In this study, one water soluble polysaccharide (IOP1-1) with a weight average molecular weight of 6886 Da was obtained from the black crystal region of Inonotus obliquus by hot water extraction, DEAE-52 cellulose extraction and Sephadex-100 column chromatography purification. Structural analysis indicated that IOP1-1 was a glucan with a main chain composed of α-Glcp-(1 → 4)-α-Glcp-(1 → 4)-β-Glcp-(1 → 4)-β-Glcp-(1 → 4)-α-Glcp-(1 → 6)-β-Glcp-(1 → 4)-α-Glcp-(1 → 3)-β-Glcp-(1→. The CCK-8 assay results showed that IOP1-1 inhibited AsPC-1 and SW1990 pancreatic cancer cell proliferation in a concentration-dependent manner. Flow cytometric analysis revealed that IOP1-1 induced cell cycle arrest in AsPC-1 and SW1990 cells. Hoechst 33342 staining and Annexin V-FITC/PI double staining analysis showed that IOP1-1 could induce apoptosis in AsPC-1 and SW1990 cells. Furthermore, western blot analysis confirmed that IOP1-1 could induce apoptosis in AsPC-1 and SW1990 pancreatic cancer cells through three pathways: the mitochondrial pathway, the death receptor pathway, and endoplasmic reticulum stress. According to these research data, IOP1-1 may be utilized as an adjuvant treatment to anticancer medications, opening up new application prospects and opportunities.

Hyperlipidemia is a multifaceted metabolic disease, which is the major risk factor for atherosclerosis and cardiovascular diseases. Traditional Chinese medicine provides valuable therapeutic strategies in the treatment of hyperlipidemia. Inonotus obliquus has been used in traditional medicine to treat numerous diseases for a long time. To screen and isolate the fractions of I. obliquus polysaccharides (IOP) that can reduce blood lipid in the hyperlipemia animals and cell models, and investigate its mechanisms. The active component IOP-A2 was isolated, purified, and identified. In vivo, rats were randomly divided into blank control group (NG), the high-fat treatment group (MG), lovastatin group (PG), and IOP-A group. Compared with MG, the hyperlipidemic rats treated with IOP-A2 had decreased body weight and organ indexes, with the level of serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) significantly decreased (p < .05), and level of serum high-density lipoprotein cholesterol (HDL-C) significantly increased (p < .05). Hepatocyte steatosis in hepatic lobules was significantly reduced. In vitro, the accumulation of lipid droplets in the model of fatty degeneration of HepG2 cells was significantly alleviated, and cellular TC and TG content was significantly decreased (p < .01). Moreover, the expression of recombinant cytochrome P450 7A1 (CYP7A1) and Liver X Receptor α (LXRα) were up-regulated (p < .05) both in vivo and in vitro. The results showed that IOP-A2 may exert its hypolipidemic activity by promoting cholesterol metabolism and regulating the expression of the cholesterol metabolism-related proteins CYP7A1, LXRα, SR-B1, and ABCA1.

