Crocin's role in modulating MMP2/TIMP1 and mitigating hypoxia-induced pulmonary hypertension in mice.

To explore the molecular pathogenesis of pulmonary arterial hypertension (PAH) and identify potential therapeutic targets, we performed transcriptome sequencing of lung tissue from mice with hypoxia-induced pulmonary hypertension. Our Gene Ontology analysis revealed that "extracellular matrix organization" ranked high in the biological process category, and matrix metallopeptidases (MMPs) and other proteases also played important roles in it. Moreover, compared with those in the normoxia group, we confirmed that MMPs expression was upregulated in the hypoxia group, while the hub gene Timp1 was downregulated. Crocin, a natural MMP inhibitor, was found to reduce inflammation, decrease MMPs levels, increase Timp1 expression levels, and attenuate hypoxia-induced pulmonary hypertension in mice. In addition, analysis of the cell distribution of MMPs and Timp1 in the human lung cell atlas using single-cell RNAseq datasets revealed that MMPs and Timp1 are mainly expressed in a population of fibroblasts. Moreover, in vitro experiments revealed that crocin significantly inhibited myofibroblast proliferation, migration, and extracellular matrix deposition. Furthermore, we demonstrated that crocin inhibited TGF-ß1-induced fibroblast activation and regulated the pulmonary arterial fibroblast MMP2/TIMP1 balance by inhibiting the TGF-ß1/Smad3 signaling pathway. In summary, our results indicate that crocin attenuates hypoxia-induced pulmonary hypertension in mice by inhibiting TGF-ß1-induced myofibroblast activation.

Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase.

Food-derived nattokinase has strong thrombolytic activity and few side effects. In the field of medicine, nattokinase has been developed as an adjuvant drug for the treatment of thrombosis, and nattokinase-rich beverages and health foods have also shown great potential in the field of food development. At present, the poor thermostability of nattokinase limits its industrial production and application. In this study, we used several thermostability-prediction algorithms to predict nattokinase from Bacillus mojavensis LY-06 (AprY), and screened two variants S33T and T174V with increased thermostability and fibrinolytic activity. The t1/2 of S33T and T174V were 8.87-fold and 2.51-fold those of the wild type AprY, respectively, and their enzyme activities were also increased (1.17-fold and 1.28-fold, respectively). Although the thermostability of N218L was increased by 2.7 times, the fibrinolytic activity of N218L was only 73.3% of that of wild type AprY. The multiple-point mutation results showed that S33T-N218L and S33T-T174V-N218L variants lost their activity, and the T174V-N218L variant did not show any significant change in catalytic performance, while S33T-T174V increased its thermostability and activity by 21.3% and 24.8%, respectively. Although the S33T-T174V variant did not show the additive effect of thermostability, it combined the excellent transient thermostability of S33T with the better thrombolytic activity of T174V. Bioinformatics analysis showed that the overall structure of S33T and T174V variants tended to be stable, while the structure of S33T-T174V variant was more flexible. Local structure analysis showed that the increased rigidity of the active center region (positions 64-75) and the key loop region (positions 129-130, 155-163, 187-192, 237-241, and 268-270) determined the increased thermostability of all variants. In addition, the enhanced flexibility of S33T-T174V variant in the Ca1 binding region (positions 1-4, 75-82) and the peripheral region of the catalytic pocket (positions 210-216) may account for the inability to superpose its thermostability. We explored the effective strategy to enhance the thermostability of nattokinase, and the resulting variants have potential industrial production and application.