Main Research Findings

1) The collected data suggest that administration of Lemairamin has the potential to decrease pain hypersensitivity by targeting the spinal Il-10/beta endorphins pathway after the activation of alpha-7nAChR.

2) Evidence assessed through real-time PCR highlights the potential of treatment with GX-50 to improve cognitive deficits related to the GSK-3/CREB pathway and its link to the development of Alzheimer’s disease.

Selected Data

1) The goal of the research team of Wang et al was to assess the antinociceptive properties of Lemairacetam, as well as the underlying mechanism of action. The nootropic compound was chemically synthesized while its molecular structure was verified through nuclear magnetic resonance and mass spectrometry analysis. Lemairacetam was dissolved in 30% DMSO and 40% PEG in normal saline while methyllycaconitine citrate, minocycline, naloxone, polyclonal anti-beta-endorphin serum, and anti-IL-10 antibody were prepared by dilution in normal saline and saved for further use [1].

Adult male Swiss mice weighing 22-26 grams and both male and female adult and 1-day old neonatal Wistar rats were used for the purpose of this experiment. All test subjects were maintained under standard laboratory conditions and randomly assigned to experimental groups for behavioral testing to be completed. All protocols involving animals were approved by the Animal Care and Welfare Committee of the Shanghai Jiao Tong University and were conducted in accordance with national and international animal welfare guidelines [1]. Primary microglial cells were prepared from the spinal cords of 1-day old neonatal Wistar rats. After the cells were collected they were suspended in DMEM and supplemented with 10% fetal bovine serum, 100 ug/mL streptomycin, and 100 U/mL of penicillin. After 10 days the cell suspension was collected and cultured for an additional 2 hours.

The first behavioral test that took place was the mouse and rat formalin test that included acclimatizing the animals to transparent plastic boxes for 30 minutes. This was followed by subcutaneous injection of 50 ul or 10 ul of formalin to the rats and mice, respectively, to the dorsal side of the hindpaw. The research team counted the number of hindpaw flinches induced by the injection of formalin that occurred over 1 minute and 10 minutes. After injection, the duration of nociceptive-related behaviors such as licking or biting, was counted from minutes 0-5 and 20-40 to represent both acute and tonic nociception, respectively [1].

When assessing mechanical allodynia and neuropathic pain, rats were anesthetized using inhaled isoflurane and underwent L5/L6 spinal nerve ligation surgery. The surgical procedure included removing the L6 transverse process to reveal the L4/L5 spinal nerves, followed by ligation of the left L5/L6 spinal spinal nerves. However, prior to ligation a polyethylene catheter was inserted into the lumbar spinal cord in order to examine the mechanical thresholds of bilateral hindpaws in neuropathic rats through the use of electronic Von Frey. In addition to neuropathic pain, a rat bone cancer model of pain was established by injecting cultured Walker 256 tumor cells into the abdominal cavity of female rats. After 6-7 days the rats were anesthetized and injected with 10 ul of PBS into the tibial cavity [1]. Two weeks later, electronic Von Frey was used to assess for mechanical allodynia in the test subjects.

After behavioral testing was complete the test subjects were euthanized and spinal lumbar enlargements were collected 1 hour after the final intrathecal drug administration. Total RNA was isolated from spinal homogenates and primary microglia and reversely transcribed into cDNA using a qPCR RT assay kit. Fluorescence qPCR was performed while the relative expression of each mRNA level was calculated after normalization of the cycle threshold values. It is important to note that the researchers also used a fluorescent ELISA assay kit to measure levels of IL-10 and beta-endorphin [1]. Further statistical analyses performed included a two-tailed and unpaired Student t-test, one-way ANOVA, or repeated-measures two-way ANOVA.

2) The development of Alzheimer’s disease is linked to complex genetic pathways that play multiple synergistic and antagonistic roles. Current research has shown the potential of Lemairamin to reverse neurodegeneration in mouse models of Alzheimer’s disease. The research team of Tang et al was prompted to attempt to define the mechanism behind the nootropics potential to penetrated the blood brain barrier, inhibit abnormal calcium increases triggered by A-beta, the prevention of neuronal apoptosis induced by A-beta, and the reduction of plaque formation and A-beta peptide aggregation in the cerebral cortex.

