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. 2024 Mar 19;16(2):370-379.
doi: 10.3390/neurolint16020027.

Hyperglycaemia Aggravates Oxidised Low-Density Lipoprotein-Induced Schwann Cell Death via Hyperactivation of Toll-like Receptor 4

Wataru Nihei  1 , Ayako Kato  1 , Tatsuhito Himeno  2 , Masaki Kondo  2 , Jiro Nakamura  3 , Hideki Kamiya  2 , Kazunori Sango  4 , Koichi Kato  1
Affiliations

Hyperglycaemia Aggravates Oxidised Low-Density Lipoprotein-Induced Schwann Cell Death via Hyperactivation of Toll-like Receptor 4

Wataru Nihei et al. Neurol Int. .

Abstract

Increased low-density lipoprotein levels are risk factors for diabetic neuropathy. Diabetes mellitus is associated with elevated metabolic stress, leading to oxidised low-density lipoprotein formation. Therefore, it is important to investigate the mechanisms underlying the pathogenesis of diabetic neuropathy in diabetes complicated by dyslipidaemia with increased levels of oxidised low-density lipoprotein. Here, we examined the effects of hyperglycaemia and oxidised low-density lipoprotein treatment on Schwann cell death and its underlying mechanisms. Immortalised mouse Schwann cells were treated with oxidised low-density lipoprotein under normo- or hyperglycaemic conditions. We observed that oxidised low-density lipoprotein-induced cell death increased under hyperglycaemic conditions compared with normoglycaemic conditions. Moreover, hyperglycaemia and oxidised low-density lipoprotein treatment synergistically upregulated the gene and protein expression of toll-like receptor 4. Pre-treatment with TAK-242, a selective toll-like receptor 4 signalling inhibitor, attenuated hyperglycaemia- and oxidised low-density lipoprotein-induced cell death and apoptotic caspase-3 pathway. Our findings suggest that the hyperactivation of toll-like receptor 4 signalling by hyperglycaemia and elevated oxidised low-density lipoprotein levels synergistically exacerbated diabetic neuropathy; thus, it can be a potential therapeutic target for diabetic neuropathy.

Keywords: OxLDL; TLR4; apoptosis; diabetic neuropathy.

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Conflict of interest statement

Hideki Kamiya received lecture fees from Daiichi Sankyo, Eli Lilly Japan, Kissei Pharmaceutical, Kowa, Mitsubishi Tanabe Pharma, MSD, Nippon Boehringer Ingelheim, Novartis Pharma, Novo Nordisk Pharma, Ono Pharmaceutical, Sanofi, Sanwa Kagaku Kenkyusho, and Sumitomo Pharma. Jiro Nakamura received lecture fees from Daiichi Sankyo, MSD, Novartis Pharma, Novo Nordisk Pharma, Ono Pharmaceutical, Sanofi, Taisho Pharmaceutical, Takeda Pharmaceutical, and Terumo. Hideki Kamiya and Jiro Nakamura received research funding from Eli Lilly Japan, Kissei Pharmaceutical, and Ono Pharmaceutical. Koichi Kato received research funding from Eli Lilly. Hideki Kamiya and Jiro Nakamura received donations from Japan Tobacco, Mitsubishi Tanabe Pharma, MSD, Novo Nordisk Pharma, Ono Pharmaceutical, Sumitomo Pharma, Taisho Pharmaceutical, and Takeda Pharmaceutical. Hideki Kamiya and Jiro Nakamura received endowed departments by commercial entities from Abbot Japan, Kowa, Ono Pharmaceutical, Sanwa Kagaku Kenkyusho, and Terumo.

Figures

Figure 1
Figure 1
Hyperglycaemia potentiates oxLDL-induced cell death. IMS32 cells were cultured in either normoglycaemic (5.5 mM glucose) or hyperglycaemic (25 mM glucose) conditions in the presence of oxLDL (150 and 300 g/mL) for 24 h. Cell viability was determined by the MTT assay (n = 5 per group). Data are representative of at least three independent experiments, mean ± SE. *** p < 0.001, **** p < 0.0001, comparisons between indicated groups. IMS32, immortalised mouse Schwann; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; n.s., not significant; oxLDL, oxidised low-density lipoprotein; SE, standard error.
Figure 2
Figure 2
Expression and localisation of TLR4 in IMS32 cells. (A) TLR4 mRNA expression in IMS32 cells. (B) IMS32 cells were grown on coverslips for 24 h. The cells were fixed with 4% paraformaldehyde, permeabilised, and immunostained with anti-TLR4 antibody (green). The nuclei were stained with DAPI (blue). DIC, differential interference contrast scale bar, 5 μm.
Figure 3
Figure 3
Hyperglycaemia and oxLDL synergistically upregulate TLR4 expression in IMS32 cells. IMS32 cells were cultured in either normo- or hyperglycaemic conditions in the presence or absence of oxLDL (150 μg/mL) for 24 h. (A) Relative mRNA expressions of TLR4 were tested by RT-qPCR (n = 6 per group). (B) Immunoblot analysis of TLR4 expression (n = 12 per group). Data are presented as the means of two (A) or three independent experiments. * p < 0.05, ** p < 0.01. IMS32, immortalised mouse Schwann; oxLDL, oxidised low-density lipoprotein; RT-qPCR, quantitative reverse transcription polymerase chain reaction; TLR4, toll-like receptor 4.
Figure 4
Figure 4
TLR4 inhibition attenuated the hyperglycaemia and oxLDL-mediated cell death. IMS32 cells were pre-treated with or without TAK-242 (100 nM) for 2 h and cultured in normo- or hyperglycaemic conditions in the presence or absence of oxLDL (150 μg/mL) for 24 h. Cell viability was quantified by the MTT assay (n = 5 per group). Data are representative of at least three independent experiments. * p < 0.05, ** p < 0.01, comparisons between indicated groups. IMS32, immortalised mouse Schwann; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; n.s., not significant; oxLDL, oxidised low-density lipoprotein; TLR4, toll-like receptor 4.
Figure 5
Figure 5
TLR4 inhibition suppressed caspase-3-dependent apoptosis. IMS32 cells were pre-treated with or without TAK-242 (100 nM) for 2 h and cultured in either normo- or hyperglycaemic conditions in the presence or absence of oxLDL (150 μg/mL) for 3 h. Caspase-3 activity was measured. * p < 0.05, ** p < 0.01, comparisons between indicated groups. n.s., not significant; IMS32, immortalised mouse Schwann; TLR4, toll-like receptor 4.
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