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Measurement of radon/thoron and its daughter nuclides in room air

Suppian R, Vegandraj S, Kandaiya S

Int J Rad Appl Instrum A, 1992 Jul;43(7):937-8.
PMID: 1321104

Pumping air through a soft tissue which acts as a membrane is a relatively easy and quick method to collect and measure radon/thoron and its daughter nuclides in air. Analysis of the activity of the radionuclides can be calculated using an alpha counter which has been calibrated. In this method the activity of radon/thoron cannot be separated from the activity of radionuclides already present in the aerosol or dust particles.
Fulltext Induction of an Antibody Response against Plasmodium falciparum F2RIIEBA by Heterologous Prime-boost Immunisation

Suppian R, Nor NM

Trop Life Sci Res, 2013 Aug;24(1):9-18.
PMID: 24575238 MyJurnal

Heterologous prime-boost immunisation strategies can evoke powerful antibody responses and may be of value in developing an improved malaria vaccine. Herein, we show that an immunisation protocol that primes Balb/c mice with a recombinant Bacille Calmette-Guérin (rBCG) vaccine consisting of a plasmid encoding a synthetic fragment of the ESAT-6 epitope of Mycobacterium tuberculosis, the fragment 2 region II of erythrocyte-binding antigen (F2RIIEBA) and the three repeat sequences of the circumsporozoite protein (NANP)3 of Plasmodium falciparum before subsequently boosting the mice with either two doses of the rBCG clone or with a DNA vaccine expressing the native form of F2RIIEBA generating higher serum anti-F2RIIEBA antibody levels than an immunisation protocol that calls for a homologous prime-boost with two doses of rBCG. These results demonstrate the potential of DNA vaccination in boosting the antibody response to a recombinant vaccine expressing multiple epitopes.
Fulltext The role of TLR-4 in the immunomodulatory effects of recombinant BCG expressing MSP-1C of Plasmodium falciparum

Abbas MA, Suppian R

J Infect Dev Ctries, 2019 11 30;13(11):1057-1061.
PMID: 32087079 DOI: 10.3855/jidc.11331

INTRODUCTION: An earlier constructed recombinant BCG expressing the MSP-1C of Plasmodium falciparum, induced inflammatory responses leading to significant production of nitric oxide (NO) alongside higher expression of the enzyme inducible nitric oxide synthase (iNOS) and significant production of the regulatory cytokine, IL-10, indicating significant immunomodulatory effects of the construct. The mechanism of these responses had not been established but is thought to involve toll-like receptor 4 (TLR-4).
METHODOLOGY: The present study was carried out to determine the role of TLR-4 on eliciting the immunomodulatory effects of recombinant BCG expressing MSP-1C of Plasmodium falciparum leading to the production of NO and IL-10, as well as the expression of iNOS. Six groups of mice (n = 6 per group) were immunised thrice, three weeks apart with intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of a TLR-4 inhibitor; TAK-242, given one hour prior to each immunisation. Peritoneal macrophages were harvested from the mice and cultured for the determination of NO, iNOS and IL-10 via Griess assay, ELISA and Western blot respectively.
RESULTS: The results showed significant inhibition of the production of NO and IL-10 and the expression of iNOS in all groups of mice in the presence of TAK-242.
CONCLUSIONS: These results presented evidence of the role of TLR-4/rBCG attachment mechanism in modulating the production of NO and IL-10 and the expression of iNOS in response to our rBCG-based malaria vaccine candidate expressing MSP-1C of P. falciparum.
Fulltext Immunomodulatory effects of recombinant BCG expressing MSP-1C of Plasmodium falciparum on LPS- or LPS+IFN-γ-stimulated J774A.1 cells

