Culture with Dextrose Reduced Macrophage Viability in A Dose Dependent Manner: Implications for Prolotherapy

Purpose: Prolotherapy, coined from proliferant therapy, is an alternative injection-based therapy that has been used in clinical practice for over 80 years to treat various chronic musculoskeletal conditions. Modern hypotheses suggest prolotherapy promotes growth of normal cells and tissues to improve ligament mechanics, and decrease pain through inflammatory mechanisms. The most common injectant contains dextrose (D-glucose), a natural form of glucose found in the body. Recent literature indicates adults with symptomatic knee pain received the most relief from intra-articular injection of dextrose, compared to saline injections, or exercise. This study aims to begin to explain the mechanism of action of dextrose in the inflammatory response. Specifically, we hypothesized that dextrose would suppress the release of inflammatory mediators from LPS-activated macrophages. Methods: To test this hypothesis, murine RAW 264.7 macrophages were cultured in vitro with phosphate-buffered saline (PBS) or dextrose solution at 2.5%, 5%, and 10% of total volume per well, with or without 500ng lipopolysaccharide (LPS). Twenty-four hours after incubation at 37°C with 5% CO2, culture supernatants were stored and assayed for nitrite (NO2-) using Griess reagent. Macrophage viability was measured using flow cytometry with the markers Annexin V and Propidium Iodide. Results: Dextrose did not significantly alter the production of NO2- in macrophages cultured without LPS. However, in LPS-activated macrophages, dextrose significantly (p2- compared to respective PBS controls. Specifically, 2.5% dextrose suppressed NO2-release by 78.31%, while 5% and 10% dextrose completely inhibited production of NO2- . Cell viability was also significantly (p Conclusions: Dextrose reduced viability and suppressed the production of NO2- by macrophages in vitro. Prolotherapy may protect against inflammation by reducing the inflammatory activities of macrophages. Future studies will examine the effect of dextrose on macrophage function in vivo using animal models.