI mice

To further confirm the effect of Comp 6d on neuropathic pain, we established a longer duration SNI mouse model and administered Comp 6d via intraperitoneal injection for 28 days, while resveratrol and pregabalin were used as positive control drugs (Fig. 2A). The results showed that during the 28 days of administration, low, medium and high doses of Comp 6d improved neuropathic pain at different degrees in SNI model mice. On day 14, the low dose of Comp 6d did not improve the cold allodynia in SNI model mice and the different doses of Comp 6d did not obviously change the mechanical withdrawal threshold. On day 21, low, medium and high doses of Comp 6d reduced cold pain scores in SNI model mice, and high doses of Comp 6d attenuated mechanical hypersensitivity. On day 28, Comp 6d significantly improved cold hypersensitivity in SNI model mice in all dose groups, and high doses of Comp 6d also reduced mechanical pain (Fig. 2B, C). Moreover, after 28 days of administration, the SIRT1 protein expression in the spinal cord of mice in the Comp 6d low-dose and mid-dose groups were not significantly altered compared with the SNI + DMSO group, whereas the mRNA levels in the spinal cord of mice in the mid- and high-dose groups were upregulated obviously (Fig. 2D, E). In addition, the Comp 6d high-dose group also caused a notable increase in SIRT1 activity in the spinal cords of SNI mice (Fig. 2F).

Fig. 2.

Fig. 2

Open in a new tab

Intraperitoneal injection of Comp 6d reversed the decrease of SIRT1 level and activity and alleviated neuropathic pain in SNI mice. A Experimental timeline. B, C Mechanical allodynia and cold hyperalgesia were determined in mice before SNI surgery and on days 1, 7, 14, 21 and 28 after surgery (n = 7–8). D-F Effect of intraperitoneal injection of Comp 6d for 28 days on the protein (D), mRNA (E) and activity (F) of SIRT1 in the spinal cords of SNI mice (n = 6). All data are expressed as mean ± SEM. **P < 0.01 versus Sham + DMSO;#P < 0.05, ##P < 0.01 versus SNI + DMSO

Intraperitoneal injection of Comp 6d had no significant side effects on heart, liver and kidney in SNI mice

In addition, we also observed the effect of Comp 6d intraperitoneally on cardiac, hepatic and renal pathomorphology and function. The results of serum biochemical indexes showed that Comp 6d had no effect on the liver and kidney functions of mice (Fig. 3B-F), and there was no significant difference in the body weight of mice in each group before and after administration of Comp 6d (Fig. 3A). HE staining showed that the heart structure of mice in each treatment group was intact, the myocardial fibers were arranged neatly, the striated muscles were clear, and the stroma was normal; the hepatocyte cords of the liver were neatly arranged with the central vein as the center, with clear cellular morphology and no abnormal interstitial changes; the structure of the kidney cortex and medulla remained intact, and the morphology of the glomerulus and tubules was clear and normal, with no obvious interstitial changes (Fig. 3G). These results suggest that intraperitoneal injection of Comp 6d has no significant side effects on heart, liver and kidney.

Fig. 3.

Fig. 3

Open in a new tab

Intraperitoneal injection of Comp 6d had no significant side effects on heart, liver and kidney in SNI mice. A Changes in body weight of mice during drug administration (n = 6–8). B-F Effect of intraperitoneal injection of Comp 6d for 28 days on liver and kidney function indexes in SNI mice (n = 5–7). G Effect of Comp 6d on heart, liver and kidney morphology was observed using HE staining. All data are expressed as mean ± SEM

Intrathecal injection of Comp 6d did not reduce pain hypersensitivity in SIRT1flox/flox, Cre mice

To further confirm whether Comp 6d relieves neuropathic pain by specifically activating spinal SIRT1, we observed the effect of Comp 6d administered intrathecally for 5 consecutive days on pain behaviors in SIRT1flox/flox, Cre mice (Fig. 4A). The results showed that compared with SIRT1flox/flox mice, the MWT and TWL of SIRT1flox/flox, Cre mice were obviously reduced (Fig. 4B, C), suggesting that knockdown of spinal SIRT1 induces pain-like behavior. Meanwhile, SIRT1 protein and mRNA levels and SIRT1 activity were significantly reduced in mice after knockdown of spinal SIRT1 (Fig. 4D-F). However, intrathecal injection of Comp 6d or SRT1720 did not reverse the pain hypersensitivity and the decrease of SIRT1 in SIRT1flox/flox, Cre mice. These results indicate that Comp 6d does improve pain by specifically activating SIRT1.