Mohamad D, Suppian R, Mohd Nor N

Hum Vaccin Immunother, 2014;10(7):1880-6.
PMID: 25424796 DOI: 10.4161/hv.28695

Macrophage phagocytosis is the first line of defense of the innate immune system against malaria parasite infection. This study evaluated the immunomodulatory effects of BCG and recombinant BCG (rBCG) strains expressing the C-terminus of the merozoite surface protein-1 (MSP-1C) of Plasmodium falciparum on mouse macrophage cell line J774A.1 in the presence or absence of lipopolysaccharide (LPS) or LPS + IFN-γ. The rBCG strain significantly enhanced phagocytic activity, production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitric oxide (NO), and inducible nitric oxide synthase (iNOS) as compared with parental BCG strain, and these activities increased in the presence of LPS and LPS+IFN-γ. Furthermore, the rBCG strain also significantly reduced the macrophage viability as well as the rBCG growth suggesting the involvement of macrophage apoptosis. Taken together, these data indicate that the rBCG strain has an immunomodulatory effect on macrophages, thus strengthen the rational use of rBCG to control malaria infection.
Fulltext The Effects of Pre-emptive Administration of Ketamine and norBNI on Pain Behavior, c-Fos, and Prodynorphin Protein Expression in the Rat Spinal Cord after Formalin-induced Pain Is Modulated by the DREAM Protein

Long I, Suppian R, Ismail Z

Korean J Pain, 2013 Jul;26(3):255-64.
PMID: 23861999 DOI: 10.3344/kjp.2013.26.3.255

We investigated the effects of pre-emptive administration of ketamine and norBNI on pain behavior and the expression of DREAM, c-Fos, and prodynorphin proteins on the ipsilateral side of the rat spinal cord at 2 and 4 hours after formalin injection.
Fulltext Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans

Hajissa K, Zakaria R, Suppian R, Mohamed Z

Parasit Vectors, 2015;8:315.
PMID: 26062975 DOI: 10.1186/s13071-015-0932-0

Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens.
Epitope-based vaccine as a universal vaccination strategy against Toxoplasma gondii infection: A mini-review

Hajissa K, Zakaria R, Suppian R, Mohamed Z

J Adv Vet Anim Res, 2019 Jun;6(2):174-182.
PMID: 31453188 DOI: 10.5455/javar.2019.f329

Despite the significant progress in the recent efforts toward developing an effective vaccine against toxoplasmosis, the search for new protective vaccination strategy still remains a challenge and elusive goal because it becomes the appropriate way to prevent the disease. Various experimental approaches in the past few years showed that developing a potential vaccine against the disease can be achievable. The combination of multi-epitopes expressing different stages of the parasite life cycle has become an optimal strategy for acquiring a potent, safe, and effective vaccine. Epitope-based vaccines have gained attention as alternative vaccine candidates due to their ability of inducing protective immune responses. This mini-review highlights the current status and the prospects of Toxoplasma gondii vaccine development along with the application of epitope-based vaccine in the future parasite immunization as a novel under development and evaluation strategy.
Immunogenicity of Multiepitope Vaccine Candidate against Toxoplasma gondii Infection in BALB/c Mice

Hajissa K, Zakaria R, Suppian R, Mohamed Z

Iran J Parasitol, 2018 8 3;13(2):215-224.
PMID: 30069205

Background: Toxoplasma gondii is a widely prevalent intracellular protozoan parasite which causes serious clinical and veterinary problems. Development of an effective vaccine for controlling toxoplasmosis is an extremely important aim. In the present study, the protective efficacy of recombinant multiepitope antigen (USM.TOXO1) expressing nine potential epitopes identified from SAG1, GRA2, and GRA7 of Toxoplasma gondii was evaluated in BALB/c mice.
Methods: Mice were immunized subcutaneously with three doses of USM.TOXO1 antigen (10 μg/ml). Following the immunization, the IgG antibody, IgG subclass, IFN-γ and IL-4 production were evaluated using ELISA, the study was conducted at Animal Research and Service Center (ARASC), USM Health Campus in 2016.
Results: Mice immunized with USM.TOXO1 significantly induced a mixed Th1/Th2 response polarized toward the IgG1 antibody isotype. While the cytokine analysis revealed a significant release of IFN-γ cytokines.
Conclusion: USM.TOXO1 is a potential vaccine candidate that elicits strong immunity in BALB/c mice. The proven immunogenicity of the generated antigen can serve as a premise for further use of epitope-based vaccine in the immunoprevention of human and animal toxoplasmosis.
Increases in mRNA and DREAM protein expression in the rat spinal cord after formalin induced pain