UNASSIGNED: The nonenzymatic glycation of fibroblasts causes functional downregulation and behavioral disorders in the skin.
UNASSIGNED: To investigate the effect of Inonotus obliquus on the nonenzymatic glycation of skin, we examined the inhibition of advanced glycation end products (AGEs) using four extraction methods: n-butanol, ethyl acetate, n-hexane and aqueous alcohol precipitation. The physical properties and chemical structure of the most effective, purified, crude I. obliquus polysaccharide (IOP) were examined. The effects of IOP on carboxymethyl lysine (CML) accumulation, inflammatory factor release, reactive oxygen species (ROS) production, key extracellular matrix (ECM) protein (MMP 1, 2 and 9; FN-1, LM-5 and COL-1) mRNA expression, and cell survival, migration and adhesion were also examined via cellular assays.
UNASSIGNED: IOP is a polysaccharide with a molecular weight (Mw) of 2.396 × 104 (±6.626%) that is composed mainly of glucose, galactose, xylose, mannose and arabinose (29.094:21.705:14.857:9.375:7.709). In addition, a cellular antiglycation assay showed that IOP, which can promote ECM formation by inhibiting the accumulation of CML, inhibiting the release of inflammatory factors (IL-1β, IL-6, and TNF-α), inhibiting the production of reactive oxygen species (ROS), inhibiting the expression of matrix metalloproteinases (MMP-1\\-2\\-9), promoting the synthesis of ECMs (COL1, FN1, and LM5), and improving cellular dysfunction, had strong antiglycation activity at concentrations in the range of 6-24 μg/mL.
UNASSIGNED: IOP effectively reduced the levels of inflammatory factors and reactive oxygen species produced by AGEs, further preventing the impairment of cell behavior (decreased migration and reduced cell adhesion) and preventing the downregulation of the expression of key extracellular matrix proteins induced by AGEs. The results indicate the potential application of IOP as an AGE inhibitor in skin care.

The study aimed to explore the protective effects of Inonotus obliquus polysaccharide (IOP) on Neospora caninum (N. caninum) infection. Our data showed that the survival rate of the mice was the highest and the survival time was the longest when the IOP was 2 mg/10 g. In agreement with these observations, IOP alleviated the pathological damage in the various organs and tissues of the mice. Compared with that in the Neosporidium infection model group, the content of N. caninum in the heart, liver, spleen, lung, kidney and brain, determined through HE staining, was significantly lower. In addition, IOP inhibited the levels of immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2a) from the 21st to 42nd day of the administration group, whereas the levels of interleukin-12 (IL-12) and serum tumor necrosis factor alpha (TNF-α) were down-regulated at 7 d - 42 d. The production of CD4+ T lymphocytes was promoted, the number of CD8+ T lymphocytes were significantly lower and the CD4+/CD8+ ratio was significantly elevated. Furthermore, IOP effectively balanced the levels of hormones including gonadotropin-releasing hormone (GnRH), luteotropic hormone (LH) and testosterone (T) in male mice, and progesterone (PROG), estradiol (E2) and prolactin (PRL) in female mice. These findings demonstrate that IOP exerts protective effects against pathological damage caused by N. caninum infection in mice, and improve the immune function of the organism and regulate the secretion balance of sex hormones.

UNASSIGNED: The macromolecular polysaccharide Inonotus obliquus polysaccharide (IOP) is composed of various monosaccharides, and it could modulate the composition and diversity of intestinal flora. However, its impact on the intestinal flora in rats of different genders remains unclear. Therefore, this study aims to investigate the structural changes of IOP and its effects on the intestinal flora after administration in male and female rats.
UNASSIGNED: In this study, the molecular weight and purity of IOP were analyzed by high-performance gel permeation chromatography (HPGPC) and phenol sulfuric acid method, and NMR was used to confirm the chemical structure of IOP. Sex hormone [testosterone (T) and estradiol (E2)] levels and intestinal microbial changes were detected by enzyme-linked immunosorbent assay (ELISA) and 16S rRNA, respectively, after gavage of IOP (100 mg/kg) in male and female Sprague Dawley (SD) rats.
UNASSIGNED: HPGPC analysis showed that the average molecular weight (Mw) of IOP was 4,828  Da, and the total sugar content of the purified IOP was 96.2%, indicating that the polysaccharide is of high purity. NMR revealed that IOP is a linear macromolecule with an α-D-type glucose backbone. The results of ELISA and 16S rRNA showed that the IOP increased the abundance of beneficial bacteria, such as Clostridia_UCG-014 and Prevotellaceae_NK3B31, and reduced that of harmful bacteria, such as Colidextribacter and Desulfobacterota in the intestine of both male and female rats, and IOP changed the levels of sex hormones in male and female rats. Further analyses revealed that the increase in alpha diversity was higher in male than female rats. α diversity and β diversity revealed a significant difference in the composition of cecal microbiota between male and female rats in the control group, but IOP intake reduced this difference. Meanwhile, α analysis revealed a change in the composition of bacterial flora was more stable in male than female rats.
UNASSIGNED: This study enhances our comprehension of the IOP structure and elucidates the alterations in intestinal flora following IOP administration in rats of varying genders. Nonetheless, further investigation is warranted to explore the specific underlying reasons for these discrepancies.

