RJX is a proprietary composition of naturally occurring antioxidants and anti-inflammatory agents, which, in combination, provide potent and immediate tissue protection. Its ingredients include ascorbic acid, magnesium sulfate heptahydrate, cyanocobalamin, thiamine hydrochloride, riboflavin 5' phosphate, niacinamide, pyridoxine hydrochloride, and calcium D-pantothenate. RJX is a two-vial system, and A and B are each of the two vials. Vial A contains the active ingredients and minerals, whereas Vial B contains the buffer, sodium bicarbonate, as the Vial A content is acidic . RJX is being developed as an anti-inflammatory and antioxidant treatment platform for patients with sepsis, including COVID-19 patients with viral sepsis and ARDS .
LPS-GalN Model of Fatal Cytokine Storm, Sepsis, and Multi-organ Failure
The ability of RJX, DEX, and RJX plus DEX to prevent fatal shock, ARDS, and multi-organ failure was examined in the well-established LPS-GalN model . In this model, LPS is combined with GalN, which further sensitizes mice to LPS-induced systemic inflammatory syndrome and multi-organ failure. Male BALB/c mice (6–8 weeks old) were obtained from the Firat University Experimental Animal Center. Mice had ad libitum access to standard rodent chow and water throughout the study. The care and treatment of the animals were in accordance with the Guide for the Care and Use of Laboratory Animals. The research protocol was approved by the Animal Care and Use Committee of Firat University. BALB/c mice were randomly divided into different treatment groups. All mice were genetically identical, of the same age, and the LPS-GalN challenged mice were injected with the same amount of LPS-GalN. This statistical equivalency of mice allowed using a pseudo-randomization convenience allocation to assign mice to identified cages. For random treatment allocation, cages were randomly selected to receive one of the specified treatments. We applied the concealment of treatment allocation and blind outcome assessment to reduce the risk of bias in our conclusions. Health care assessments were performed by animal care technicians not involved in treatment assignments or treatments. Investigators did not participate in individual health status or outcome assessments. Untreated normal control mice did not receive any treatments.
All mice except for the untreated normal control mice were challenged with an otherwise lethal dose of LPS mixed with GalN. Specifically, all mice were challenged with an i.p. injection of LPS plus D-galactosamine (Sigma, St. Louis, MO). D-Galactosamine (Sigma Chemicals), which was dissolved at a 32 mg/ml final concentration in phosphate-buffered saline (PBS), was mixed with an equal volume of diluted, sonicated LPS immediately before dosing. This freshly prepared LPS-galactosamine mixture (LPS-GalN) was used immediately after preparation. Each mouse received a 500 µL i.p. injection of LPS-GalN (consisting of 100 ng of LPS plus 8 mg of D-galactosamine). In Part A of the study, vehicle control mice were treated with 0.5 mL normal saline (NS), i.e., an aqueous solution of 0.9% NaCl instead of RJX. NS was administered intraperitoneally (i.p) 2 hours before and 2 hours after the i.p injection of LPS-GalN. Test mice received 0.7 mL/kg RJX dose (2 hours before and 2 hours after LPS-GalN. Mice were monitored for mortality for 24 h. The Kaplan-Meier method, log-rank chi-square test, was used to analyze the 24 h survival outcomes of mice in the different treatment groups. At the time of death, lungs and liver were harvested, fixed in 10% buffered formalin, and processed for histopathologic examination. 3 µm sections were cut, deparaffinized, dehydrated, and stained with hematoxylin and eosin (H & E) and examined with light microscopy.
Lipid peroxidation as a biomarker of oxidative stress was determined and expressed as the amount of malondialdehyde (MDA, nmol/g tissue) in the brain, as previously described . The enzymatic activities of SOD, CAT, and GSH-Px in the brain specimens were determined using the commercially available kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s procedures. In MDA assays, tissue samples (0.3 g) were analyzed for MDA using high-performance liquid chromatography (HPLC, Shimadzu, Kyoto, Japan) . Specifically, an HPLC system equipped with the LC solution Software (Shimadzu, Kyoto, Japan), a pump (LC-20AD), a UV Detector (SPD-20A), a column oven (CTO-10ASVP), an autosampler (SIL-20 A), a degasser unit (DGU-20A5), and a column (Inertsil ODS-3, 250x 46 mm, 5 mm) was used. Tissue samples were homogenized on ice in a glass–glass homogenizer in a mixture of 0.5 ml of HClO4 (0.5 M), 2.5 ml distilled water, and 2-di-tert-butyl-p-cresol (BHT). Then, the samples were centrifuged at 4500 rpm for 5 min, and supernatants were injected into the HPLC system. The addition of acid was necessary to precipitate proteins and release the MDA bound to the amino groups of proteins and other amino compounds. The mobile phase was 30 mM KH2PO4–methanol (82.5 + 17.5, v/v %, pH 3.6), and the flow rate was 1 ml min-1. The injection volume was 30µL, and chromatograms were scanned at 250 nm.
Statistical analyses employed standard methods, including analysis of variance (ANOVA) and/or, nonparametric analysis of variance (Kruskal-Wallis) using the SPSS statistical program (IBM, SPPS Version 21), as reported . Furthermore, the Kaplan–Meier method, log-rank chi-square test, was used to investigate survival and fatality in each group. P-values < 0.05 were considered significant.