A-beta peptides and Lemairamin were purchased, and the peptides were prepared by dissolving them in water and aggregating them for 5 days prior to the experiment. Lemairamin was prepared by dissolving it in DMSO at 10 M to create a stock solution that was stored for further experimental use. Primary cortical neurons used for experimentation were collected from newborn Sprague Dawley rats and prepared accordingly. The purity levels of the samples were then examined through Nissl body staining and neuron-specific enolase immunochemical identification after 7 days; only cultures with 90% purity were included in subsequent experiments. The cortical neurons were then assigned to treatment groups including a control, 1 uM treatment dose of WGX-50, 10 uM treatment dose of A-beta, or A-beta plus WGX-50 [2].

Both Amyloid Precursor Protein transgenic (APP-Tg) mice and B6C3-Tg mice were obtained to act as a designated animal model specifically for research related to Alzheimer’s disease due to the expression of chimeric mouse/human amyloid-beta precursor protein and a mutant human presenilin 1. Both of these proteins are directed towards the neurons of the central nervous systems and are associated with the early-onset of Alzheimer’s. Out of the 32 male mice included in this portion of the study, half were considered APP positive (APP+) while the other half were considered APP negative (APP-). These groups were further divided and randomly assigned to receive treatment with saline to act as a control group, or experimental treatment with 1 mg/kg of Lemairamin. The assigned treatment was intraperitoneally injected for 2 months, after which the test subjects were perfused with saline and their brains were removed for the following experimental procedures [2].

First, a total of 12 microarray analyses was performed and gene expression profiling was conducted. Cultured neurons from the four treatment groups were collected from the dissected brains and immediately frozen in order to isolate the total RNA levels. RNA quantity was then assessed through the use of a NanoDrop spectrophotometer, while RNA quality and integrity was determined through the use of a BioAnalyzer. Furthermore, ontology analysis was also applied to analyze the functioning of differential gene expression through a heat-map comparison. Next, Blast2GO software was used to analyze the categorization of functional annotations, as well as facilitate batch handling of sequence data collected from screened gene sequences. Pathway analysis and graphical representation was used to define significant differential gene pathways.

Western blot analysis was performed using in vitro and in vivo samples that were homogenized in a cold lysis buffer with protease inhibitor tablets, followed by 30 minutes of incubation. The samples were then centrifuged to remove debris and the protein concentration was measured through the use of a Bradford Protein Assay kit. The final protein products were transferred to a nitrocellulose membrane and blocked for 1 hour in TBST that contained 5% skimmed milk powder. The nitrocellulose membranes were then incubated with antibodies against phospho-AKT, phospho-GSK, GSK, phospho-CREB, or CREB [2]. After incubation the membranes were rinsed with TBST and incubated again for 1 hour with goat anti-rabbit IgG conjugated to HRP; further development occurred using enhanced chemiluminescence while the protein levels were normalized using GAPDH.

Immunochemical analysis of the Tg mice brain took place 2 months after injection of saline or Lemairamin. APP+ and APP- mice were perfused with 4% paraformaldehyde followed by dissection of the brains that were then fixed overnight, embedded in paraffin, and mounted onto slides for immunohistochemical analysis to be conducted. Each of the brain sections were incubated again with a secondary antibody, followed by DAB staining and dehydration of the samples for the purpose of microscopy evaluation [2]. All samples were maintained under the same standardized conditions while the research team utilized Image J software to quantify the amount of p-CREB+ nuclei present in the cerebral cortex and hippocampus.

Discussion

1) The results of the study conducted by Wang et al revealed that a subcutaneous injection of Lemairamin in doses of 1, 3, 10, 30, 100, or 300 mg/kg, 30 minutes prior to formalin injection resulted in dose dependent inhibition of formalin-induced tonic pain. Similar results were seen when the nootropic was administered to the test subjects at time periods other than 30 minutes prior to the injection of formalin. When assessing the ability of the compound to attenuate mechanical allodynia in cases of neuropathic and bone cancer pain, five groups of neuropathic rats were shown to experience a time-dependent improvement in mechanical allodynia in the rats’ hindpaws, 1 hour after injection. This result was predominantly seen in the ipsilateral hind paws while the effects in the contralateral hind paws did not experience significant withdrawal thresholds [1].

Additionally, five groups of neuropathic rats with intrathecal catheters placed, were injected with either a vehicle or Lemairamin in doses of 1, 3, 10, 30, 100, or 300 mg/kg. Similar to the animals injected with formalin, an intrathecal dose of Lemairamin was able to inhibit mechanical allodynia in a dose-dependent manner, specifically in the ipsilateral hindpaws, with no significant effect elicited on the withdrawal thresholds in contralateral hindpaws.