Fig. 4.

Fig. 4

Open in a new tab

Intrathecal injection of Comp 6d did not improve pain hypersensitivity in SIRT1flox/flox, Cre mice. A Experimental timeline. B, C Effect of intrathecal injection of Comp 6d on mechanical hypersensitivity and thermal allodynia in SIRT1flox/flox, Cre mice. D-F Effect of intrathecal injection of Comp 6d on the protein (D), mRNA (E) and activity (F) of SIRT1 in the spinal cords of SIRT1flox/flox, Cre mice. All data are expressed as mean ± SEM. n = 6–8, **P < 0.01 versus SIRT1flox/flox + DMSO

Comp 6d relieved neuropathic pain by decreasing mGluR1/5 expression and inhibiting spinal neuronal activation

Our previous study has demonstrated that SIRT1 alleviates neuropathic pain in type 2 diabetic rats by epigenetically regulating the expression of mGluR1/5 (Zhou et al. 2017). In order to further investigate the potential mechanism of the new SIRT1 activator Comp 6d in relieving neuropathic pain, we determined the effect of Comp 6d on mGluR1/5 expressions. As shown in Fig. 5A-D, the levels of mGluR1/5 protein and mRNA were upregulated markedly in the spinal cords of CCI rats, which were reversed after intrathecal injection of Comp 6d. Similarly, in SNI mice, the levels of mGluR1/5 in the spinal cords were also elevated compared with the Sham + DMSO group. After 28 days of continuous intraperitoneal injection of Comp 6d, the levels of mGluR1/5 protein and mRNA in the spinal cords of SNI mice at the high dose group were significantly reduced (Fig. 5E-H). Moreover, compared with SIRT1flox/flox mice, in the spinal cords of SIRT1flox/flox, Cre mice, the levels of mGluR1/5 were markedly increased. However, intrathecal injection of Comp 6d had no significant influence on the levels of mGluR1/5 after knocking down spinal SIRT1 (Fig. 5I-L).

Fig. 5.

Fig. 5

Open in a new tab

Comp 6d downregulated mGluR1/5 expression through specific activation of SIRT1. A-D Effect of intrathecal injection of Comp 6d on the expression of mGluR1/5 in the spinal cords of CCI rats (n = 6–7, *P < 0.05, **P < 0.01 versus Sham; #P < 0.05, ##P < 0.01 versus CCI + DMSO). E–H  Effect of intraperitoneal injection of Comp 6d on the expression of mGluR1/5 in the spinal cords of SNI mice (n = 5–7, *P < 0.05, **P < 0.01 versus Sham + DMSO;#P < 0.05, ##P < 0.01 versus SNI + DMSO). I-L Effect of intrathecal injection of Comp 6d on the expression of mGluR1/5 in the spinal cords of SIRT1flox/flox, Cre mice (n = 6, *P < 0.05, **P < 0.01 versus SIRT1flox/flox + DMSO). All data are expressed as mean ± SEM

The activation of mGluR1/5 has been reported to increased neuronal excitability (Park et al. 2004; Li et al. 2011), therefore, we further observed the effect of Comp 6d on the expression of c-Fos protein, a well-known marker of neuronal activation (Zhou et al. 2017; Ren et al. 1997). We found that compared with the control animals, c-Fos expression upregulated significantly in the spinal cord of CCI rats and SNI mice, which was inhibited by administration of Comp 6d (Fig. 6A, B). However, the effect of Comp 6d on c-Fos protein was abolished after knocking down spinal SIRT1 (Fig. 6C). Moreover, the number of c-Fos and NeuN double positive neurons increased remarkably in the spinal dorsal horn of CCI rats, mainly in the laminae II and III, which was decreased by Comp 6d (Fig. 6D, E).

Fig. 6.

