Intrathecal lactate dehydrogenase A inhibitors FX11 and oxamate alleviate chronic constriction injury-induced nociceptive sensitization through neuroinflammation and angiogenesis

Abstract

Background: Neuropathic pain involves neuroinflammation and upregulation of glycolysis in the central nervous system. Unfortunately, few effective treatments are available for managing this type of pain. The overactivation of lactate dehydrogenase A (LDHA), an essential enzyme in glycolysis, can cause neuroinflammation and nociception. This study investigated the spinal role of LDHA in neuropathic pain.

Method: Using immunohistochemical analysis, nociceptive behavior, and western blotting, we evaluated the cellular mechanisms of intrathecal administration of LDHA inhibitors, including FX11 and oxamate, in chronic constriction injury (CCI)-induced neuropathic rats.

Result: FX11 and oxamate attenuated CCI-induced neuronal LDHA upregulation and nociceptive sensitization. Moreover, CCI-induced neuroinflammation, microglial polarization, and angiogenesis were attenuated by LDHA inhibitors. These inhibitors regulate the TANK binding kinase-1 (TBK1)/hypoxia-inducible factor 1 subunit alpha (HIF-1α) axis, crucial for controlling inflammation and new blood vessel growth. Additionally, CCI-induced nuclear LDHA translocation, as associated with oxidative stress resistance, was attenuated by LDHA inhibitors.

Conclusion: In conclusion, LDHA may be a potential therapeutic target for treating neuropathic pain by regulating neuroinflammation and angiogenesis.

Keywords: Angiogenesis; Lactate dehydrogenase A; Neuroinflammation; Neuropathic pain.

Fig. 1

CCI-induced upregulation of LDHA in the spinal cord. The ipsilateral SCDHs were harvested 3, 7, 14, 21, and 28 d after CCI. a Immunofluorescence staining of LDHA expression in the ipsilateral SCDH. b Quantification of LDHA immunoreactivity within lamina I to III in the ipsilateral lumbar SCDH. CCI significantly upregulated LDHA expression 7 and 14 d after CCI. Data are presented as the mean ± SEM, n = 3–4 in each group. *P < 0.05, compared with the control group. Scale bar: 100 µm

Fig. 2

The analgesic effects of intrathecal administration of LDHA inhibitors, FX11 and oxamate, on CCI-induced nociception. The dose–response curve for the intrathecal administration of (a) FX11 (0.05, 0.1, 0.25, and 1 µg) and (b) oxamate (1, 5, 10, 50, and 100 µg) assessed via paw withdrawal latency transformed into the percentage of maximum possible effect (MPE%) in the anti-thermal hyperalgesia assay. c Mechanical allodynia and (d) thermal hyperalgesia were examined using paw withdrawal threshold (g) and paw withdrawal latency, respectively, on rats with an intrathecal chronic infusion of 0.1 µg/h of FX11 or 5 µg/h of oxamate using an osmotic pump. LDHA inhibitors exhibited significant analgesic effects on CCI-induced nociceptive sensitization. Data are presented as the mean ± SEM, n = 6 in each group. * and #P < 0.05, were compared with the control and CCI groups, respectively

Fig. 3

Intrathecal administration of LDHA inhibitors inhibits CCI-induced neuronal LDHA upregulation in the ipsilateral SCDH. Rats received vehicle, FX11 (0.1 µg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. The ipsilateral lumbar SCDHs were harvested 7 and 14 d after CCI. a Immunofluorescence staining of LDHA expression in the ipsilateral SCDH. Scale bar: 100 µm. b Quantification of LDHA immunoreactivity within lamina I to III in the ipsilateral lumbar SCDH. c Results for western blot analysis of relative cytosolic LDHA protein expression in the ipsilateral spinal cord. d Immunohistochemical localization and distribution of LDHA in the ipsilateral lumbar SCDH. Immunoreactive co-localization of the red signal for LDHA and the green signal for the astrocyte-specific marker glial fibrillary acidic protein (GFAP), microglia-specific maker ionized calcium-binding adaptor molecule 1 (Iba1), and neuronal-specific marker (NeuN) in the ipsilateral lumbar SCDH in the control, 7d, and 14d groups. The merged images (yellow) indicate the main co-localization of LDHA with NeuN immunoreactive cells. Scale bar: 25 µm. e Pearson’s correlation coefficient results for quantifying the co-localization between LDHA and GFAP, Iba1, or NeuN via double immunofluorescence staining. LDHA inhibitors significantly mitigate CCI-induced neuronal LDHA upregulation in the ipsilateral SCDH. *P < 0.05, compared with control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group