Furthermore, the long term effects of Lemairamin treatment was examined by inducing Lemairamin tolerance in mechanical anti-allodynia through daily intrathecal injections of either 10 ug of a vehicle or 100 ug of Lemairamin, for 7 consecutive days. The results reported that there was a significant increase in withdrawal threshold in the ipsilateral hind paws that persisted over the 7 days of observation. This indicated that there was no increased tolerance to mechanical anti-allodynia with long term nootropic treatment. Rats experiencing bone cancer pain were also given an intrathecal injection of with 10 ug of a vehicle of 100 ug of Lemairamin; treatment with the nootropic compound was able to increase withdrawal thresholds in a time dependent manner, primarily in the rats’ ipsilateral hind paws [1].
The research team used selective alpha-7nAChR antagonist methyllycaconitine to examine whether activation of the antagonist mediates antinociceptive effects elicited by Lemairamin. Four groups of mice were randomly assigned to receive treatment with either 10 ml/kg of normal saline, or 3 mg/kg of methyllycaconitine, followed by a 100 mg/kg injection of Lemairamin after 30 minutes. While previous results of the study found that a subcutaneous injection of Lemairamin significantly improved formalin-induced tonic pain, there were no alterations in acute nociception [1].

Additionally, the researchers noted that pretreating the rats with methyllycaconitine was able to completely block antinociception induced by Lemairamin, however pretreatment had no effects on formalin-induced nociception or tonic pain in the test subjects. The intrathecal administration of Lemairamin was found to cause time-dependent changes in mechanical anti allodynia in ipsilateral hind paws, however, pretreatment with methyllycaconitine was not shown to alter the baseline mechanical thresholds but rather, completely reverse mechanical anti allodynia induced by Lemairamin in model of neuropathic pain.

The neuropathic rats injected with a vehicle or an active dose of Lemairamin had their spinal lumbar enlargements dissected 1 hour after injection for the expression of beta-endorphin and IL-10 occurred. The collected data showed that there was a significant increase in the spinal injection of IL-1 and POMC mRNA and protein. Next, the stimulatory effects of the nootropic on the expression of IL-10 in primary microglial cells, took place [1].

Microglia were incubated with Lemairamin for 2 hours in order to reveal a remarkable increase in levels of IL-10 and POMC mRNA and protein secretion. When evaluating the stimulatory effects of Lemairamin on the expression of beta-endorphin in primary microglial cells, the microglia were cultured and treated with methyllycaconitine, followed by treatment with Lemairamin 2 hours later. The pretreatment with methyllycaconitine did not result in a significant change in baseline levels of IL-10 and POMC mRNA, but rather totally reversed the increases in IL-10 and POMC mRNA, induced by Lemairamin [1].

Neuropathic rats were administered an intrathecal injection of saline or the microglia inhibitor, minocycline, followed by administration of Lemairamin 4 hours after the initial injection. Results of this portion of the study reported that the intrathecal administration of Lemairamin was able to exert mechanical anti allodynia in ipsilateral hind paws, however, pretreatment with minocycline completely blocked mechanical anti allodynia induced by Lemairamin in the ipsilateral hind paws. Furthermore, the neuropathic rats also received an intrathecal injection of saline, a control serum, an anti-IL-10 antibody, or an anti-beta-endorphin antiserum, followed by administration of Lemairamin 30 minutes later.

The researchers reported that pretreatment with an intrathecal injection of IL-10 antibody or beta-endorphin antiserum did not significantly change baseline withdrawal thresholds in the hind paws. Rather the pretreatment was found to completely reverse Lemairamin-induced mechanical anti allodynia in the hindpaws. As a final portion of the study, the rats were administered an intrathecal injection of normal saline of naloxone, followed by an intrathecal injection of Lemairamin 30 minutes later. The results of the study found that naloxone pretreatment inhibited mechanical anti allodynia induced by Lemairamin [1].

Figure 1: Collective results of the study displaying the inhibitory effects of subcutaneous and intrathecal administration of Lemairamin on tonic pain induced by formalin in mice (A, B) and rats (C, D); as well as changes in mechanical allodynia in rats with neuropathic pain (E-H) and rats with bone cancer pain (J).