Fig. 6

Open in a new tab

Comp 6d inhibited spinal neuronal activation through specific activation of SIRT1. A  Effect of intrathecal injection of Comp 6d on the expression of c-Fos in the spinal cords of CCI rats (n = 6–7, **P < 0.01 versus Sham; #P < 0.05, ##P < 0.01 versus CCI + DMSO). B Effect of intraperitoneal injection of Comp 6d on the expression of c-Fos in the spinal cords of SNI mice (n = 5–7, **P < 0.01 versus Sham + DMSO;#P < 0.05 versus SNI + DMSO). C  Effect of intrathecal injection of Comp 6d on the expression of c-Fos in the spinal cords of SIRT1flox/flox, Cre mice (n = 6, **P < 0.01 versus SIRT1flox/flox + DMSO). D, E Effect of intrathecal injection of Comp 6d on the activation of spinal neurons in CCI rats. The c-Fos- and NeuN-positive cells present the activated neurons (n = 3, **P < 0.01 versus Sham; ##P < 0.01 versus CCI + DMSO). All data are expressed as mean ± SEM

Discussion

Neuropathic pain is a common clinical disease, however, there is still a lack of effective treatments. Therefore, developing new drugs for the prevention and treatment of neuropathic pain is crucial (Singh et al. 2017; Ferraro et al. 2016). Recently studies have indicated that changes in pain-related genes mediated by epigenetic modifications play key parts in the occurrence and development of neuropathic pain. The common mechanisms of epigenetic modifications mainly include DNA methylation, histone modifications, etc. (Ziemka-Nalecz et al. 2018). Multiple studies have demonstrated that histone acetylation modification participates in the regulation of pain-related factors (Denk et al. 2013; Uchida et al. 2010, 2013).

SIRT1, an NAD+-dependent histone deacetylase (Xu et al. 2016; Abe-Higuchi et al. 2016; Baur et al. 2012), has been shown in our previous studies to play a role in the development of DNP (Zhang et al. 2019). In neuropathic pain models induced by type 1 and type 2 diabetes, SIRT1 expression and activity are significantly decreased in spinal cords, and intrathecal injection of SRT1720 (a SIRT1 activator) significantly improves the pain-like behavior of animals (Zhang et al. 2019). It has also been shown that intrathecal injection of SRT1720 or resveratrol can upregulate SIRT1 expression in the spinal cords of CCI rats and relieve neuropathic pain induced by CCI (Ferraro et al. 2016; Chen et al. 2018; Crow et al. 2013; Danaher et al. 2018; Falkenberg and Johnstone 2014). Therefore, SIRT1 is an important target to develop new drugs for neuropathic pain. In our previous study, we have successfully identified several new SIRT1 activators, among which Comp 6d has the strongest activity (Gao et al. 2021). We first observed the effect of Comp 6d on CCI-induced neuropathic pain, and we found that intrathecal injection of Comp 6d significantly upregulated SIRT1 expression and activity, and reduced CCI-induced nociceptive hyperalgesia. As a SIRT1 activator, our previous study has demonstrated that Comp 6d can directly bind with the N-terminal domain of SIRT1, therefore increasing its deacetylating activity (Gao et al. 2021). Interestingly, in this study, we also found that Comp 6d upregulated the expressions of SIRT1 mRNA and protein, which may also contribute to the enhancement of SIRT1 activity. However, the underlying mechanism for Comp 6d to upregulate SIRT1 expression is still unclear. It has been shown that increased SIRT1 activity can promote the deacetylation of P53 and subsequently inhibit the transcription of miR-34a, while miR-34a can target and inhibit SIRT1 mRNA, reducing its protein expression (Audrito et al. 2011; Wu et al. 2018). Consistent with this opinion, some other SIRT1 activators including SRT1720 and SRT2104 have also been shown to upregulate SIRT1 expression (Liu et al. 2024, 2019; Zhou et al. 2017). However, further studies are needed to determine whether Comp 6d upregulated SIRT1 expression through the P53- miR-34a pathway or other mechanisms.