Long I, Suppian R, Ismail Z

Neurochem Res, 2011 Mar;36(3):533-9.
PMID: 21188515 DOI: 10.1007/s11064-010-0375-0

Downstream Regulatory Element Antagonist Modulator (DREAM) protein modulates pain by regulating prodynorphin gene transcription. Therefore, we investigate the changes of mRNA and DREAM protein in relation to the mRNA and prodynorphin protein expression on the ipsilateral side of the rat spinal cord after formalin injection (acute pain model). DREAM like immunoreactivity (DLI) was not significantly different between C and F groups. However, we detected the upregulation of mean relative DREAM protein level in the nuclear but not in the cytoplasmic extract in the F group. These effects were consistent with the upregulation of the relative DREAM mRNA level. Prodynorphin like immunoreactivity (PLI) expression increased but the relative prodynorphin mRNA level remained unchanged. In conclusion, we suggest that upregulation of DREAM mRNA and protein expression in the nuclear compartment probably has functional consequences other than just the repression of prodynorphin gene. It is likely that these mechanisms are important in the modulation of pain.
Fulltext An evaluation of a recombinant multiepitope based antigen for detection of Toxoplasma gondii specific antibodies

Hajissa K, Zakaria R, Suppian R, Mohamed Z

BMC Infect Dis, 2017 12 29;17(1):807.
PMID: 29284420 DOI: 10.1186/s12879-017-2920-9

BACKGROUND: The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated.
METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.
RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.
CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.
Cloning and expression of malaria and tuberculosis epitopes in mycobacterium bovis bacille calmette-guérin

Suppian R, Zainuddin ZF, Norazmi MN

Malays J Med Sci, 2006 Jan;13(1):13-20.
PMID: 22589585

Mycobacterium bovis bacille Calmette-Guèrin (BCG) represents one of the most promising live vectors for the delivery of foreign antigens to the immune system. A recombinant BCG containing a synthetic gene coding for the malarial epitopes namely, the fragment 2 of region II of EBA-175 (F2R(II)EBA) and the repeat sequence of the circumsporozoite protein NANP generated in favour of mycobacterium codon usage using assembly PCR was constructed. Two T-cell epitopes of the 6-kDa M. tuberculosis early-secreted antigenic target (ESAT-6) antigen were also clone in the same construct. Expression of the synthetic gene was driven by the heat shock protein 65 (hsp65) promoter from M. tuberculosis and the signal peptide from the MPT63 antigen of M. tuberculosis. Expression of the composite epitopes was detected by Western blotting of the cell extract and culture supernatant of the recombinant clones using a specific rabbit polyclonal antibody against F2R(II)EBA. This study demonstrates the possibility of cloning and expressing immunogenic epitopes from causative agents of two important diseases: malaria and tuberculosis (TB) in a single recombinant BCG construct.
Fulltext Immunomodulatory Effects of Asiaticoside Against Shigella flexneri-Infected Macrophages

Michael S, Zakaria NM, Abbas MA, Abdullah H, Suppian R

Trop Life Sci Res, 2021 Jun;32(2):29-44.
PMID: 34367513 DOI: 10.21315/tlsr2021.32.2.3

