Eight-week-old CD-1 mice (male) were obtained from GemPharmatech (Nanjing, China). All mice were housed in polypropylene cages under 12–12 hour light-dark cycles at 24 °C with 70% humidity. Mice were fed with standard chow diet and sterile filtered water. The animal study and treatment protocols were approved by Quanzhou First Hospital Affiliated to Fujian Medical University. The CIPN mouse model was established as described previously [19]. Mice were divided into six different groups randomly. One group of mice was intraperitoneally (i.p.) injected with saline, which was set as normal control (NC). The mice in CIPN groups were injected 2 mg/kg/day Paclitaxel (PTX, #T7191, Sigma, St. Louis, MO, USA) from Day 0 to Day 6. The daily administration of Apigenin (#10798, Sigma, St. Louis, MO, USA) was started at Day 4. Three doses (25, 50, 75 mg/kg) were used in this study. The treatment ended at Day 20. Gabapentin (GABA, #1287303, Sigma, St. Louis, MO, USA) injection (100 mg/kg) was set as positive control. The pain behaviors of treated mice were monitored during the treatment, and surgical spinal cord tissues were collected after the treatment for the following analysis.

The basal pain behaviors of the normal control and CIPN mice related to allodynia and hyperalgesia were assessed by hot plate and tail prick latency tests. Hot plate test was carried out to measure the response latencies by using the Perspex cylinder as described previously [20, 21]. Tail flick test was carried out to assess the anti-nociceptive effect of certain treatment through detecting the response latencies. The plantar test device (7370 plantar test) was used to performed tail flick test as described previously [22, 23]. Tests were carried out on Day 0 (started PTX administration), Day 6, Day 11, and Day 18.

Mice were euthanized using CO${}_2$ and spinal cord was isolated through hydraulic extrusion method. In brief, the fur was cut open along the spinal column in a distal direction. The spinal column was isolated by cutting on both sides along the spinal column past the pelvic bone. Then, the spinal column was straightened. Phosphate-buffered saline was inserted into the canal through a 18G cannula. The isolated spinal tissue was homogenized using liquid nitrogen.

The inflammatory factors (TNF-$\alpha$ and IL-1$\beta$) and oxidative stress-related parameters (malondialdehyde (MDA), oxidized glutathione (GSSG), ratio of glutathione (GSH) and GSSG) in surgical spinal cord tissues were determined using the commercial kits ordered from Beyotime Biotechnology (Shanghai, China) following the protocols provided by the manufacturer.

Total RNA was extracted from the surgical spinal cord tissues, and isolated with Beyotime RNAeasy™ Kit (Beyotime, Shanghai, China). The cDNA was synthesized using the Vazyme Script RT SuperMix (Vazyme, Nanjing, China). ABI StepOne™ System (Applied Biosystems, Foster City, CA, USA) was used to perform qRT-PCR with Vazyme SYBR Green Master Mix (Vazyme, Nanjing, China). The 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{CT}}$ method was used to calculate the relative expression levels of target genes.

Total protein of surgical spinal cord tissue was extracted using RIPA buffer containing phosphatase/protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Nuclear and cytoplasmic protein was extracted by using the nuclear extract kit (Active Motif, Shanghai, China). The 12% SDS-PAGE gel and nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) were used for Western blot as described previously [24]. Primary antibodies of CD11b (ab133357, 1:1000 dilution in blocking buffer (5% nonfat milk)), CD68 (ab283654, 1:1000 dilution), and CD206 (ab64693, 1:1000 dilution) were purchased from Abcam (Cambridge, MA, USA). Primary antibodies of NRF2 (#12721, 1:1000 dilution), HDAC1 (#34589, 1:1500 dilution), $\alpha$-Tubulin (#2125, 1:3000 dilution), and GAPDH (#5174, 1:1500 dilution) were ordered from Cell Signaling (Danvers, MA, USA). The corresponding second antibodies, rabbit (611-005-215, 1:2000 dilution) and goat (805-005-180, 1:2000 dilution), were purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA).