To further verify the effect of systemic administration of Comp 6d on neuropathic pain, we established an SNI mouse model and administered Comp 6d via intraperitoneal injection for 28 days. The results showed that intraperitoneal injection of Comp 6d also significantly improved the pain hyperalgesia and upregulated spinal SIRT1 level and activity in SNI mice. In the present experiment, we found that resveratrol could also relieve neuropathic pain and upregulate SIRT1 expression and activity in the spinal cords, which is in agreement with the previous studies (Yin et al. 2013; Jia et al. 2020). However, it has been suggested that resveratrol has poor bioavailability and is not a direct activator of SIRT1, and so it may not depend on SIRT1 for pain relief (Pacholec et al. 2010; Sinclair and Guarente 2014). Interestingly, we unexpectedly found that pregabalin, a drug commonly used in the clinical treatment of pain, also upregulated SIRT1 level and activity, however, further research is needed to determine whether the analgesic effect of pregabalin depends on SIRT1. Furthermore, the present study showed no significant changes in serum markers of liver and kidney function (ALT, AST, ALB, CRE and BUN) after intraperitoneal injection of Comp 6d. HE staining of the liver, heart and kidney indicated that Comp 6d did not exhibit significant toxicity. These results suggest that long-term intraperitoneal injection of Comp 6d has no significant side effects in the dose range of this experiment.

We have demonstrated that Comp 6d significantly ameliorated nociceptive hyperalgesia in animal models of CCI- or SNI-induced neuropathic pain, so does it indeed modulate pain by targeting spinal SIRT1 but not other related proteins? To test this hypothesis, we used mice specifically knocked out of spinal SIRT1, that is, SIRT1flox/flox, Cre mice, to see whether intrathecal injection of Comp 6d can still ameliorate neuropathic pain. The results showed that knockdown of SIRT1 induced neuropathic pain in mice, which is consistent with our previous study (Zhang et al. 2019). However, Comp 6d had no significant effect on pain-like behavior and spinal SIRT1 level and activity in SIRT1flox/flox, Cre mice after 5 days of continuous intrathecal injection, indicating that Comp 6d ameliorates neuropathic pain by specifically activating spinal SIRT1.

Metabotropic glutamate receptors (mGluRs) are widely distributed in the central nervous system (CNS) and are classified into three classes according to their functions, while mGluR1 and mGluR5 belong to type I mGluRs (Blacker et al. 2017; Ferraguti et al. 2008; Jakaria et al. 2018). Numerous studies have shown that type I mGluRs are widely distributed at excitatory synapses in the CNS as important regulators of synaptic transmission and have significant impacts on synaptic plasticity, neuroprotection and neurodegenerative diseases (Yu et al. 2013; Bagot et al. 2012; Okubo et al. 2019; Pin and Duvoisin 1995). Moreover, mGluR1/5 has also been shown to be a new research direction and target for the treatment of epilepsy, depression, drug addiction and pain. It has also been shown that spinal mGluR1/5 can induce a pain response by enhancing the excitability of spinal excitatory neurons (Okubo et al. 2019; Niu et al. 2020). Intrathecal injection of the mGluR1/5 antagonists has been reported to produce analgesic effects in neuropathic pain animal models (Yashpal et al. 2001). In addition, the results from our previous study and other groups have demonstrated that the upregulation of SIRT1 in the spinal cords improves diabetic neuropathic pain and bone cancer pain by decreasing mGluR1/5 expression (Yang et al. 2019; Zhou et al. 2017). Therefore, we explored the effect of Comp 6d on mGluR1/5 expression. As expected, we found that both intrathecal and intraperitoneal injections of Comp 6d significantly downregulated mGluR1/5 level in the spinal cords of animals with neuropathic pain. In addition, our previous study has demonstrated that SIRT1 promotes histone H3 deacetylation in the promoter region of the mGluR1/5 gene through epigenetic mechanisms, thereby regulating mGluR1/5 expression and improving neuropathic pain (Zhou et al. 2017). Therefore, we speculate that Comp 6d may regulate mGluR1/5 expression by upregulating SIRT1 and enhancing its deacetylating effect. However, further studies are needed to confirm this point of view.

Since the activation of mGluR1/5 is associated with the increased neuronal excitability (Park et al. 2004; Li et al. 2011), we observed the effect of Comp 6d on the expression of c-Fos, a marker protein of neuronal activation. We found that Comp 6d downregulated the expression of c-Fos and decreased the number of c-Fos-positive neurons in neuropathic pain animals. However, knockdown of SIRT1 reversed the role of Comp 6d on mGluR1/5 level and c-Fos expression. These results suggest that Comp 6d alleviates neuropathic pain by specifically activating SIRT1 in spinal cords, which in turn downregulates mGluR1/5 expression and inhibits spinal neuronal activation.