Macrophages provide the first line of defense against Shigella flexneri infection in the gastrointestinal tract by inducing a variety of inflammatory and antimicrobial responses. Secondary metabolites of plants are used as drugs against infections that are resistant to common antibiotics. In this study, the innate effects of asiaticoside on the proinflammatory activity of mouse macrophages infected with S. flexneri were investigated. The viability of the infected mouse macrophages were examined using viability assay, while the pro-inflammatory cytokines productions were determined using the enzyme-linked immunosorbent assay (ELISA) for determination of IL-1β, IL-12 p40 and TNF-α levels. The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) protein were determined using the Griess assay and western blot, respectively. Statistical analyses were performed using the Statistical Package of Social Sciences (SPSS) software, version 20. The data obtained from independent experiments (n = 3) were presented as the mean ± standard error of mean (SEM). The results showed that, asiaticoside stimulated the infected macrophages by stimulating increased production of TNF-α, IL-12 p40 and NO as well as increased expression of iNOS in a dose-dependent manner. In contrast the viability of the cells and the production of IL-1β and were reduced also in a dose-dependent manner when compared to untreated cells. These results indicate that asiaticoside has immunomodulatory effects on the innate immune function of infected macrophages, showing the potential use of this compound to reduce the clinical symptoms of the infections.
Ifenprodil Reduced Expression of Activated Microglia, BDNF and DREAM Proteins in the Spinal Cord Following Formalin Injection During the Early Stage of Painful Diabetic Neuropathy in Rats

Ismail CAN, Suppian R, Ab Aziz CB, Long I

J Mol Neurosci, 2021 Feb;71(2):379-393.
PMID: 32671697 DOI: 10.1007/s12031-020-01661-1

The pharmacological inhibition of glial activation is one of the new approaches for combating neuropathic pain in which the role of glia in the modulation of neuropathic pain has attracted significant interest and attention. Neuron-glial crosstalk is achieved with N-methyl-D-aspartate-2B receptor (NMDAR-2B) activation. This study aims to determine the effect of ifenprodil, a potent noncompetitive NMDAR-2B antagonist, on activated microglia, brain-derived neurotrophic factors (BDNF) and downstream regulatory element antagonist modulator (DREAM) protein expression in the spinal cord of streptozotocin-induced painful diabetic neuropathy (PDN) rats following formalin injection. In this experimentation, 48 Sprague-Dawley male rats were randomly selected and divided into four groups: (n = 12): control, PDN, and ifenprodil-treated PDN rats at 0.5 μg or 1.0 μg for 7 days. Type I diabetes mellitus was then induced by injecting streptozotocin (60 mg/kg, i.p.) into the rats which were then over a 2-week period allowed to progress into the early phase of PDN. Ifenprodil was administered in PDN rats while saline was administered intrathecally in the control group. A formalin test was conducted during the fourth week to induce inflammatory nerve injury, in which the rats were sacrificed at 72 h post-formalin injection. The lumbar enlargement region (L4-L5) of the spinal cord was dissected for immunohistochemistry and western blot analyses. The results demonstrated a significant increase in formalin-induced flinching and licking behavior with an increased spinal expression of activated microglia, BDNF and DREAM proteins. It was also shown that the ifenprodil-treated rats following both doses reduced the extent of their flinching and duration of licking in PDN in a dose-dependent manner. As such, ifenprodil successfully demonstrated inhibition against microglia activation and suppressed the expression of BDNF and DREAM proteins in the spinal cord of PDN rats. In conclusion, ifenprodil may alleviate PDN by suppressing spinal microglia activation, BDNF and DREAM proteins.
Fulltext Imbalanced oxidative stress and pro-inflammatory markers differentiate the development of diabetic neuropathy variants in streptozotocin-induced diabetic rats

Ismail CAN, Aziz CBA, Suppian R, Long I

J Diabetes Metab Disord, 2018 Dec;17(2):129-136.
PMID: 30918846 DOI: 10.1007/s40200-018-0350-x

