Cystathionine-Gamma-Lyase-Derived Hydrogen Sulfide-Regulated Substance P Modulates Liver Sieve Fenestrations in Caecal Ligation and Puncture-Induced Sepsis

Abstract

Cystathionine-γ-lyase (CSE) isa hydrogen sulfide (H₂S)-synthesizing enzyme that promotesinflammation by upregulating H₂S in sepsis. Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells (liver sieve) that undergo alteration during sepsis and H₂S plays a role in this process. Substance P (SP) is encoded by the preprotachykinin A (PPTA) gene, and promotes inflammation in sepsis; however, its regulation by H₂S is poorly understood. Furthermore, the interaction between H₂S and SP in modulating LSEC fenestrations following sepsis remains unclear. This study aimed to investigate whether CSE/H₂S regulates SP and the neurokinin-1 receptor (NK-1R) andmodulates fenestrations in LSECs following caecalligation and puncture (CLP)-induced sepsis. Here we report thatthe absence of either CSE or H₂S protects against liver sieve defenestration and gaps formation in LSECsin sepsis by decreased SP-NK-1R signaling. Following sepsis, there is an increased expression of liver CSE and H₂S synthesis, and plasma H₂S levels, which were aligned with higher SP levels in the liver, lungs and plasma and NK-1R in the liver and lungs. The genetic deletion of CSE led to decreased sepsis-induced SP and NK-1R in the liver, lungs and plasma SP suggesting H₂S synthesized through CSE regulates the SP-NK-1R pathway in sepsis. Further, mice deficient in the SP-encoding gene (PPTA) preservedsepsis-induced LSEC defenestrationand gaps formation, as seen by maintenance of patent fenestrations and fewer gaps. In conclusion, CSE/H₂S regulates SP-NK-1R and modulates LSEC fenestrations in sepsis.

Keywords: cystathionine-gamma-lyase; hydrogen sulfide; liver sieve; neurokinin-1 receptor; substance P.

Figure 1

CSE-derived H₂S regulates SP following CLP-induced sepsis. (a–d) Increased liver CSE expression (a) and quantification of CSE in ‘a’ (b), liver H₂S-synthesizing activity (c) and plasma H₂S levels (d) following CLP-induced sepsis. (e–g) Genetic deletion of CSE significantly decreased SP levels in liver (e), lung (f) and plasma (g) compared to WT mice following sepsis. Data represented as mean ± S.E.M. (n = 8). The significance of differences among groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as p < 0.05 (* p < 0.05 vs. WT sham; # p < 0.05 vs. WT sepsis).

Figure 2

CSE-derived H₂S regulates NK-1R following CLP-induced sepsis. (a–d) Genetic deletion of CSE significantly reduced liver (a) and lung (c) NK-1R protein expression compared to WT mice following sepsis and quantification of liver (b) and lung (d) NK-1R protein expression in ‘a’ and ‘c’, respectively. Data represented as mean ± S.E.M. (n = 8). The significance of differences among groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as p < 0.05 (* p < 0.05 vs. WT sham; # p < 0.05 vs. WT sepsis).

Figure 3

PPTA KO mice protect against sepsis-induced LSEC defenestration and gaps formation. (a,b) Representative images of liver sieve fenestration micrographs (a) and average gap area of liver sieve fenestrae (b). LSEC injury significantly increased (as evidenced by gaps formation) following CLP-induced sepsis in WT mice. Genetic deletion of PPTA in mice showed fewer gaps following sepsis compared to WT sepsis mice. Data represented as mean ± S.E.M. (n = 4). The significance of differences among groups was evaluated by ANOVA with post-hoc Tukey’s test. Statistical significance was assigned as p < 0.05 (* p < 0.05 vs. WT sham; # p < 0.05 vs. WT sepsis).