Conclusion

In conclusion, our present results suggest that Comp 6d is a specific activator of SIRT1 and can effectively alleviate neuropathic pain by specifically activating SIRT1 in the spinal cord, which in turn downregulates mGluR1/5 expression and inhibits spinal neuronal activation (Fig. 7). Our study provides a theoretical basis for preclinical studies of Comp 6d, and Comp 6d is expected to be an effective therapeutic agent for neuropathic pain.

Fig. 7.

Fig. 7

Open in a new tab

Schematic plot for the effect of Comp 6d on neuropathic pain. Comp 6d attenuates neuropathic pain by inhibiting spinal neuronal activation via the SIRT1-mGluR1/5 pathway

Supplementary information

Below is the link to the electronic supplementary material.

ESM 1 (25.7KB, docx)

(DOCX 25.6 KB)

Acknowledgements

We sincerely thank Dr. Fuxing Dong from the Public Experimental Research Center for his enthusiastic help in the experiment of laser scanning confocal microscopy.

Author contributions

CZ and YW designed the study. JG synthesized Comp 6d. XD, GW and YL performed the experiments. WH and CC assisted in the completion of the behavioral test. GW wrote the manuscript. CZ revised the paper. All authors have read and agree to the published version of the manuscript.

Funding

National Natural Science Foundation of China, Grant/Award Number: 82071232 and 82371240; the Key Subject of Colleges and Universities Natural Science Foundation of Jiangsu Province, Grant/Award Number: 23KJA310009; Qing Lan Project of Jiangsu Province; Xuzhou Medical University Scientific Research Funding, Grant/Award Number: D2023020.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiaobao Ding, Guizhi Wang and Yuwen Lin contributed equally to this work.

Contributor Information

Jian Gao, Email: gaojian@aust.edu.cn.

Yuqing Wu, Email: xzmcyqwu@163.com.

Chenghua Zhou, Email: xzhmuchzhou@163.com.

