Unlocking the Gut's Treasure: Lipase-Producing Bacillus subtilis Probiotic from the Intestine of Microstomus kitt (Lemon sole).

The main objective of this research was to identify potential probiotic candidates belonging to the Bacillus species that could demonstrate tolerance to bile salt and acidic conditions. The study focused on isolating Bacillus strains from the intestine of marine fish-Microstomus kitt. The isolation process involved the use of selective MRS media through the pour plate method. After 24 h, one particular isolate was identified based on its morphological and biochemical traits as Bacillus species. To confirm the identity, molecular characterization of the 16S RNA from the isolated strain was performed, and the sequence analysis verified it as Bacillus subtilis strain ACL_BS 001. With the molecular confirmation, the next step was to assess the probiotic characteristics of this B. subtilis strain. Various tests were conducted to evaluate its acid/pH tolerance, NaCl tolerance, and bile salt tolerance. The results indicated that B. subtilis exhibited high viability percentages even under acidic pH, in the presence of 1.5% bile salt, and at high salt concentrations. Subsequently, we investigated the strain's ability to produce lipase, an important enzyme with potential industrial applications. B. subtilis was grown in MRS agar amended with olive oil as a lipase substrate. After incubation, the presence of lipase activity was confirmed, and the enzymatic assay revealed a significant lipase enzyme activity of 100.23 µmoles/ml of the sample. In conclusion, the study successfully isolated and identified B. subtilis from the intestine of Microstomus kitt, and the strain exhibited promising probiotic characteristics, including resistance to bile salt and acidic conditions. Furthermore, the strain was found to produce lipase, which opens up possibilities for future research focusing on isolating and purifying the lipase from this potential probiotic B. subtilis strain.

Anti-Atherogenic Protection by Oligomeric Proanthocyanidins via Regulating Collagen Crosslinking Against CC Diet-Induced Atherosclerosis in Rats.

The synthesis of collagen and its turnover remained as critical determinants for the progression of atherosclerosis. During this condition, proteases secreted by SMCs and foam cells in the necrotic core degrade collagen. Growing evidences demonstrated that consumption of antioxidant rich diet is highly associated with a reduced risk of atherosclerosis. Oligomeric proanthocyanidins (OPC) have been proved to possess promising antioxidant, anti-inflammatory and cardioprotective activity, based on our previous studies. The present study aims to investigate the efficacy of OPC isolated from Crataegus oxyacantha berries as a natural collagen crosslinker and anti-atherogenic agent. Spectral studies like FTIR, ultraviolet and circular dichroism analysis confirmed the in vitro crosslinking ability of OPC with rat tail collagen when compared to the standard epigallocatechin gallate. The administration of cholesterol:cholic acid (CC) diet induces proteases-mediated collagen degradation that could result in plaque instability. Further, the CC diet fed rats showed significantly increased levels of total cholesterol and triacylglycerols which, in turn, increases the activities of collagen degrading proteases-MMPs (MMP 1, 2 and 9) and Cathepsin S and D. Upon OPC treatment, marked reduction in the lipid content, activation of proteases with concomitant increase in the mRNA levels of collagen Type I and Type III as similar to atorvastatin treatment were observed .Thus, OPC supplementation may contribute to the prevention of atherosclerotic plaque instability by acting as a natural crosslinker of collagen.

Amelioration of C-Reactive Protein and Lectin Like Oxidized Low Density Lipoprotein Receptor Complex Induced Endothelial Dysfunction by Oligomeric Proanthocyanidins.