Purpose: Diabetic neuropathy is a prolonged symptom of diabetes mellitus that affect a number of diabetes mellitus patients. So far, the variants of diabetic neuropathy, either painful (PDN) or non-painful (or painless, non-PDN) response have distinctive clinical entities. This study aims to determine the effects of oxidative stress parameters and pro-inflammatory factors at spinal cord level of streptozotocin-induced diabetic neuropathy rat model.
Methods: Thirty Sprague-Dawley rats were randomly assigned to control (non-diabetic), PDN and non-PDN groups (n = 10). The rats were induced with diabetes by streptozotocin injection (60 mg/kg). Tactile allodynia and thermal hyperalgesia were assessed on day 0, 14 (week 2) and 21 (week 3) in the rats. The rats were sacrificed and the spinal cord tissue was collected for the measurement of oxidative stress (malondialdehyde (MDA), superoxide dismutase (SOD) and catalase) and pro-inflammatory markers (interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α)).
Results: PDN rats demonstrated a marked tactile allodynia with no thermal hyperalgesia whilst non-PDN rats exhibited a prominent hypo-responsiveness towards non-noxious stimuli and hypoalgesia towards thermal input. The MDA level and pro-inflammatory TNF-α was significantly increased in PDN rats whilst catalase was reduced in these rats. Meanwhile, non-PDN rats demonstrated reduced SOD enzyme activity and TNF-α level and increased MDA and catalase activity.
Conclusion: The changes in oxidative stress parameters and pro-inflammatory factors may contribute to the changes in behavioural responses in both PDN and non-PDN rats.
Fulltext Minocycline attenuates the development of diabetic neuropathy by modulating DREAM and BDNF protein expression in rat spinal cord

Ismail CAN, Suppian R, Aziz CBA, Long I

J Diabetes Metab Disord, 2019 Jun;18(1):181-190.
PMID: 31275889 DOI: 10.1007/s40200-019-00411-4

Aim: This study investigates the effects of minocycline (an inhibitor of microglial activation) administration on the expression level of spinal BDNF and DREAM proteins in diabetic neuropathic pain (DNP) rats.
Methods: The rats were divided into four groups (n = 16): non-diabetic control, diabetic control and diabetic rats receiving minocycline (80 μg/day or 160 μg/day). The diabetic rat model was induced by intraperitoneal injection of streptozotocin (60 mg/kg STZ). Tactile allodynia was assessed on day-0 (baseline), day-14 (pre-intervention) and day-22 (post-intervention). Minocycline at doses of 80 μg and 160 μg were given intrathecally from day-15 until day-21. On day-23, formalin test was conducted to assess nociceptive behaviour response. The spinal expression of OX-42 and level of BDNF and DREAM proteins were detected by immunohistochemistry and western blot analyses.
Results: Diabetes rats showed significant tactile allodynia and nociceptive behaviour. These were accompanied by augmented expression of spinal OX-42, BDNF and DREAM protein levels. Both doses of minocycline attenuated tactile allodynia and nociceptive behaviour and also suppressed the diabetic-induced increase in spinal expressions of OX-42, BDNF and DREAM proteins.
Conclusion: This study revealed that minocycline could attenuate DNP by modulating spinal BDNF and DREAM protein expressions.
Expressions of spinal microglia activation, BDNF, and DREAM proteins correlated with formalin-induced nociceptive responses in painful and painless diabetic neuropathy rats

Ismail CAN, Suppian R, Ab Aziz CB, Long I

Neuropeptides, 2020 Feb;79:102003.
PMID: 31902597 DOI: 10.1016/j.npep.2019.102003