References

  1. Abe-Higuchi N, Uchida S, Yamagata H, Higuchi F, Hobara T, Hara K, Kobayashi A, Watanabe Y. Hippocampal Sirtuin 1 Signaling Mediates Depression-like Behavior. Biol Psychiatry. 2016;80(11):815–26. [DOI] [PubMed] [Google Scholar]
  2. Alcántara Montero A, Ibor Vidal PJ, Alonso Verdugo A, Trillo Calvo E. Update in the pharmacological treatment of neuropathic pain. SEMERGEN. 2019;45(8):535–45. [DOI] [PubMed] [Google Scholar]
  3. Audrito V, Vaisitti T, Rossi D, Gottardi D, D’Arena G, Laurenti L, Gaidano G, Malavasi F, Deaglio S. Nicotinamide blocks proliferation and induces apoptosis of chronic lymphocytic leukemia cells through activation of the p53/miR-34a/SIRT1 tumor suppressor network. Cancer Res. 2011;71(13):4473–83. [DOI] [PubMed] [Google Scholar]
  4. Bagot, R. C., T. Y. Zhang, X. Wen, T. T. Nguyen, H. B. Nguyen, J. Diorio, T. P. Wong, and M. J. Meaney. 2012. Variations in postnatal maternal care and the epigenetic regulation of metabotropic glutamate receptor 1 expression and hippocampal function in the rat. Proc Natl Acad Sci U S A 109 Suppl 2 (Suppl 2):17200–17207. [DOI] [PMC free article] [PubMed]
  5. Baur JA, Ungvari Z, Minor RK, Le Couteur DG, de Cabo R. Are sirtuins viable targets for improving healthspan and lifespan? Nat Rev Drug Discov. 2012;11(6):443–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33(1):87–107. [DOI] [PubMed] [Google Scholar]
  7. Blacker CJ, Lewis CP, Frye MA, Veldic M. Metabotropic glutamate receptors as emerging research targets in bipolar disorder. Psychiatry Res. 2017;257:327–37. [DOI] [PubMed] [Google Scholar]
  8. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53(1):55–63. [DOI] [PubMed] [Google Scholar]
  9. Chen K, Fan J, Luo ZF, Yang Y, Xin WJ, Liu CC. Reduction of SIRT1 epigenetically upregulates NALP1 expression and contributes to neuropathic pain induced by chemotherapeutic drug bortezomib. J Neuroinflammation. 2018;15(1):292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chiechio S, Nicoletti F. Metabotropic glutamate receptors and the control of chronic pain. Curr Opin Pharmacol. 2012;12(1):28–34. [DOI] [PubMed] [Google Scholar]
  11. Crow M, Denk F, McMahon SB. Genes and epigenetic processes as prospective pain targets. Genome Med. 2013;5(2):12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Curry RJ, Peng K, Lu Y. Neurotransmitter- and Release-Mode-Specific Modulation of Inhibitory Transmission by Group I Metabotropic Glutamate Receptors in Central Auditory Neurons of the Mouse. J Neurosci. 2018;38(38):8187–99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Danaher RJ, Zhang L, Donley CJ, Laungani NA, Hui SE, Miller CS, Westlund KN. Histone deacetylase inhibitors prevent persistent hypersensitivity in an orofacial neuropathic pain model. Mol Pain. 2018;14:1744806918796763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Denk F, Huang W, Sidders B, Bithell A, Crow M, Grist J, Sharma S, Ziemek D, Rice ASC, Buckley NJ, McMahon SB. HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain. Pain. 2013;154(9):1668–79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ding X, Lin Y, Chen C, Yan B, Liu Q, Zheng H, Wu Y, Zhou C. DNMT1 Mediates Chronic Pain-Related Depression by Inhibiting GABAergic Neuronal Activation in the Central Amygdala. Biol Psychiatry. 2023;94(8):672–84. [DOI] [PubMed] [Google Scholar]
  16. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov. 2014;13(9):673–91. [DOI] [PubMed] [Google Scholar]
  17. Ferraguti F, Crepaldi L, Nicoletti F. Metabotropic glutamate 1 receptor: current concepts and perspectives. Pharmacol Rev. 2008;60(4):536–81. [DOI] [PubMed] [Google Scholar]
  18. Ferraro, F., M. Jacopetti, V. Spallone, L. Padua, M. Traballesi, S. Brunelli, C. Cantarella, C. Ciotti, D. Coraci, E. Dalla Toffola, S. Mandrini, G. Morone, C. Pazzaglia, M. Romano, A. Schenone, R. Togni, and S. Tamburin. 2016. Diagnosis and treatment of pain in plexopathy, radiculopathy, peripheral neuropathy and phantom limb pain. Evidence and recommendations from the Italian Consensus Conference on Pain on Neurorehabilitation. Eur J Phys Rehabil Med 52 (6):855–866. [PubMed]
  19. Finnerup NB, Kuner R, Jensen TS. Neuropathic Pain: From Mechanisms to Treatment. Physiol Rev. 2021;101(1):259–301. [DOI] [PubMed] [Google Scholar]