Fig. 4

Intrathecal administration of LDHA inhibitors inhibits CCI-induced spinal microglia and astrocyte activation. Rats received vehicle (1 µL/h), FX11 (0.1 μg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. The ipsilateral lumbar SCDHs were harvested 7 and 14 d after CCI. a Immunofluorescence staining of microglia-specific maker ionized calcium-binding adaptor molecule 1 (Iba1, green) and astrocyte-specific marker glial fibrillary acidic protein (GFAP, green) in the ipsilateral SCDH. Quantification of (b) Iba1 and (c) GFAP immunoreactivity within lamina I to lamina III in the ipsilateral lumbar SCDH. CCI-induced Iba1 and GFAP activation attenuated via intrathecal administration of LDHA inhibitors. *P < 0.05, compared with control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group. Scalebar: 100 µm

Fig. 5

Intrathecal administration of LDHA inhibitors reduces CCI-induced spinal M1 microglia upregulation. Rats received vehicle (1 µL/h), FX11 (0.1 μg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. The ipsilateral lumbar SCDHs were harvested 7 and 14 d after CCI. a Immunofluorescence staining of CD86 (M1 microglia, red) expression and the co-localization (insert, yellow) of Iba1 and CD86 in the ipsilateral SCDH. The inserted merged images (yellow) indicate the co-localization of CD86 with microglia-specific maker-ionized calcium-binding adaptor molecule 1 (Iba1) in the ipsilateral SCDH. Scale bar: 100 and 10 µm (insert). b Quantification of CD86 immunoreactivity within lamina I to IV in the ipsilateral lumbar SCDH. c Western blotting analysis of relative CD86 protein expression in the ipsilateral spinal cord. LDHA inhibitors mitigate CCI-induced M1 microglia upregulation. *P < 0.05, compared with control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group

Fig. 6

Intrathecal administration of LDHA inhibitors downregulate CCI-induced neuronal phosphor-TBK1 upregulation. Rats received vehicle (1 µL/h), FX11 (0.1 µg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. The ipsilateral lumbar SCDHs were harvested 7 and 14 d after CCI. a Immunofluorescence staining of phosphorylated TBK1 (pTBK1) expression in the ipsilateral SCDH. Scale bar: 100 µm. b Quantification of pTBK1 immunoreactivity within lamina I to III in the ipsilateral lumbar SCDH. c Western blot analysis of relative pTBK1 expression in the ipsilateral spinal cord. d Immunohistochemical localization and distribution of pTBK1 in the ipsilateral lumbar SCDH. Immunoreactive co-localization of the red signal for pTBK1 and the green signal for the astrocyte-specific marker glial fibrillary acidic protein (GFAP), microglia-specific maker ionized calcium-binding adaptor molecule 1 (Iba1), and neuronal-specific marker (NeuN) in the ipsilateral lumbar SCDH in the control, 7d, and 14d groups. The merged images (yellow) indicate the main co-localization of pTBK1 with NeuN immunoreactive cells. Scale bar: 25 µm. e Pearson’s correlation coefficient results for quantifying the co-localization between pTBK1 and GFAP, NeuN, or Iba1 via double immunofluorescence staining. LDHA inhibitors significantly alleviate CCI-induced neuronal pTBK1 expression in the ipsilateral SCDH. *P < 0.05, compared with control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group

