iNOS Mediates High-Fat Diet-Associated Aggravation of Complete Freund's Adjuvant-Induced Inflammatory Pain

Abstract

Chronic inflammatory pain (IP) remains a therapeutic challenge under the worldwide prevalence of the high-fat dietary lifestyle. This study aimed at identifying mediators of the IP augmented by short-term high-fat diet (HFD). IP was induced on C57BL/6J mice by unilateral, intra-plantar, injection of Complete Freund's Adjuvant (CFA). Von Frey test for mechanical hyperalgesia and Hargreaves' test for thermal hyperalgesia were performed at pre-injection baseline and post-injection 6th h. and days 1/3/5/7/10/14. Ad libitum HFD feeding started 2 weeks pre-injection in assigned groups. Body weight and random blood glucose levels were measured. RT-qPCR and ELISA helped quantify expression levels of the selected candidate genes at manipulated hind-paws. After CFA injection, at 1400 W, a highly selective inducible nitric oxide synthase (iNOS) inhibitor was administered regularly to elicit differences in CFA-induced pain behaviors and gene expression in HFD-fed mice. Results showed that HFD-fed mice were heavier (p < 0.001) and relatively hyperglycemic (p = 0.013) at baseline. HFD aggravated CFA-induced mechanical and thermal pain (mechanical: p = 0.0004, thermal: p = 0.003), showing prolonged hyperalgesic durations and reduced pain thresholds at multiple timepoints. HFD-influenced paws showed accentuated overexpression of pro-inflammatory cytokines and iNOS (RT-qPCR for IL-1β: p = 0.015, IL-6: p = 0.019, TNF: p = 0.04; ELISA for iNOS: p = 0.011). At 1400 W, exertion of analgesic effects (mechanical: p < 0.0001, thermal: p < 0.0001) but pro-inflammatory (RT-qPCR for IL-1β: p = 0.004, IL-6: p = 0.03, TNF: p = 0.04) were exerted on the inflamed paw on day 5 post-injection. In conclusion, short-term HFD aggravated CFA-induced inflammatory pain. Pharmacological inhibition of iNOS attenuated the CFA-induced pain in HFD-fed mice. Future research might uncover signaling pathways mediating such effects, potentially benefiting obese patients with chronic IP.

Keywords: iNOS; inflammatory pain; obesity; short-term high-fat diet.

Figure 1

Comparing behavioral data from CFA and saline groups. (A): Log-transformed PWT from von Frey test (CFA group: n = 7; saline group: n = 8 for all time points); (B): PWL from Hargreaves’ test (CFA group: n = 6; saline group: n = 6 for all time points); ns (not significant) p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, from two-way ANOVA with Šidák’s post hoc test.

Figure 2

Relative transcription of inflammatory markers in CFA group. Log10-transformed 2^−ΔΔCt of transcribed mRNA on post-injection day 5, relative to saline group. (A): IL-1β (CFA group: n = 5, saline group: n = 6); (B): IL-6 (CFA group: n = 7, saline group: n = 8); (C): TNF (CFA group: n = 6, saline group: n = 8); (D): IFN-γ (CFA group: n = 6, saline group: n = 6); (E): IL-4 (CFA group: n = 5, saline group: n = 6); (F): IL-10 (CFA group: n = 5, saline group: n = 8); (G): TGF-β1 (CFA group: n = 6, saline group: n = 8). ns (not significant) p ≥ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, from unpaired Student’s t-test.

