Baricitinib protects mice from sepsis-induced cardiac dysfunction and multiple-organ failure

Abstract

Sepsis is one of the major complications of surgery resulting in high morbidity and mortality, but there are no specific therapies for sepsis-induced organ dysfunction. Data obtained under Gene Expression Omnibus accession GSE131761 were re-analyzed and showed an increased gene expression of Janus Kinase 2 (JAK2) and Signal Transducer and Activator of Transcription 3 (STAT3) in the whole blood of post-operative septic patients. Based on these results, we hypothesized that JAK/STAT activation may contribute to the pathophysiology of septic shock and, hence, investigated the effects of baricitinib (JAK1/JAK2 inhibitor) on sepsis-induced cardiac dysfunction and multiple-organ failure (MOF). In a mouse model of post-trauma sepsis induced by midline laparotomy and cecal ligation and puncture (CLP), 10-week-old male (n=32) and female (n=32) C57BL/6 mice received baricitinib (1mg/kg; i.p.) or vehicle at 1h or 3h post-surgery. Cardiac function was assessed at 24h post-CLP by echocardiography in vivo, and the degree of MOF was analyzed by determination of biomarkers in the serum. The potential mechanism underlying both the cardiac dysfunction and the effect of baricitinib was analyzed by western blot analysis in the heart. Trauma and subsequent sepsis significantly depressed the cardiac function and induced multiple-organ failure, associated with an increase in the activation of JAK2/STAT3, NLRP3 inflammasome and NF- κβ pathways in the heart of both male and female animals. These pathways were inhibited by the administration of baricitinib post the onset of sepsis. Moreover, treatment with baricitinib at 1h or 3h post-CLP protected mice from sepsis-induced cardiac injury and multiple-organ failure. Thus, baricitinib may be repurposed for trauma-associated sepsis.

Keywords: Janus Kinase; baricitinib; cardiac dysfunction; multiple-organ failure; sepsis.

Figure 1

JAK2 and STAT3 gene expression is elevated in post-operative septic shock patients. Data were taken from the Gene Expression Omnibus under dataset accession number GSE131761, published by Martínez-Paz and colleagues. RNA was extracted from whole blood of (total n=81 patients, of which n=48 male and n=33 female patients) post-operative septic shock patients (Septic Shock group) and from (total n=15, of which n=10 male and n=5 female healthy volunteers) healthy volunteers (Healthy group) of mixed age. (A, B) Differences in JAK2 (A) and STAT3 (B) gene expression between healthy volunteers and post-operative septic shock patients of both genders. (C, D) Differences in JAK2 (C) and STAT3 (D) gene expression between male healthy volunteers and post-operative septic shock patients and female healthy volunteers and post-operative septic shock patients. Statistical differences between the groups (Healthy and Septic Shock) were analyzed by unpaired t-test or using one-way ANOVA followed by a Bonferroni’s post-hoc test, as appropriate. A value of ****P<0.0001 was statistically significant.

Figure 2

Baricitinib reduces sepsis-induced cardiac dysfunction when administered 1h or 3h post-CLP. Mice were treated with vehicle or baricitinib (1mg/kg; i.p.) either 1h or 3h after CLP. (A) Experimental design. (B) Schematic representations of the left ventricle taken in M-mode of the four groups. (C–H) The graphs display the differences between the groups in terms of ejection fraction (%), fractional shortening (%), cardiac output, stroke volume, body temperature at 24h and murine sepsis score (MSS). The following groups containing equal number of male and female mice were studied: sham+vehicle (n=16), CLP+vehicle (n=16), CLP+1hBaricitinib (n=16) and CLP+3hBaricitinib (n=16). A value of ****P<0.0001 was statistically significant when compared to CLP+vehicle by one-way ANOVA followed by a Bonferroni’s post hoc test. (I) Correlation between MSS and EF%, (J) correlation between temperature at 24h and EF%. Correlations coefficients were determined by Pearson’s correlation with P-value based on two-tailed tests.

Figure 3

Baricitinib attenuates sepsis-induced multi-organ dysfunction. Mice were treated with vehicle or Baricitinib (1mg/kg; i.p.) either 1h or 3h after. 24h after CLP, blood samples were collected to evaluate the amount of (A) aspartate transaminase (AST), (B) alanine aminotransferase (ALT), (C) creatinine, (D) creatine kinase (CK) and E) lactate dehydrogenase (LDH). The following groups containing equal number of male and female mice were studied: sham+vehicle (n = 16), CLP+vehicle (n = 16), CLP+1hBaricitinib (n=16) and CLP+3hBaricitinib (n=16). All data are expressed as mean ± SEM for n number of observations. A value of ****P<0.0001 was statistically significant when compared to CLP+vehicle by one-way ANOVA followed by a Bonferroni’s post hoc test.

Figure 4

Baricitinib reduces JAK/STAT and abolishes NF-κB and NLRP3 activation in both genders CLP-animals. Mice were treated with vehicle or baricitinib (1mg/kg; i.p.) 1h after CLP. Western blot analyses were conducted on cardiac tissue of animals of both genders to determine (A) phosphorylation of JAK2 at Tyr^1007-1008; (B) phosphorylation of STAT3 at Tyr⁷⁰⁵; (C) expression of NLRP3; (D) activation of caspase-1; (E) phosphorylation of IκBα at Ser32/36; (F) nuclear translocation of p65. Protein expression was measured as relative optical density (O.D.) and normalized to the sham band. The following groups of male and female animals were studied: sham+vehicle (n=4), CLP+vehicle (n=5) and CLP+1hBaricitinib (n=5). All data are expressed as mean ± SEM for n number of observations. A value of ****P<0.0001, *** P<0.001, **P<0.01 was statistically significant when compared to CLP+vehicle by one-way ANOVA followed by a Bonferroni’s post hoc test.

Figure 5

Baricitinib protects mice from CLP-induced sepsis in both genders. Mice were treated with vehicle or baricitinib (1mg/kg; i.p.) either 1h or 3h after CLP. (A–D) The graphs display the differences between the groups devided by genders in terms of Ejection Fraction % (EF%), alanine aminotransferase (ALT), creatinine and lactate dehydrogenase (LDH). The following groups of male and female animals were studied: sham+vehicle (n=8), CLP+vehicle (n = 8), CLP+1hBaricitinib (n = 8) and CLP+3hBaricitinib (n = 8). All data are expressed as mean ± SEM for n number of observations A value of ****P<0.0001, *** P<0.001, **P<0.01, *P<0.05 was statistically significant when compared to the CLP+vehicle by one-way ANOVA followed by a Bonferroni’s post hoc test.