This study aims to investigate the effects of Janus kinase/signal transducers and activators of the transcription (JAK/STAT) pathway inhibition on collagen biosynthesis in fibroblast cell culture by tofacitinib.

BJ-CRL-1474® (skin) and BRL3A® (hepatic) fibroblast cell cultures were proliferated in a suitable medium. Tofacitinib was administered to fibroblast cells proliferating in 96-well flasks at concentrations of 25, 50, 100, 200, 400, and 800 nM. Tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-3 (MMP-3), transforming growth factor beta 1 (TGF-β1), and hydroxyproline levels were measured using the enzyme-linked immunosorbent assay method.

Tofacitinib showed cytotoxic effect on skin and liver cell culture. The cytotoxic effect of tofacitinib started at 100 nM (p<0.05). The highest effect was obtained at 800 nM. The time-dependent cytotoxic effect of tofacitinib was significantly higher at all concentrations after 72 hours than at 24 and 48 hours (p<0.05). The level of TGF-β1 was significantly lower even at a tofacitinib concentration of 25 nM (p<0.05). There were significant decreases in MMP-3, TIMP-1, and hydroxyproline levels after tofacitinib administration (p<0.05).

Tofacitinib inhibited fibroblast cell proliferation in a concentration-dependent manner in a fibroblast cell culture. However, further extensive animal and human studies are necessary to determine the clinical significance of this effect.

Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated.

To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis.

In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated.

Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice.

Extracellular histones potentially enhance fibrogenesis via the TLR4-MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

Hot-water extracts of the mycelial culture and fruiting bodies of Agaricus blazei Murill were fractionated by ethanol precipitation, using various ethanol concentrations. The mycelial fraction (A-4) inhibited abnormal collagen fiber formation, and fractions A-1 to A-3 showed a small inhibitory effect. The strongest inhibition was obtained by fraction A-4, and no significant inhibition was observed with fractions A-5 and A-6. With the fruiting bodies, fractions B-1 to B-6 showed no inhibitory effects on collagen fiber formation in HCC. The reverse transcription-polymerase chain reaction (RT-PCR) demonstrates that Agaricus blazei mycelial fraction A-4 did not inhibit the type I, II or III procollagen gene expression.

Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli, Klebsiella, Proteus, and Vibrio alginolyticus) was identical. However, the amino acid composition of Staphylococcus aureus and Bacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium, Chroococidiopsis) was quite similar to that of E. coli. A marked difference in the amino acid composition was observed between E. coli and green alga (Chlorella), and significant differences were observed between E. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.