Despite the tremendous biological activity of polysaccharides from the mushroom Dictyophora indusiata, its role in the restoration of gut microbiota has not yet been explored. The present study aimed to investigate whether D. indusiata polysaccharide (DIP) could modulate the recovery of gut microbiota composition and intestinal barrier function after broad-spectrum antibiotic-driven dysbiosis. Alteration and restoration in the microbial communities were elucidated by the Illumina MiSeq platform. Colon histology, expression of tight-junction associated proteins, and serum/tissue endotoxin and cytokine levels were evaluated. Two-week daily oral administration of clindamycin and metronidazole resulted in reduced bacterial diversity and richness, and perturbed the microbial flora at various taxonomic levels (altered Firmicutes/Bacteroidetes ratio and increased relative abundance of harmful flora (Proteobacteria, Enterococcus, and Bacteroides)), whereas DIP administration reversed the dysbiosis and increased beneficial flora, including Lactobacillaceae (lactic acid-producing bacteria), and Ruminococaceae (butyrate-producing bacteria). In addition, it resulted in the reduction of endotoxemia (through lipopolysaccharides (LPSs)) and pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β)) levels, with the increased expression of tight-junction associated proteins (claudin-1, occludin, and zonula occludens-1). These findings not only suggested a comprehensive understanding of the protective effects of a DIP in the restoration of gut microbiota but also highlighted its role in the enhancement of gut barrier integrity, reduction of inflammation and lowering of endotoxin levels in mice.

Obesity is a multifactorial metabolic disorder characterized by low-grade chronic inflammation, hyper-permeability of the gut epithelium, and perturbation of the intestinal microbiome. Despite the numerous therapeutic efficacies of Dictyophora indusiata mushroom, its biological activity in alleviating obesity through regulation of the gut microbiota and inflammatory cascades remain obscure. Henceforth, we determined the modulatory impact of D. indusiata polysaccharide (DIP) in the high-fat diet (HFD)-induced obesity mice model. The experimental subjects (BALB/C mice) were supplemented with chow diet (Control group), high-fat diet (HFD group), or HFD along with DIP at a low dose [HFD + DIP(L)] and high dose [HFD + DIP(H)]. Obesity-related parameters, including body weight gain, epididymal adipocyte size, fat accumulation, adipogenic markers, lipogenic markers, inflammatory associated markers, intestinal integrity, and intestinal microbiome, were elucidated. Our findings demonstrated that the oral administration of DIP at low dose partially and at high dose significantly reversed HFD-induced obesity parameters. Furthermore, the body weight, fat accumulation, adipocyte size, adipogenic and liver associated markers, glucose levels, inflammatory cytokines, and endotoxin (Lipopolysaccharide, LPS) levels were reduced considerably. Moreover, the study revealed that DIP treatment reversed the dynamic alterations of the gut microbiome community by decreasing the Firmicutes to Bacteroidetes ratio. These findings led us to infer the therapeutic potential of DIP in alleviating HFD-induced obesity via regulating inflammatory cascades, modulating intestinal integrity and intestinal microbiome community.

Lp16-PSP (Latcripin 16-Perchloric acid Soluble Protein) from Lentinula edodes strain C_91-3 has been reported previously in our laboratory to have selective cytotoxic activity against a panel of human cell lines. Herein, we have used several parameters in order to characterize the Lp16-PSP-induced cell death using human acute promyeloid leukemia (HL-60) as a model cancer. The results of phase contrast microscopy, nuclear examination, DNA fragmentation detection and flow cytometry revealed that high doses of Lp16-PSP resulted in the induction of apoptosis in HL-60 cells. The colorimetric assay showed the activation of caspase-8, -9, and -3 cascade highlighting the involvement of Fas/FasL-related pathway. Whereas, Western blot revealed the cleavage of caspase-3, increased expression of Bax, the release of cytochrome c and decreased expression of Bcl-2 in a dose-dependent manner, suggesting the intrinsic pathway might be involved in Lp16-PSP-induced apoptosis as well. Low doses of Lp16-PSP resulted in the anchorage-independent growth inhibition, induction of G₁ phase arrest, accompanied by the increased expression of p21^WAF1/CIP1, along with the decreased expression of cyclin D, E, and cdk6. In addition, Lp16-PSP resulted in constitutive translocation inhibition of transcription factor nuclear factor kappa B (NF-κB) into the nucleus by decreasing the phosphorylation of IκBα. All these findings suggested Lp16-PSP as a potential agent against acute promyeloid leukemia; however, further investigations are ultimately needed.

Latcripin-16 (Lp16-PSP) is a gene that was extracted as a result of de novo characterization of the Lentinula edodes strain C _91-3 transcriptome. The aim of the present study was to clone, express and investigate the selective in vitro anticancer potential of Lp16-PSP in human cell lines. Lp16-PSP was analyzed using bioinformatics tools, cloned in a prokaryotic expression vector pET32a (+) and transformed into E. coli Rosetta gami. It was expressed and solubilized under optimized conditions. The differential scanning fluorometry (DSF)-guided refolding method was used with modifications to identify the proper refolding conditions for the Lp16-PSP protein. In order to determine the selective anticancer potential of Lp16-PSP, a panel of human cancerous and non-cancerous cell lines was used. Lp16-PSP protein was identified as endoribonuclease L-PSP protein and a member of the highly conserved YjgF/YER057c/UK114 protein superfamily. Lp16-PSP was expressed under optimized conditions (37°C for 4 h following induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside). Solubilization was achieved with mild solubilization buffer containing 2M urea using the freeze-thaw method. The DSF guided refolding method identified the proper refolding conditions (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 400 mM Arginine, 0.2 mM GSH and 2 mM GSSG; pH 8.0) for Lp16-PSP, with a melting transition of ~58°C. A final yield of ~16 mg of purified Lp16-PSP from 1 L of culture was obtained following dialysis and concentration by PEG 20,000. A Cell Counting Kit-8 assay revealed the selective cytotoxic effect of Lp16-PSP. The HL-60 cell line was demonstrated to be most sensitive to Lp16-PSP, with an IC ₅₀ value of 74.4±1.07 μg/ml. The results of the present study suggest that Lp16-PSP may serve as a potential anticancer agent; however, further investigation is required to characterize this anticancer effect and to elucidate the molecular mechanism underlying the action of Lp16-PSP.