Individual and combined walnut oil and blueberry attenuate lipopolysaccharide-induced neuroinflammation in rat microglia

Authors: CAREY, AMANDA - Simmons University; Fisher, Derek; CAHOON, DANIELLE - Tufts University; Shukitt Hale, Barbara

Submitted to: Frontiers in Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2025
Publication Date: 5/26/2025
Citation: Carey, A., Fisher, D.R., Cahoon, D.S., Shukitt Hale, B. 2025. Individual and combined walnut oil and blueberry attenuate lipopolysaccharide-induced neuroinflammation in rat microglia. Frontiers in Nutrition. 28(11), 1360-1371. https://doi.org/10.1080/1028415X.2025.2509753.
DOI: https://doi.org/10.1080/1028415X.2025.2509753

Interpretive Summary: Walnuts and blueberries are two foods which provide protection to the aging brain, but it is not known if their protection is greater if they are combined rather than given individually. This study aimed to examine the individual and potentially combined beneficial effects of walnut oil and blueberries in a cell model of inflammation. Cells were treated at different timepoints with different doses of blueberry, walnut oil, or their combination before being exposed to an inflammatory compound. Results showed that all of the treatments were protective against the inflammatory compound in that they reduced three different markers of inflammation relative to control. Also, higher concentrations and longer treatment durations typically were the most beneficial. However, the combination of blueberries and walnuts was not more effective than either individual treatment, suggesting that they do not act together to attenuate inflammation in this cell model.

Technical Abstract: Objectives Neuroinflammation has been associated with cognitive decline and neurodegenerative disease. Walnuts and blueberries (BB) each provide neuroprotective anti-inflammatory components. However, it is unknown whether they act synergistically to enhance the effects seen with individual supplementation. This study aimed to examine the individual and potentially synergistic beneficial effects of walnut oil (WO) and BB on lipopolysaccharide (LPS)-induced neuroinflammation in rat microglial cells. The effects of pretreatment duration and concentration were also explored. Methods Rat microglial cells were pretreated for 48 hours, one, two, or four weeks with freeze-dried BB extract, WO or WOBB diluted in media at 0.05, 0.1, 0.2, 0.5 and 1.0 mg/mL. At the end of each treatment time point, cells were exposed to LPS (100 ng/mL) overnight. Standard immunochemical techniques were used to measure levels of inflammatory biomarkers: nitrite, inducible nitrous oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Data were analyzed using three-way analysis of variance with treatment type, concentration, and duration as independent variables, and Tukey’s post-hoc test was used to determine differences among groups. Results Results showed that BB, WO, and WOBB reduced LPS-induced nitrite, COX-2 and iNOS relative to control. All three treatments showed similar ability to reduce iNOS expression. BB had a stronger effect on reducing nitrite production than WO and WOBB. However, there were no significant differences between treatment effects on COX-2 expression. All three treatments attenuated LPS-induced nitrite, COX-2, and iNOS, with higher concentrations and longer treatment durations typically being most beneficial. Conclusion Although BB, WO and WOBB reduced LPS-induced neuroinflammation, WOBB was not more effective than either individual treatment. This result suggests that BB and WO do not act synergistically, and may work through similar mechanisms, to attenuate inflammation in microglia. Overall, the reduction in neuroinflammation following all treatments shows that the addition of BBs or walnuts to the diet may attenuate the neurodegenerative effects of inflammation.