Figure 3

Effects of short-term high-fat diet. HFD + CFA: mice fed with HFD and injected with CFA; ND + CFA: mice fed with ND and injected with CFA; ND + saline: mice fed with ND and injected with normal saline. (A): body weight (in grams) of mice after being fed ad libitum with HFD or ND for 14 days (n = 4 per group); *** p < 0.001 from unpaired Student’s t-test. (B): random blood glucose (in mmol/L) of mice after being fed ad libitum with HFD or ND for 14 days (n = 4 per group); * p < 0.05 from unpaired Student’s t-test. (C): log-transformed PWT from von Frey test at various time points before and after CFA or saline injection, in mice with or without 14-day pre-injection dietary modification using ad libitum HFD (for all time points: n = 7 for HFD + CFA group, n = 7 for ND + CFA group, n = 8 for ND + saline group); ns (not significant) p ≥ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, from two-way ANOVA with Šidák’s post hoc test. (D): PWL from Hargreaves’ test at various time points before and after CFA or saline injection, in mice with or without 14-day pre-injection dietary modification using ad libitum HFD (for all time points: n = 6 per group); ns (not significant) p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, from two-way ANOVA with Šidák’s post hoc test. (E–J): Log10-transformed 2^−ΔΔCt of transcribed mRNA on day 5 after CFA injection, in mice with 14-day pre-injection dietary modification using ad libitum HFD, relative to normal diet group, in regards to common inflammatory marker genes of IL-1β ((E), HFD + CFA group: n = 5, ND + CFA group: n = 5), IL-6 ((F), HFD + CFA group: n = 5, ND + CFA group: n = 7), TNF ((G), HFD + CFA group: n = 6, ND + CFA group: n = 6), IFN-γ ((H), HFD + CFA group: n = 6, ND + CFA group: n = 6), IL-4 ((I), HFD + CFA group: n = 5, ND + CFA group: n = 5), IL-10 ((J), HFD + CFA group: n = 6, ND + CFA group: n = 5), and TGF-β1 ((K), HFD + CFA group: n = 6, ND + CFA group: n = 6). ns (not significant) p ≥ 0.05, * p < 0.05, ** p < 0.01 from unpaired Student’s t-test.

Figure 4

mRNA transcription and protein expression of iNOS gene under HFD and CFA influence. HFD + CFA: mice fed with HFD and injected with CFA; ND + CFA: mice fed with ND and injected with CFA; ND + saline: mice fed with ND and injected with normal saline. (A): Obtained from RT-qPCR assay, log10-transformed 2^−ΔΔCt of transcribed iNOS mRNA on post-injection day 5, in mice fed with HFD or ND and injected with CFA or normal saline, relative to normal diet—saline group (n = 5 for each group); * p < 0.05, **** p < 0.0001, from one-way ANOVA with Šidák’s post hoc test. (B): Obtained from ELISA, on post-injection day 5, protein expression level of iNOS in mice fed with HFD or ND and injected with CFA, relative to the ND + CFA group (n = 5 for each group). * p < 0.05, from unpaired Student’s t-test.

Figure 5

Effects of 1400 W on HFD-fed, CFA-injected mice. HFD + CFA + 1400 W: mice fed with HFD and injected with CFA, followed by post-injection intervention with 1400 W; HFD + CFA + Saline: mice fed with HFD and injected with CFA, followed by post-injection normal saline control. (A): log-transformed PWT from von Frey test on day 5 after CFA injection, in mice fed with HFD, with post-injection 1400 W intervention or saline control (n = 6 for each group); **** p < 0.0001, from unpaired Student’s t-test. (B): PWL from Hargreaves’ test on day 5 after CFA injection, in mice fed with HFD, with post-injection 1400 W intervention or saline control (n = 6 for each group); **** p < 0.0001, from unpaired Student’s t-test. (C): body weight (in grams) of mice after being fed ad libitum with HFD for 14 days and injected with CFA, followed by 1400 W intervention or saline control (n = 12 for each group); ns (not significant) p ≥ 0.05, from two-way ANOVA with Šidák’s post hoc test. (D): change in body weight (in grams) of mice since receiving CFA injection, in HFD-fed mice given with 1400 W intervention or saline control following CFA injection (n = 12 for each group); ns (not significant) p ≥ 0.05, from unpaired Student’s t-test. (E–I): Log10-transformed 2^−ΔΔCt of transcribed mRNA on day 5 after CFA injection, in mice fed with HFD, with post-injection 1400 W intervention or saline control, with regards to genes including iNOS (E), IL-1β (F), IL-6 (G), TNF (H), and IFN-γ (I) (for all genes, n = 5 for each group); * p < 0.05, ** p < 0.01, from unpaired Student’s t-test.

Figure 6

Experimental time points.