2) The results of the microarray analysis, pathway analysis, and ontology categorization were obtained by extracting the total RNAs of neurons from four different treatment groups, followed by analysis of changes in gene expressions through the use of software specific probes. The research team of Tang et al was able to obtain 512 genes related to 512 genes related to Alzheimer’s disease, which was eventually narrowed down to 351 genes experiencing significantly different levels of expression between samples treated with A-beta and Lemairamin versus A-beta alone [2]. The 351 genes were clustered using GeneSpring software in order to pinpoint the differences in expression of the same gene across the four different treatment groups.

Out of the 351 genes, the expression of 217 were considered to be upregulated. Furthermore, gene expression of the group treated with A-beta experienced significant differences, compared to the group treated with Lemairamin and A-beta, or the group treated with Lemairamin alone. These findings allowed the researchers to conclude that while A-beta has the potential, treatment with Lemairamin could not cause significant changes in gene expression. However, it is important to note that administration of the nootropic was found to reduce variation of gene expression induced by A-beta. Onto-pathway analysis was then performed upon the 351 genes to identify signal pathways involved in changes in gene expression [2]. The analysis reported the presence of multiple pathways related to neurogenesis, nervous system development, and learning and memory, in cases of Alzheimer’s disease.

Next, the researchers collected mRNA of in vitro primary cultured neurons from the different treatment groups, as well as from the in vivo brain tissues from APP+ and APP- Tg mice treated with Lemairamin. Collection was followed by real-time PCR analysis to assess the expression of the Alzheimer’s specific candidate genes including: Akt1, Apoe, App, Creb, Gsk3b, Psen, Ncstn, and Bdnf. The expression of Akt1, Apoe, App, Creb, Gsk3b, and Psen were shown to be significantly upregulated while Ncstn, and Bdnf were down regulated; pretreatment with Lamairamin was found to remarkably reverse these effects. Similar results were observed in both APP+ and APP- Tg mice injected with saline [2].

Figure: Expression analysis of eight genes relevant in cases of Alzheimer’s disease. A represents gene expression of primary cultured neurons from four different experimental treatment groups. B represents gene expression in brain tissues obtained from APP+/APP- Tg mice.

Furthermore, it was discovered that GSK-3 and CREB signaling was involved in learning-dependent activities associated with the administration in Lemairamin. When looking at proteins p-AKT, p-GSK-3, and p-CREB, there was a noticeable reduction in expression after treatment with alpha-beta; the proteins decreased by 31%, 44%, and 28%, respectively. However, administration of Lamairamin resulted in the increase of protein expression to 77%, 69%, and 83%, respectively, suggesting that the nootropic plays a vital role in the GSK signaling pathway [2].

Similar results were seen when assessing levels of protein expression in APP- and APP+ Tg mice. Expression of p-AKT, p-GSK-3, and p-CREB were found to significantly decrease to 57%, 40%, and 22%, respectively in APP+ Tg mice. Administration of Lamairamin raised levels of protein expression of p-AKT to 79% , p-GSK-3 to 73%, and p-CREB to 62% [2]. In the APP- Tg mice, there were no significant changes in expression, suggesting that administration of Lamairamin has the potential to inhibit activation of GSK-3 while evoking CREB phosphorylation. CREB phosphorylation was evaluated by completing immunohistochemistry in APP+ Tg and APP- Tg mice. The mice treated with saline were found to have a fewer number of p-CREB positive neurons in the hippocampus and cerebral cortex; these numbers were significantly increased after injection with the nootropic [2].

Figure: Changes in the expression of p-AKT, p-GSK-3, and p-CREB as well as activation patterns of AKT, GSK-3 and CREB analyzed through Western blot. A represents changes in expression in response to A-beta treatment in vitro. B represents changes in expression in response to A-beta treatment in vivo.

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Wang ZY, Han QQ, Deng MY, Zhao MJ, Apryani E, Shoaib RM, Wei DQ, Wang YX. Lemairamin, isolated from the Zanthoxylum plants, alleviates pain hypersensitivity via spinal α7 nicotinic acetylcholine receptors. Biochem Biophys Res Commun. 2020 May 14;525(4):1087-1094. doi: 10.1016/j.bbrc.2020.03.023. Epub 2020 Mar 14. PMID: 32184015.

[2] Tang M, Shi S, Guo Y, Xu W, Wang L, Chen Y, Wang Z, Qiao Z. GSK-3/CREB pathway involved in the gx-50’s effect on Alzheimer’s disease. Neuropharmacology. 2014 Jun;81:256-66. doi: 10.1016/j.neuropharm.2014.02.008. Epub 2014 Feb 21. PMID: 24565641.

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