Fig. 7

Intrathecal administration of LDHA inhibitors downregulate CCI-induced neuronal HIF-1α upregulation. Rats received vehicle (1 µL/h), FX11 (0.1 µg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. The ipsilateral lumbar SCDHs were harvested 7 and 14 d after CCI. a Immunofluorescence staining of HIF-1α expression in the ipsilateral SCDH. Scale bar: 100 µm. b Quantification of HIF-1α immunoreactivity within lamina I to III in the ipsilateral lumbar SCDH. c Immunohistochemical localization and distribution of HIF-1α in the ipsilateral lumbar SCDH. Immunoreactive co-localization of the red signal for HIF-1α and the green signal for the astrocyte-specific marker glial fibrillary acidic protein (GFAP), microglia-specific maker ionized calcium-binding adaptor molecule 1 (Iba1), and neuronal-specific marker (NeuN) in the ipsilateral lumbar SCDH in the control, 7d, and 14d groups. The merged images (yellow) indicate the main co-localization of HIF-1α with NeuN immunoreactive cells. Scale bar: 25 µm. d Pearson’s correlation coefficient results for quantifying the co-localization between HIF-1α and GFAP, NeuN, or Iba1 via double immunofluorescence staining. LDHA inhibitors attenuate CCI-induced HIF-1α expression in the ipsilateral SCDH. *P < 0.05, compared with the control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group

Fig. 8

LDHA inhibitors attenuate CCI-induced spinal Ang2 and CD31 upregulation. Rats were intrathecally administered vehicle (1 µL/h), FX11 (0.1 µg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. Spinal tissue sections were harvested 7 and 14 d after CCI. a Immunofluorescence staining of Ang2 and CD31 expression in the ipsilateral SCDH. Quantification of (b) Ang2 and (c) CD31 immunoreactivity within lamina I to III in the ipsilateral lumbar SCDH. LDHA inhibitors attenuate CCI-induced Ang2 and CD31 upregulation. *P < 0.05, compared with the control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group. Scale bar: 100 µm

Fig. 9

Intrathecal administration of LDHA inhibitors mitigate CCI-induced neuronal nuclear LDHA upregulation. Rats received vehicle (1 µL/h), FX11 (0.1 µg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. The ipsilateral lumbar SCDHs were harvested 7d and 14d after CCI. a Immunofluorescent microscopy captured double immunofluorescence staining of the nucleus (DAPI, blue) and LDHA (red) in the ipsilateral SCDH and the Mander’s correlation coefficient result of nuclear LDHA expression. b Triple immunofluorescence staining of the nucleus (DRAQ5, blue), neuronal-specific marker (NeuN, green), and LDHA (red) in the ipsilateral lumbar SCDH were captured using the confocal system. c Western blot analysis of relative nuclear LDHA expression in the ipsilateral spinal cord after CCI. LDHA inhibitors attenuate CCI-induced neuronal nuclear LDHA upregulation in the ipsilateral lumbar SCDH *P < 0.05, compared with the control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14d group. Scale bar: 25 µm

Fig. 10

Intrathecal administration of LDHA inhibitors reduce CCI-induced spinal superoxide dismutase 1 (SOD1) expression. Rats received vehicle (1 µL/h), FX11 (0.1 µg/h), or oxamate (5 µg/h) for 7 or 14 d after CCI surgery using osmotic pumps. Spinal cord tissue sections were harvested 7 and 14d after CCI. a Immunofluorescence staining of SOD1 expression in the ipsilateral SCDH. b Quantification of SOD1 immunoreactivity within lamina I to III in the ipsilateral lumbar SCDH. c Western blot analysis of relative SOD1 expression in the ipsilateral spinal cord. LDHA inhibitors alleviate CCI-induced SOD1 upregulation in the ipsilateral SCDH. *P < 0.05, compared with the control group; #P < 0.05, compared with the 7d group; $P < 0.05, compared with the 14 d group. Scale bar: 100 µm

Fig. 11

The hypothetical graphic illustrating how LDHA regulates neuroinflammation and angiogenesis in CCI-induced neuropathic pain