The Prism 8.0 (GraphPad, San Diego, CA, USA) was used to assess significant differences among different treated groups in this study. Results were shown as mean $\pm$ standard deviation (SD). Differences between treatments were analyzed by a one-way or two-way analysis of variance (ANOVA) followed by a post hoc test.

To investigate the therapeutic effects of Apigenin on chemotherapy-induced pain, we established the CIPN mouse model using the PTX treatment as described previously [8], and then performed hot plate and tail prick latency tests. As shown in Fig. 1A, CIPN reduced latency to half in all treated groups except the NC group which did not receive PTX chemotherapy at Day 6. Latency increased significantly in the CIPN-treated groups over time. At Day 11, the latency of GABA group (as a positive control) and Apigenin treated group increased significantly compared to that of disease control (DC) group (Fig. 1A). At Day 18, the latency of high dose of Apigenin treated group significantly increased compared to DC group. The group receiving the medium dose of Apigenin had increase in latency during this period too, which indicated the dose-dependent effect of Apigenin administration in hot plate test. To further assess the modulation of pain sensation and relief of mechanical hyperalgesia by Apigenin treatment, we performed tail prick latency test, and similar results to hot plate test were observed (Fig. 1B).

Fig. 1.

Apigenin alleviated CIPN-induced nociceptive behavior of CIPN mice. Nociceptive behavior was determined by the latency time measured by hot plate (A) and Tail Flick (B). *p 0.05, **p 0.01, ***p 0.001.

Next, we examined the effects of Apigenin treatment on the levels of the pro-inflammatory cytokines TNF-$\alpha$ and IL-1$\beta$ in the spinal cord of control and CIPN mice. Compared with the NC group, on Day 7, the TNF-$\alpha$ level of DC mice was significantly higher, while TNF-$\alpha$ level was significantly decreased in GABA and high Apigenin treated groups (Fig. 2A). Interestingly, Apigenin treatment decreased TNF-$\alpha$ level in a dose-dependent pattern, which were all statistically significant with three doses and were more obvious on Day 14 (Fig. 2A). Similar trend of IL-1$\beta$ level was obtained after Apigenin treatment (Fig. 2B). These results indicated that Apigenin reduced the production of inflammatory cytokines in CIPN mice.

Fig. 2.

Anti-inflammatory effect of Apigenin treatment was evaluated through the measurements of TNF-$\alpha$ (A) and IL-1$\beta$ (B) in the spinal cord of treated mice. N = 6 in all groups. Data are represented as mean $\pm$ SD. ###p 0.001, when significantly different in comparison to NC; *p 0.05, **p 0.01, ***p 0.001, when significantly different in comparison to DC. Api, Apigenin; NC, normal control; DC, disease control; GABA, Gabapentin.

The elevated oxidative stress is another important feature of CIPN [25]. Therefore, we examined GSSG and MDA levels and GSH:GSSG ratio in surgical spinal cord tissue to assess the effects of Apigenin treatment on oxidative stress in CIPN mice. In comparison with the control group, the lipid peroxidation in the CIPN group was enhanced, and the level of MDA was significantly increased, while GABA and Apigenin treatments significantly decreased MDA levels in each treatment group (Fig. 3A). Interestingly, GSSG level was significantly increased in CIPN group compared that of the control group. GABA treatment showed a slight decrease in GSSG levels, however, GSSG levels were significantly decreased in all Apigenin treated groups compared to DC group (Fig. 3B). Likewise, GSH:GSSG ratio was significantly elevated in all treatment groups compared to the DC group (Fig. 3C). The above data indicated that Apigenin alleviated CIPN-induced oxidative stress of CIPN mice.

Fig. 3.

