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. 2021 Feb 27:2021:8854461.
doi: 10.1155/2021/8854461. eCollection 2021.

Impact of Hepatoma-Derived Growth Factor Blockade on Resiniferatoxin-Induced Neuropathy

Chieh-Hsin Wu  1   2 , Ming-Kung Wu  3 , Chun-Ching Lu  4   5 , Hung-Pei Tsai  1 , Ying-Yi Lu  6   7   8 , Chih-Lung Lin  1   2
Affiliations

Impact of Hepatoma-Derived Growth Factor Blockade on Resiniferatoxin-Induced Neuropathy

Chieh-Hsin Wu et al. Neural Plast. .

Abstract

Resiniferatoxin is an ultrapotent capsaicin analog that mediates nociceptive processing; treatment with resiniferatoxin can cause an inflammatory response and, ultimately, neuropathic pain. Hepatoma-derived growth factor, a growth factor related to normal development, is associated with neurotransmitters surrounding neurons and glial cells. Therefore, the study aims to investigate how blocking hepatoma-derived growth factor affects the inflammatory response in neuropathic pain. Serum hepatoma-derived growth factor protein expression was measured via ELISA. Resiniferatoxin was administrated intraperitoneally to induce neuropathic pain in 36 male Sprague-Dawley rats which were divided into three groups (resiniferatoxin+recombinant hepatoma-derived growth factor antibody group, resiniferatoxin group, and control group) (n = 12/group). The mechanical threshold response was tested with calibration forceps. Cell apoptosis was measured by TUNEL assay. Immunofluorescence staining was performed to detect apoptosis of neuron cells and proliferation of astrocytes in the spinal cord dorsal horn. RT-PCR technique and western blot were used to measure detect inflammatory factors and protein expressions. Serum hepatoma-derived growth factor protein expression was higher in the patients with sciatica compared to controls. In resiniferatoxin-group rats, protein expression of hepatoma-derived growth factor was higher than controls. Blocking hepatoma-derived growth factor improved the mechanical threshold response in rats. In dorsal root ganglion, blocking hepatoma-derived growth factor inhibited inflammatory cytokines. In the spinal cord dorsal horn, blocking hepatoma-derived growth factor inhibited proliferation of astrocyte, apoptosis of neuron cells, and attenuated expressions of pain-associated proteins. The experiment showed that blocking hepatoma-derived growth factor can prevent neuropathic pain and may be a useful alternative to conventional analgesics.

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Conflict of interest statement

All the authors declare they have no conflicts of interest.