  20. Flatters SJ, Bennett GJ. Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy. Pain. 2004;109(1–2):150–61. [DOI] [PubMed] [Google Scholar]
  21. Gao J, Chen QQ, Huang Y, Li KH, Geng XJ, Wang T, Lin QS, Yao RS. Design, Synthesis and Pharmacological Evaluation of Naphthofuran Derivatives as Potent SIRT1 Activators. Front Pharmacol. 2021;12: 653233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jakaria M, Park SY, Haque ME, Karthivashan G, Kim IS, Ganesan P, Choi DK. Neurotoxic Agent-Induced Injury in Neurodegenerative Disease Model: Focus on Involvement of Glutamate Receptors. Front Mol Neurosci. 2018;11:307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jia, Q., W. Dong, L. Zhang, and X. Yang. 2020. Activating Sirt1 by resveratrol suppresses Nav1.7 expression in DRG through miR-182 and alleviates neuropathic pain in rats. Channels (Austin) 14 (1):69–78. [DOI] [PMC free article] [PubMed]
  24. Li Z, Ji G, Neugebauer V. Mitochondrial reactive oxygen species are activated by mGluR5 through IP3 and activate ERK and PKA to increase excitability of amygdala neurons and pain behavior. J Neurosci. 2011;31(3):1114–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu Y, Ni Y, Zhang W, Sun YE, Jiang M, Gu WJ, Ma ZL, Gu XP. Anti-nociceptive effects of caloric restriction on neuropathic pain in rats involves silent information regulator 1. Br J Anaesth. 2018;120(4):807–17. [DOI] [PubMed] [Google Scholar]
  26. Liu ZH, Zhang Y, Wang X, Fan XF, Zhang Y, Li X, Gong YS, Han LP. SIRT1 activation attenuates cardiac fibrosis by endothelial-to-mesenchymal transition. Biomed Pharmacother. 2019;118:109227. [DOI] [PubMed]
  27. Liu Y, Liu Q, Wang H, Qiu Y, Lin J, Wu W, Wang N, Dong W, Wan J, Chen C, Li S, Zheng H, Wu Y. Hippocampal synaptic plasticity injury mediated by SIRT1 downregulation is involved in chronic pain-related cognitive dysfunction. CNS Neurosci Ther. 2024;30(2): e14410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Michán S, Li Y, Chou MM, Parrella E, Ge H, Long JM, Allard JS, Lewis K, Miller M, Xu W, Mervis RF, Chen J, Guerin KI, Smith LE, McBurney MW, Sinclair DA, Baudry M, de Cabo R, Longo VD. SIRT1 is essential for normal cognitive function and synaptic plasticity. J Neurosci. 2010;30(29):9695–707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Napper AD, Hixon J, McDonagh T, Keavey K, Pons JF, Barker J, Yau WT, Amouzegh P, Flegg A, Hamelin E, Thomas RJ, Kates M, Jones S, Navia MA, Saunders JO, DiStefano PS, Curtis R. Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem. 2005;48(25):8045–54. [DOI] [PubMed] [Google Scholar]
  30. Niu Y, Zeng X, Zhao L, Zhou Y, Qin G, Zhang D, Fu Q, Zhou J, Chen L. Metabotropic glutamate receptor 5 regulates synaptic plasticity in a chronic migraine rat model through the PKC/NR2B signal. J Headache Pain. 2020;21(1):139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Okubo M, Yamanaka H, Kobayashi K, Noguchi K. Differential expression of mGluRs in rat spinal dorsal horns and their modulatory effects on nocifensive behaviors. Mol Pain. 2019;15:1744806919875026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Onakpoya IJ, Thomas ET, Lee JJ, Goldacre B, Heneghan CJ. Benefits and harms of pregabalin in the management of neuropathic pain: a rapid review and meta-analysis of randomised clinical trials. BMJ Open. 2019;9(1): e023600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS, Griffith D, Griffor M, Loulakis P, Pabst B, Qiu X, Stockman B, Thanabal V, Varghese A, Ward J, Withka J, Ahn K. SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem. 2010;285(11):8340–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Park YK, Galik J, Ryu PD, Randic M. Activation of presynaptic group I metabotropic glutamate receptors enhances glutamate release in the rat spinal cord substantia gelatinosa. Neurosci Lett. 2004;361(1–3):220–4. [DOI] [PubMed] [Google Scholar]
  35. Pin JP, Duvoisin R. The metabotropic glutamate receptors: structure and functions. Neuropharmacology. 1995;34(1):1–26.> [DOI] [PubMed] [Google Scholar]