The complications of diabetic polyneuropathy (DN) determines its level of severity. It may occur with distinctive clinical symptoms (painful DN) or appears undetected (painless DN). This study aimed to investigate microglia activation and signalling molecules brain-derived neurotrophic factor (BDNF) and downstream regulatory element antagonist modulator (DREAM) proteins in spinal cord of streptozotocin-induced diabetic neuropathy rats. Thirty male Sprague-Dawley rats (200-230 g) were randomly assigned into three groups: (1) control, (2) painful DN and (3) painless DN. The rats were induced with diabetes by single intraperitoneal injection of streptozotocin (60 mg/kg) whilst control rats received citrate buffer as a vehicle. Four weeks post-diabetic induction, the rats were induced with chronic inflammatory pain by intraplantar injection of 5% formalin and pain behaviour responses were recorded and assessed. Three days later, the rats were sacrificed and lumbar enlargement region of spinal cord was collected. The tissue was immunoreacted against OX-42 (microglia), BDNF and DREAM proteins, which was also quantified by western blotting. The results demonstrated that painful DN rats exhibited increased pain behaviour score peripherally and centrally with marked increase of spinal activated microglia, BDNF and DREAM proteins expressions compared to control group. In contrast, painless DN group demonstrated a significant reduction of pain behaviour score peripherally and centrally with significant reduction of spinal activated microglia, BDNF and DREAM proteins expressions. In conclusions, the spinal microglia activation, BDNF and DREAM proteins correlate with the pain behaviour responses between the variants of DN.
Fulltext Natural Gallic Acid and Methyl Gallate Induces Apoptosis in Hela Cells through Regulation of Intrinsic and Extrinsic Protein Expression

Abdullah H, Ismail I, Suppian R, Zakaria NM

Int J Mol Sci, 2023 May 09;24(10).
PMID: 37239840 DOI: 10.3390/ijms24108495

Induction of apoptosis is one of the targeted approaches in cancer therapies. As previously reported, natural products can induce apoptosis in in vitro cancer treatments. However, the underlying mechanisms of cancer cell death are poorly understood. The present study aimed to elucidate cell death mechanisms of gallic acid (GA) and methyl gallate (MG) from Quercus infectoria toward human cervical cancer cell lines (HeLa). The antiproliferative activity of GA and MG was characterised by an inhibitory concentration using 50% cell populations (IC50) by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Cervical cancer cells, HeLa, were treated with GA and MG for 72 h and calculated for IC50 values. The IC50 concentration of both compounds was used to elucidate the apoptotic mechanism using acridine orange/propidium iodide (AO/PI) staining, cell cycle analysis, the Annexin-V FITC dual staining assay, apoptotic proteins expressions (p53, Bax and Bcl-2) and caspase activation analysis. GA and MG inhibited the growth of HeLa cells with an IC50 value of 10.00 ± 0.67 µg/mL and 11.00 ± 0.58 µg/mL, respectively. AO/PI staining revealed incremental apoptotic cells. Cell cycle analysis revealed an accumulation of cells at the sub-G1 phase. The Annexin-V FITC assay showed that cell populations shifted from the viable to apoptotic quadrant. Moreover, p53 and Bax were upregulated, whereas Bcl-2 was markedly downregulated. Activation of caspase 8 and 9 showed an ultimate apoptotic event in HeLa cells treated with GA and MG. In conclusion, GA and MG significantly inhibited HeLa cell growth through apoptosis induction by the activation of the cell death mechanism via extrinsic and extrinsic pathways.
Reduced nitric oxide-mediated relaxation and endothelial nitric oxide synthase expression in the tail arteries of streptozotocin-induced diabetic rats

Mokhtar SS, Vanhoutte PM, Leung SW, Suppian R, Yusof MI, Rasool AH

Eur J Pharmacol, 2016 Feb 15;773:78-84.
PMID: 26825543 DOI: 10.1016/j.ejphar.2016.01.013

