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Abstract Immunoparalysis is an important pathological mechanism in sepsis. However, an effective small molecule therapy is lacking. Here, we show that ouabain, a Na+,K+‐ATPase ligand, can reverse immunoparalysis in vitro, in vivo, and in clinical samples. Notably, the effect of ouabain was critically dependent on TNF‐α expression. However, ouabain had opposing effects on the stability of TNF‐α mRNA: Ouabain triggered miR‐181 transcription, which promoted TNF‐α mRNA degradation and induced immunoparalysis, and ouabain triggered the nuclear export of human antigen R (HuR), which stabilized TNF‐α mRNA and suppressed immuno‐paralysis. Interestingly, because the miR‐181 binding site is located within the HuR binding site in the 3′‐untranslated region of TNF‐α, in ouabain‐treated cells, HuR competed with miR‐181 for binding to TNF‐α mRNA and recruited TNF‐α mRNA to stress granules, thereby stabilizing TNF‐α mRNA and reversing immunoparalysis. Ouabain also induced GM‐CSF and interferon‐γ expression in a HuR‐dependent manner. Hence, the fine‐tuning of TNF‐α mRNA stability by HuR and miR181 plays a crucial role in immunoparalysis, and Na+,K+‐ATPase ligands are promising agents for immunoparalysis therapy.

Immunoparalysis is an important pathological mechanism in sepsis. However, an effective small molecule therapy is lacking. Here, we show that ouabain, a Na+,K+- ATPase ligand, can reverse immunoparalysis in vitro, in vivo, and in clinical samples. Notably, the effect of ouabain was critically dependent on TNF-α expression. However, ouabain had opposing effects on the stability of TNF-α mRNA: Ouabain triggered miR-181 transcription, which promoted TNF-α mRNA degradation and induced immunoparalysis, and ouabain triggered the nuclear export of human antigen R (HuR), which stabilized TNF-α mRNA and suppressed immuno-paralysis. Interestingly, because the miR-181 binding site is located within the HuR binding site in the 3′-untranslated region of TNF-α, in ouabain-treated cells, HuR competed with miR-181 for binding to TNF-α mRNA and recruited TNF-α mRNA to stress granules, thereby stabilizing TNF-α mRNA and reversing immunoparalysis. Ouabain also induced GM- CSF and interferon-γ expression in a HuR-dependent manner. Hence, the fine-tuning of TNF-α mRNA stability by HuR and miR181 plays a crucial role in immunoparalysis, and Na+,K+- ATPase ligands are promising agents for immunoparalysis therapy. [ABSTRACT FROM AUTHOR]

High purity polysaccharide of pachyman was isolated from the powder of Poria cocos sclerotium with a yield of 77.8%. The intrinsic viscosity of polysaccharide was found to be 78.95 mL/g in DMSO solution at 25°C. The isolated polysaccharide was reacted with chlorosulfonic acid to obtain pachyman sulfate using the improved Wolfrom method. The results of the orthogonality experiment on the sulfation reaction identified that the effectiveness of the reaction conditions on the degree of sulfation and the value of intrinsic viscosity is in the following order: molar ratio of chlorosulfonic acid to glucoside (3–5) > reaction temperature (60–80°C) > reaction time (1–2 h). The kinetic studies of the pachyman sulfation indicated that the hydrolysis is accompanied with the sulfation process. The decrease in intrinsic viscosity of the sulfated pachyman is proportional to the increase in the degree of sulfation under the mild reaction conditions of < 80°C, chlorosulfonic acid/glucoside mole ratio < 5, and reaction time < 2 h. Beyond the above reaction conditions, excessive loss of –OH group occurs during hydrolysis. The NMR results indicated a complete sulfation on C-6 and a partial sulfation on the C-2 and C-4 of glucoside. [ABSTRACT FROM AUTHOR]