C-reactive protein (CRP) is a well-established biochemical marker for atherosclerosis. Modification of LDL inside the artery wall favors the elevation of this acute phase protein. Hence, this mechanism is considered an important factor to trigger the monocyte to macrophages differentiation which results in the formation of foam cells. Therefore, this key event should be targeted and focused on how this complex (OxLDL + CRP) proceeds to endothelial dysfunction. Oligomeric proanthocyanidins (OPC) is a well-known cardioprotective flavon-3-ols. The present study is challenged between the cardioprotective roles of OPC against the deleterious effect of OxLDL + CRP complex upon endothelial cells. Protein-protein docking was carried out between CRP and LOX-1. This docked protein complex was again docked with OPC to show the inhibitory mechanism of CRP binding with LOX-1. OPC showed a promising inhibitory mechanism against OxLDL + CRP complex. Docking studies showed that in the absence of ligands (OPC), binding of CRP and LOX-1 was greater and vice versa in the presence of ligands. Based on these molecular docking results, in vitro studies have been carried out. The monolayer of endothelial cells was incubated with THP-1 monocytes for 48 h, induced with OxLDL (10 µg/ml) + CRP (15 µg/ml) and cotreated with OPC (100 µg/ml). Morphological changes, cell migration assay, and capillary tube forming assay were carried out. Myeloperoxidase levels were estimated to determine the adhesion of monocytes onto EC monolayer. RT-PCR analysis of L-Selectin was also done. The quantification of NO levels and analysis of mRNA expressions of eNOS was to determine the nitric oxide demand caused due to OxLDL + CRP complex. LOX-1, scavenger receptor levels were analyzed by mRNA expression. Proinflammatory markers such as IL-6, MCP-1, and IL-1ß were studied. Accumulation of ROS levels was measured fluorimetrically using DCF-DA staining. Mitochondrial membrane potential was determined by JC-1 dye and cell cycle analysis was done by FACS analysis. To emphasis the results, the OPC-treated group showed decreased levels of proinflammatory markers, LOX-1 and L-selectin levels. Endothelial nitric oxide levels were increased upon OPC treatment and reduction in the ROS levels was also observed. Endothelial cells apoptosis was prevented by OPC. To conclude, OxLDL + CRP complex inhibitory effects of OPC could maintain the normal homeostasis.

Oligomeric proanthocyanidins and epigallocatechin gallate aggravate autophagy of foam cells through the activation of Class III PI3K/Beclin1-complex mediated cholesterol efflux.

Foam cells are specialized types of cells which predominate the necrotic core of atherosclerotic plaque. Recently, autophagy-mediated cholesterol efflux from foam cells has been proposed as a beneficial therapy for atherosclerosis. The purpose of this study was to delineate the underlying molecular mechanism of oligomeric proanthocyanidins (OPC) and epigallocatechin gallate (EGCG) induced autophagy of foam cells and associated cholesterol efflux. The oxidized low-density lipoprotein induced foam cells demonstrated impaired autophagy flux through the downregulated expressions of LC3BII/LC3BI, autophagy related gene-5, Class III phosphoinositide 3 kinase (Class III PI3K), Beclin1, ABCA1, and ABCG1 with concomitant increase in the expressions of protein 62, Class I phosphoinositide 3 kinase, Akt, and mammalian target of rapamycin. However, these effects were significantly abolished by treatment with OPC and EGCG through activation of autophagy flux via Class III PI3K/Beclin1 and with upregulated expression of transporter proteins ABCA1 and ABCG1. Furthermore, the cholesterol efflux process in the foam cells was activated by lysosomal acid lipase and cathepsin D facilitated lipolysis of lipid droplets. Taken together, our data demonstrate that OPC and EGCG treatment stimulated the coordinated activation of autophagy and cholesterol efflux through Class III PI3K/Beclin1 pathway in foam cells, suggesting a promising therapeutic strategy against atherosclerosis.

Vitexin protects isoproterenol induced post myocardial injury by modulating hipposignaling and ER stress responses.

The molecular mechanisms involved in ER stress-induced post myocardial injury remain elusive. In this study, we have investigated the molecular mechanism of ER stress-mediated myocyte death in Isoproterenol (ISO) induced myocardial infarction and its inhibition by a potent anti oxidant and anti-apoptotic bioflavonoid, Vitexin. ISO mediated apoptosis was found to be associated with ER permeabilization and characterized by enhanced production of ROS, activation of caspase-3, modulation of Bcl2 family proteins and activation of bnip3. Moreover, post treatment with Vitexin inhibits the ISO induced translocation of CHOP to nucleus during MI. Further results have demonstrated that, activation of Mst1 through ER stress was diminished upon treatment with Vitexin. In addition to this, Vitexin treatment significantly downregulated the expression of p-Yap and p-Mst1 which were enhanced during post myocardial injury. Taken together, our data indicate that co-ordinated activation of ER stress and hipposignaling by ISO was ameliorated by the potent cardioprotective effects of Vitexin.