Apigenin alleviated CIPN-induced oxidative stress and inflammation of CIPN mice. Effects of Apigenin treatments on the oxidative stress status in surgical spinal cord tissue were evaluated through MDA level (A), GSSG level (B), and GSH:GSSG ratio (C). Data are represented as mean $\pm$ SD. N = 6 in all groups. ##p 0.01, ###p 0.001, when significantly different in comparison to NC; *p 0.05, **p 0.01, ***p 0.001, when significantly different in comparison to DC. Api, Apigenin; NC, normal control; DC, disease control; GABA, Gabapentin.

To further explore the underlying mechanism of Apigenin on CIPN, we assessed changes in pro-/anti-inflammatory phenotypes of microglia from protein and gene levels through detecting the expression of microglia markers in the surgical spinal cord tissue. CIPN promoted the expression of CD11b, CD68 protein, but had no significant effect on CD206, Apigenin administration significantly decreased the expression of CD68 and CD11b (Fig. 4A–D), which was further verified at the gene expression levels (Fig. 4E–G). Notably, the CD68/CD206 ratio was elevated in the CIPN group and significantly decreased after Apigenin administration (Fig. 4H). These data suggested that Apigenin suppressed pro-inflammatory and anti-inflammatory phenotypes of microglia markers in the surgical spinal cord tissue of CIPN mice.

Fig. 4.

Apigenin altered pro-inflammatory and anti-inflammatory phenotypes microglia markers of the nerve tissue of CIPN mice. (A) A representative blot of CD11b, CD68, CD206 of the nerve tissue. (B–D) A quantitative analysis of CD11b, CD68, CD206 protein level. Fold increase of pro-inflammatory phenotype markers (CD11b, CD68) (E,F) and anti-inflammatory phenotype microglia markers (CD206) (G) mRNA. (H) Fold increase of the ratio of CD68/CD206. Data are represented as mean $\pm$ SD. N = 6 in all groups. ##p 0.01, when significantly different in comparison to NC; *p 0.05, **p 0.01, ***p 0.001, when significantly different in comparison to DC. Api, Apigenin; NC, normal control; DC, disease control.

Recent studies have shown that Apigenin affected the occurrence and development of various diseases through the NRF2/ARE signaling pathway [26]. We determined NRF2 protein levels in the cytoplasmic and nuclear fractions of microglia from lumbar spinal cord of control and CIPN mice. The results showed that cytoplasmic NRF2 protein in the all CIPN groups was decreased relative to the normal group, while the nuclear NRF2 protein was increased in microglia after Apigenin administration (Fig. 5A,B). To further verify the nuclear translocation of NRF2, we detected the expression of its downstream target genes Heme oxygenase 1 (Hmox1) and NAD (P) H quinone oxidoreductase1 (Nqo1) by RT-qPCR. The results showed that the expression of Hmox1 and Nqo1 genes was significantly upregulated in Apigenin-treated CIPN mice in a dose-dependent manner (Fig. 5C,D). All these data indicated that Apigenin induced nuclear recruitment of NRF2 and activated the NRF2/ARE pathway in microglia.

Fig. 5.

Apigenin induced nuclear recruitment of NRF2. The localization of NRF2 protein was evaluated in isolated microglia from different groups. (A,B) Cytoplasmic and nuclear NRF2 protein expression (80 kDa) was evaluated in our experimental conditions. $\alpha$-Tubulin ($\alpha$Tub, 50 kDa) and histone deacetylase 1 (HDAC1) 55 kDa were used as reference proteins. Detected proteins were quantified and normalized. (C,D) Expression of NRF2-target genes (Hmox1, Nqo1) were evaluated by real-time PCR. Gene expressions were normalized to Rpl27 expression level and control levels were fixed at 1.0. Data are represented as mean $\pm$ SD. ##p 0.01, when significantly different in comparison to NC; **p 0.01, ***p 0.001, when significantly different in comparison to DC. Api, Apigenin; NC, normal control; DC, disease control.