Ulcerative colitis (UC) is a disease characterized by chronic inflammation of the colon. Polysaccharides not only have biological activities but also can regulate gut microbiota to alleviate the symptoms of UC. In this study, polysaccharide extracted from mycelium of Inonotus obliquus (IOP) was prescribed to treat UC induced by dextran sodium sulfate (DSS) in mice. Compared to model control group (MC), IOP-Low, IOP-Medium and IOP-High (IOP-L, IOP-M and IOP-H) treatment groups increased the body weight rate by 6.0%-9.6%, colon length by 8.57%-25.14% and superoxide dismutase (SOD) activity by 53.8-110.4 U/mg, while decreased the malondialdehyde (MDA) content by 37.4%-64.8%, myeloperoxidase (MPO) activity by 29.0%-46.9%, and the concentration of nitric oxide (NO) by 24.8-35.6 μmol/L. IOP treatment also promoted the secretion of interleukin (IL)-10 but suppressed those of interleukin (IL)-6, interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Simultaneously, analysis of high-throughput sequencing indicated that IOP reduced the ratio of Firmicutes to Bacteroidetes (F/B) at phylum level, and increased the relative abundance of Bacteroides and Lactobacillus at genus level. In brief, IOP may be a promising alternative medicine for UC remedy by regulating the anti-inflammatory level, the anti-oxidative ability and the gut microbiota composition.

OBJECTIVE: Endometritis bacterial pathogenic condition that affects both humans and animals develops in the inner lining of the uterus. Inonotus obliquus polysaccharide (IOP), an active cocktail of Inonotus obliquus, has been shown to have a relatively wide range of biological activities and can play a role in various diseases. However, from the currently reported article, there is no information about the anti-inflammatory effect of IPO in the symptoms of lipopolysaccharide (LPS)-induced endometritis. Therefore, this study carefully observed the phenomenon of IOP on the symptoms of endometritis induced by LPS in mice, elucidated the protective mechanism of IOP on the body, and clarified the potential mechanism of IOP.
METHODS: A total of 72 BALB/c female experimental mice were divided into several groups for comparison. They were the blank control group, the LPS group, the LPS+ IOP group (the effect of IOP dose on mice was also explored, divided into low, medium, and high) and LPS+ amoxicillin group. All groups except control group were infused with LPS into the uterus. The mice of LPS+ IOP groups and LPS+ amoxicillin group were orally administered with IOP or amoxicillin after LPS challenge for 3 hours. Histopathology and myeloperoxidase (MPO) activity were used to detect uterine tissue injury, and cytokine levels were used to measure uterine inflammation. The expression of toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB)-related proteins in the inflammatory signaling pathway was observed.
RESULTS: Pathological and MPO activity analyses revealed that IOP relieved LPS-induced uterine tissue injury. Quantitative reverse transcription-polymerase chain reaction was used to detect and quantitatively study the RNA information of mouse cells, which had high accuracy and sensitivity. From the test results, IOP does effectively control the release of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-1β, IL-8 and tumor necrosis factor-α (TNF-α), avoiding the body\'s immune response. Analysis of uterine tissue cell components also confirmed that the expression level of inflammatory mediator-induced nitric oxide synthase (iNOS) was also greatly reduced. Analysis of western blotting results of cell synthesis showed that IOP mainly inhibited the protein expression of TLR4 and myeloid differentiation factor 88 in the body.
CONCLUSIONS: This study proved that the mechanism of action of IOP is to inhibit the TLR4/NF-κB signaling pathway to reduce the release of pro-inflammatory cytokines from body cells, thereby alleviating the symptoms of endometritis induced by LPS. Thus, IOP may act as an effective drug in preventing and curing LPS-induced endometritis.