  36. Reichling DB, Levine JD. Critical role of nociceptor plasticity in chronic pain. Trends Neurosci. 2009;32(12):611–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ren K, Blass EM, Zhou Q, Dubner R. Suckling and sucrose ingestion suppress persistent hyperalgesia and spinal Fos expression after forepaw inflammation in infant rats. Proc Natl Acad Sci U S A. 1997;94(4):1471–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shao H, Xue Q, Zhang F, Luo Y, Zhu H, Zhang X, Zhang H, Ding W, Yu B. Spinal SIRT1 activation attenuates neuropathic pain in mice. PLoS ONE. 2014;9(6): e100938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sinclair DA, Guarente L. Small-molecule allosteric activators of sirtuins. Annu Rev Pharmacol Toxicol. 2014;54:363–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Singh H, Bhushan S, Arora R, Singh Buttar H, Arora S, Singh B. Alternative treatment strategies for neuropathic pain: Role of Indian medicinal plants and compounds of plant origin-A review. Biomed Pharmacother. 2017;92:634–50. [DOI] [PubMed] [Google Scholar]
  41. Sun YM, Shen Y, Huang H, Liu Q, Chen C, Ma LH, Wan J, Sun YY, Zhou CH, Wu YQ. Downregulated SIRT1 in the CeA is involved in chronic pain-depression comorbidity. Brain Res Bull. 2021;174:339–48. [DOI] [PubMed] [Google Scholar]
  42. Szok D, Tajti J, Nyári A, Vécsei L. Therapeutic Approaches for Peripheral and Central Neuropathic Pain. Behav Neurol. 2019;2019:8685954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Uchida H, Ma L, Ueda H. Epigenetic gene silencing underlies C-fiber dysfunctions in neuropathic pain. J Neurosci. 2010;30(13):4806–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Uchida H, Matsushita Y, Ueda H. Epigenetic regulation of BDNF expression in the primary sensory neurons after peripheral nerve injury: implications in the development of neuropathic pain. Neuroscience. 2013;240:147–54. [DOI] [PubMed] [Google Scholar]
  45. Wu H, Wu J, Zhou S, Huang W, Li Y, Zhang H, Wang J, Jia Y. SRT2104 attenuates diabetes-induced aortic endothelial dysfunction via inhibition of P53. J Endocrinol. 2018;237(1):1–14. [DOI] [PubMed] [Google Scholar]
  46. Xu N, Wu MZ, Deng XT, Ma PC, Li ZH, Liang L, Xia MF, Cui D, He DD, Zong Y, Xie Z, Song XJ. Inhibition of YAP/TAZ Activity in Spinal Cord Suppresses Neuropathic Pain. J Neurosci. 2016;36(39):10128–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yang C, Kang F, Wang S, Han M, Zhang Z, Li J. SIRT1 Activation Attenuates Bone Cancer Pain by Inhibiting mGluR1/5. Cell Mol Neurobiol. 2019;39(8):1165–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yashpal K, Fisher K, Chabot JG, Coderre TJ. Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats. Pain. 2001;94(1):17–29. [DOI] [PubMed] [Google Scholar]
  49. Yin Q, Lu FF, Zhao Y, Cheng MY, Fan Q, Cui J, Liu L, Cheng W, Yan CD. Resveratrol facilitates pain attenuation in a rat model of neuropathic pain through the activation of spinal Sirt1. Reg Anesth Pain Med. 2013;38(2):93–9. [DOI] [PubMed] [Google Scholar]
  50. Yu F, Zhong P, Liu X, Sun D, Gao HQ, Liu QS. Metabotropic glutamate receptor I (mGluR1) antagonism impairs cocaine-induced conditioned place preference via inhibition of protein synthesis. Neuropsychopharmacology. 2013;38(7):1308–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zeng Y, Shi Y, Zhan H, Liu W, Cai G, Zhong H, Wang Y, Chen S, Huang S, Wu W. Reduction of Silent Information Regulator 1 Activates Interleukin-33/ST2 Signaling and Contributes to Neuropathic Pain Induced by Spared Nerve Injury in Rats. Front Mol Neurosci. 2020;13:17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhang Z, Ding X, Zhou Z, Qiu Z, Shi N, Zhou S, Du L, Zhu X, Wu Y, Yin X, Zhou C. Sirtuin 1 alleviates diabetic neuropathic pain by regulating synaptic plasticity of spinal dorsal horn neurons. Pain. 2019;160(5):1082–92. [DOI] [PubMed] [Google Scholar]
  53. Zhou CH, Zhang MX, Zhou SS, Li H, Gao J, Du L, Yin XX. SIRT1 attenuates neuropathic pain by epigenetic regulation of mGluR1/5 expressions in type 2 diabetic rats. Pain. 2017;158(1):130–9. [DOI] [PubMed] [Google Scholar]
  54. Ziemka-Nalecz M, Jaworska J, Sypecka J, Zalewska T. Histone Deacetylase Inhibitors: A Therapeutic Key in Neurological Disorders? J Neuropathol Exp Neurol. 2018;77(10):855–70. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ESM 1 (25.7KB, docx)

(DOCX 25.6 KB)

Data Availability Statement

No datasets were generated or analysed during the current study.

Articles from Cell Biology and Toxicology are provided here courtesy of Springer

RESOURCES