Diabetes is associated with endothelial dysfunction, which is characterized by impaired endothelium-dependent relaxations. The present study aimed to examine the role of nitric oxide (NO), prostacyclin and endothelium-dependent hyperpolarization (EDH), in the relaxation of ventral tail arteries of rats under diabetic conditions. Relaxations of tail arteries of control and diabetic rats were studied in wire myograph. Western blotting and immunostaining were used to determine the presence of proteins. Acetylcholine-induced relaxations were significantly smaller in arteries of diabetic compared to control rats (Rmax; 70.81 ± 2.48% versus 85.05 ± 3.15%). Incubation with the combination of non-selective cyclooxygenase (COX) inhibitor, indomethacin and potassium channel blockers, TRAM 34 and UCL 1684, demonstrated that NO-mediated relaxation was attenuated significantly in diabetic compared to control rats (Rmax; 48.47 ± 5.84% versus 68.39 ± 6.34%). EDH-type (in the presence of indomethacin and NO synthase inhibitor, LNAME) and prostacyclin-mediated (in the presence of LNAME plus TRAM 34 and UCL 1684) relaxations were not significantly reduced in arteries of diabetic compared to control rats [Rmax: (EDH; 17.81 ± 6.74% versus 34.16 ± 4.59%) (prostacyclin; 15.85 ± 3.27% versus 17.23 ± 3.75%)]. Endothelium-independent relaxations to sodium nitroprusside, salbutamol and prostacyclin were comparable in the two types of preparations. Western blotting and immunostaining indicated that diabetes diminished the expression of endothelial NO synthase (eNOS), while increasing those of COX-1 and COX-2. Thus, since acetylcholine-induced NO-mediated relaxation was impaired in diabetes because of reduced eNOS protein expression, pharmacological intervention improving NO bioavailability could be useful in the management of diabetic endothelial dysfunction.
Fulltext Minocycline alleviates nociceptive response through modulating the expression of NR2B subunit of NMDA receptor in spinal cord of rat model of painful diabetic neuropathy

Ismail CAN, Ghazali AK, Suppian R, Abd Aziz CB, Long I

J Diabetes Metab Disord, 2021 Jun;20(1):793-803.
PMID: 34178864 DOI: 10.1007/s40200-021-00820-4

Background: It has been reported that neuropathic pain can be overcome by targeting the NR2B subunit of N-methyl-D-aspartate receptors (NR2B). This study aimed to investigate the effects of minocycline on phosphorylated and total expression of NR2B in the spinal cord of rats with diabetic neuropathic pain.
Methods: A total of 32 Sprague-Dawley male rats were randomly assigned into four groups (n = 8); control healthy, control diabetic (PDN), and PDN rats that received 80 µg or 160 µg intrathecal minocycline respectively. The rats were induced to develop diabetes and allowed to develop into the early phase of PDN for two weeks. Hot-plate and formalin tests were conducted. Intrathecal treatment of minocycline or normal saline was conducted for 7 days. The rats were sacrificed to obtain the lumbar enlargement region of the spinal cord (L4-L5) for immunohistochemistry and western blot analyses to determine the expression of phosphorylated (pNR2B) and total NR2B (NR2B).
Results: PDN rats showed enhanced flinching (phase 1: p
Lower Formalin-Induced Pain Responses in Painless Diabetic Neuropathy Rat Correlate with the Reduced Spinal Cord NR2B Subunit of N-Methyl-D-Aspartate Receptor Activation

Ismail CAN, Ghazali AK, Suppian R, Abd Aziz CB, Long I

J Mol Neurosci, 2021 Nov 02.
PMID: 34727325 DOI: 10.1007/s12031-021-01929-0

Diabetic neuropathy (DN) is a late complication of diabetic mellitus and may rise into painful and painless variants. Limited studies have looked at nociceptive mechanisms of painless DN variant. The study aimed to determine phosphorylation and total NR2B subunit of N-methyl-D-aspartate receptor in the spinal cord of painless DN rat during early phase following formalin injection. Thirty-six Sprague-Dawley male rats were randomly assigned into three groups: control, painful, and painless DN (n = 12). The rats were developed into the early phase of DN for 2 weeks following diabetic induction. Two weeks later, the rats were injected with 5% formalin solution and flinching and licking responses were recorded for 60 min. The rats were sacrificed 3 days later, and the spinal cord enlargement region was collected. Immunohistochemistry and Western blot procedures were conducted to determine the phosphorylated and total NR2B subunit expressions. The results showed reduced flinching and licking responses in painless DN rats compared to control and painful DN groups, followed by a significant reduction in phosphorylated and total NR2B expression at both ipsilateral and contralateral regions of the spinal cord. In conclusion, reduced pain behavior responses in painless DN rats following formalin injection is possibly contributed by the reduced expression of phosphorylated and total NR2B subunit in the spinal cord.

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