Type 2 diabetes mellitus is a global disease that endangers human health, and the need for the development of nontoxic treatment candidates is urgent. In the present work, one homogeneous polysaccharide from Inonotus obliquus (IN) was isolated, and the protective effect and mechanism of IN on type 2 diabetes mellitus were investigated from the aspects of the intestinal barrier. IN mainly consisted of 9 monosaccharides with a Mw of 373 kDa. IN attenuated body weight loss, alleviated pathological damage, and suppressed the production of proinflammatory cytokines. Additionally, IN repaired the intestinal barrier by upregulating the expression of Ki-67, ZO-1 and MUC2. Furthermore, the abundance of Firmicutes was significantly increased with IN treatment, while the levels of Bacteroidetes were significantly inhibited. In conclusion, IN protected against type 2 diabetes mellitus by ameliorating intestinal barrier dysfunction and might serve as a novel drug candidate for type 2 diabetes mellitus.

Excessive lipid intake will cause hyperlipidemia, fatty liver metabolism disease, and endanger people\'s health. Edible fungus polysaccharide is a natural active substance for lipid lowering. In this study, the HepG2 cell model induced by oleic acid and mice model induced by a high-fat diet was established. The lipid-lowering effects of Inonotus obliquus polysaccharide (IOP) was investigated in vivo and in vitro. Glucose (251.33 mg/g), rhamnose (11.53 mg/g), ribose (5.10 mg/g), glucuronic acid (6.30 mg/g), and galacturonic acid (2.95 mg/g) are present in IOP, at a ratio of 85.2:3.91:1.73:2.14:1. The molecular weight of IOP is 42.28 kDa. Treatment with 60 mg/L of IOP showed a significant lipid-lowering effect in HepG2 cells compared with the oleic acid-treated group. In the oil red O-stained images, the red fat droplets in the IOP-treated groups were significantly reduced. TC and TG levels of IOP-treated groups decreased. IOP can alleviate the lipid deposition in the mice liver due to high-fat diet, and significantly reduce their serum TC, TG, and LDL-C contents. IOP could activate AMPK but decrease the SREBP-1C, FAS, and ACC protein expression related to adipose synthesis in mice. IOP has a certain potential for lipid-lowering effects both in vivo and in vitro.

BACKGROUND: Diabetes mellitus is a systemic disease mainly caused by the disorder of metabolism, which has become huge threat to human health. Polysaccharides are the main active substance from Inonotus obliquus (I. obliquus) with hypoglycemic effect. This study aims to evaluate the hypoglycemic activity and investigate the molecular mechanism of I. obliquus polysaccharide (IOP) in streptozotocin (STZ)-induced diabetic mice using metabolomics based on UPLC-Q-Exactive-MS method.
RESULTS: The results showed that the oral administration of IOP in high dose (1.2 g/kg) can significantly reduce the blood glucose with 31% reduction comparing with the diabetic model and relieve dyslipidemia in diabetic mice. By UPLC-Q-Exactive-MS method and multivariate statistical analysis, a total of 15 differential metabolites were identified, including 4 up-regulated and 11 down-regulated biomarkers, of which L-tryptophan, L-leucine, uric acid, 12-HETE, arachidonic acid, PC(20:1(11Z)/14:1(9Z)) and SM(d18:0/24:1(15Z)) were exhibited an important variation, as the potential biomarkers in diabetes. Pathway analysis indicated that phenylalanine, tyrosine and tryptophan biosynthesis and arachidonic acid metabolism were prone to interference in diabetes. Moreover, leucine and proline were reversed and phytosphingosine was further reduced in diabetic mice under the intervention of IOP.
CONCLUSIONS: IOP has predominant hyperglycemic effect on STZ-induced diabetic mice via ameliorating serum profiling.