Figures

Figure 1
Figure 1
Serum HDGF is expressed higher in sciatica patients and rHDGF antibody increases pain threshold in RTX-treated rats. (a) Comparison of serum HDGF protein expression between sciatica patients and normal controls after ELISA analysis. The values detected from individuals were drawn as dots. The expression levels were shown as scattered plots; the middle line demonstrated the mean value. ∗∗P < 0.01 compared with normal controls. (b) The PC-12 cells were incubated with different concentrations of rHDGF antibody (0, 10, 20, 50, and 100 nM) and 500 μM H2O2. Values are expressed as percentages of viable cells. ∗∗P < 0.01, ∗∗∗P < 0.001 compared with control group (without rHDGF antibody and H2O2). ##P < 0.01 compared with the control group treated with 500 μM H2O2. Data are expressed as the mean + SEM (n = 6). (c) After the rats were divided into three groups, neuropathy was induced by intraperitoneally administration of a single dose of RTX (50 μg/kg). In the RTX+rHDGF antibody group, 100 μg/kg rHDGF antibody was administered and applied 24 hours before RTX injection. The control group received no rHDGF antibody pretreatment and no RTX injection. Calibrated forceps test was used to test the mechanical threshold for pain before and 1, 2, 3, and 7 days after RTX injection. ∗∗P < 0.01, compared with control group. ++P < 0.01 compared with RTX+rHDGF antibody group. Data are expressed as the mean + SEM (n = 6 per group).
Figure 2
Figure 2
Effect of rHDGF antibody on astrocytes in the L3/L5 spinal cord dorsal horn (laminae I-II) of RTX-treated rats. After the rats were divided into three groups, neuropathy was induced by intraperitoneally administration of a single dose of RTX (50 μg/kg). In the RTX+rHDGF antibody group, 100 μg/kg rHDGF antibody was administered and applied 24 hours before RTX injection. The control group received no rHDGF antibody pretreatment and no RTX injection. At 3 or 7 days after RTX injection and/or pretreated with rHDGF antibody, L3-L5 spinal cord dorsal horn were harvested, and the proliferation of astrocytes (GFAP) was detected through immunofluorescence staining (200x magnification). (a) Representative immunofluorescence staining image shown are from 3 to 5 sections stained at least from 6 rats for each group. White rectangle in (a) indicates the astrocyte morphology for high magnifications. (b) Changes in intensity. ∗∗P < 0.01, ∗∗∗P < 0.001. Data are expressed as the mean + SEM (n = 6 per group).
Figure 3
Figure 3
Effect of rHDGF antibody on inflammatory cytokines in RTX-treated rats. After the rats were divided into three groups, neuropathy was induced by intraperitoneally administration of a single dose of RTX (50 μg/kg). In the RTX+rHDGF antibody group, 100 μg/kg rHDGF antibody was administered and applied 24 hours before RTX injection. The control group received no rHDGF antibody pretreatment and no RTX injection. At 3 or 7 days after RTX injection and/or pretreated with rHDGF antibody, L3-L5 dorsal root ganglion and spinal cord dorsal horn were harvested. Inflammatory cytokines were examined through RT-PCR or western blot analysis. (a) Gene expression of TNF-α and (b) IL-1β in dorsal root ganglion of rats. Gene expressions are ratios relative to GAPDH. ∗P < 0.05. (c) iNOS expression in the spinal cord dorsal horn of rats. Representative Western blot results was shown. Expression levels were normalized to β-actin. ∗P < 0.05. Data are expressed as the mean + SEM (n = 6 per group).
Figure 4
Figure 4
Effect of rHDGF antibody on neuron cells in the L3/L5 spinal cord dorsal horn of RTX-treated rats. After the rats were divided into three groups, neuropathy was induced by intraperitoneally administration of a single dose of RTX (50 μg/kg). In the RTX+rHDGF antibody group, 100 μg/kg rHDGF antibody was administered and applied 24 hours before RTX injection. The control group received no rHDGF antibody pretreatment and no RTX injection. At 3 or 7 days after RTX injection and/or pretreated with rHDGF antibody, L3-L5 spinal cord dorsal horn were harvested, and the expression of apoptotic neurons were evaluated through immunofluorescence staining by TUNEL assay (green), neuron cell marker (Neu-N) (Red), and DAPI (blue) (400x magnification). (a) Representative immunofluorescence staining image shown are from 3 to 5 sections stained at least from 6 rats for each group. Apoptotic neurons visualized (indicated by arrows) in the dorsal horn of the L3/L5 spinal cord are also shown. (b) The numbers of apoptotic neurons per animal were shown. ∗∗∗P < 0.001. Data are expressed as the mean + SEM (n = 6 per group).
Figure 5
Figure 5
Effect of rHDGF antibody on expressions of HDGF, p-Akt/Akt, PI3K, substance P, and TrkB in the L3/L5 spinal cord dorsal horn of RTX-rats. After the rats were divided into three groups, neuropathy was induced by intraperitoneally administration of a single dose of RTX (50 μg/kg). In the RTX+rHDGF antibody group, 100 μg/kg rHDGF antibody was administered and applied 24 hours before RTX injection. The control group received no rHDGF antibody pretreatment and no RTX injection. At 3 or 7 days after RTX injection and/or pretreated with rHDGF antibody, L3-L5 spinal cord dorsal horn were harvested and protein expressions were measured through western blot analysis. (a) Representative Western blot results. (b) Expression levels were normalized to β-actin. ∗P < 0.05, ∗∗P < 0.01. Data are expressed as the mean + SEM (n = 6 per group).
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