Comparison of extraction processes, characterization and intestinal protection activity of Bletilla striata polysaccharides.

We optimized the extraction process of Bletilla striata polysaccharides using orthogonal design, Box-Behnken design (BBD), and genetic algorithm-back propagation (GA-BP), then compared and evaluated them to confirm that the combination of BBD and GA-BP neural networks was capable of increasing polysaccharide yields and antioxidant activity. The optimal extraction parameters were as follows: liquid-to-solid ratio of 15 mL/g, extraction power of 450 W, and extraction time of 34 min. Under these conditions, the polysaccharide yield and antioxidant activity were 8.29 ± 0.50 % and 26.20 ± 0.28 (mM FE/mg). Subsequently, the polysaccharide was purified to obtain purified Bletilla striata polysaccharides 1 (pBSP1) with a Mw of 255.172 kDa. Scanning electron microscope (SEM), ultraviolet-visible detector (UV), fourier transform infrared spectrometer (FTIR), high performance liquid chromatography (HPLC), X-ray diffraction (XRD), nuclear magnetic resonance (NMR) and periodate oxidation were used to analyze the structure of pBSP1. The results showed pBSP1 had a smooth surface and a rough interior, with a composition of α-D conformation glucose (18.23 %) and ß-D conformation mannose (53.77 %), and an amorphous crystal structure. According to the results of thermogravimetric and rheological tests, pBSP1 exhibits good thermal stability and viscoelastic behavior. Furthermore, pBSP1 protected lipopolysaccharide (LPS)-induced GES - 1 and Caco2 cells, the results showed pBSP1(400 µg/mL) lowered TEER synthesis in Caco2 cells as well as apoptosis and reactive oxygen species (ROS) production in both cells, indicating that pBSP1 may have an intestine protective effect.

The repeated administration of rhIL-12 for 14 weeks in rhesus monkeys: A toxicity assessment.

Interleukin-12 (IL-12) is known to exert antitumor immune effects by promoting the activation and proliferation of T cells and NK cells within the immune system. However, clinical trials have observed systemic toxicity associated with the administration of IL-12. This has shelved development plans for its use as a cancer therapeutic drug. Therefore, it is critical that we perform a systematic evaluation of the toxicity and safety of repeated IL-12 administration. In this study, we conducted a comprehensive evaluation of the toxicity and safety of repeated rhIL-12 (recombinant human interleukin-12) administration in rhesus monkeys by assessing its effects on the immune system, organ function, and vital signs. Rhesus monkeys were subcutaneously injected with 0.5, 2.5, and 12.5 µg/kg of rhIL-12 for up to for 14 consecutive weeks. The low dose exhibited no signs of toxicity, whereas animals receiving higher doses displayed symptoms such as loose stools, reduced activity, anemia, and elevated liver function indicators (AST and TBIL). Following three administrations of 12.5 µg/kg, high dosing was adjusted to 7.5 µg/kg due to manifestations of symptoms like loose stools, decreased activity, and huddling in the cage. Furthermore, rhesus monkeys exhibited marked immunogenic responses to recombinant human interleukin-12 (rhIL-12). However, based on overall study findings, the No Observed Adverse Effect Level (NOAEL) for the subcutaneous injection of rhIL-12, when repeatedly administered for 3 months in rhesus monkeys, was considered to be 0.5 µg/kg. The Highest Non-Severely Toxic Dose (HNSTD) was considered to be 7.5 µg/kg.

IL-12 inhibits postoperative residual tumor growth in murine models of sarcoma and renal carcinoma.

Surgical tumor removing is the most common procedure after a confirmed cancer diagnosis with no detected metastasis. Surgery can reduce tumor burden and address pathologic changes caused by local compression of tissues by the tumor. This lowers the chances of tumor cell spreading and creates more favorable conditions for further treatment. However, not all tumor cells can be eliminated through surgery. Even in the early stages of the disease, tumor cells often metastasize and cannot be identified by current detection methods. These tiny, disseminated tumors are often the cause of tumor recurrence. There is currently a lack of effective treatment options that can completely prevent tumor recurrence after surgery. To simulate the actual clinical situation, we selected murine-derived tumor cell lines S180 and Kcc853 to establish a post-transplantation residual tumor model in mice. Surgery was performed on mice inoculated with tumors. Tumor tissue was partially excised to set up the postsurgical residual tumor models. The model simulated the clinical situation where tumor cells were not completely eliminated or there were small tumors that had metastasized before surgery. IL-12 was injected to observe its effect on residual tumors or metastatic microtumors. The administration of IL-12 after surgery can significantly inhibit the growth of residual tumors and metastasis, improve the postoperative tumor-free rate and address the problem of tumor recurrence caused by the growth of residual tumors and micro-metastasis. Therefore, the use of IL-12 antitumor cytokine combined with surgery can effectively inhibit tumor recurrence. Low-dose IL-12 (1-10 ng/kg in humans) can inhibit residual tumor growth.

Transmembrane routes of cationic liposome-mediated gene delivery using human throat epidermis cancer cells.

For studying the mechanism of cationic liposome-mediated transmembrane routes for gene delivery, various inhibitors of endocytosis were used to treat human throat epidermis cancer cells, Hep-2, before transfection with Lipofectamine 2000/pGFP-N2 or Lipofectamine 2000/pGL3. To eliminate the effect of inhibitor toxicity on transfection, the RLU/survival rate was used to represent the transfection efficiency. Chlorpromazine and wortmannin, clathrin inhibitors, decreased transfection efficiency by 44 % (100 µM) and 31 % (100 nM), respectively. At the same time, genistein, a caveolin inhibitor, decreased it by 30 % (200 µM). Thus combined transmembrane routes through the clathrin and caveolae-mediated pathways were major mechanisms of cell uptake for the cationic liposome-mediated gene delivery. After entering the cells, microtubules played an important role on gene delivery as vinblastine, a microtubulin inhibitor, could reduce transfection efficiency by 41 % (200 nM).

[Research on cationic vector-mediated RNAi].

In order to study the efficiency of small interfering RNA (siRNA) transfer mediated by cationic liposome, we used luciferase siRNA to evaluate the gene silencing activity in the Hep-2 cells, which were stably transduced with a luciferase gene. The pDNA transfection was studied, and siRNA arrearage assay was conducted to determine the capability of cationic liposome with siRNA. Different concentrations of siRNA was used to silence luciferase gene' activity, and then the result was examined by microplate reader. Cell viability was analyzed after transfection by MTT assay. The results suggested that Lipofectamine 2000 could transfer the pDNA efficiently, and have strong binding capacity with siRNA. The silencing efficiency of luciferase was obtained with low concentration of siRNA. The cell viability was influenced by RNA interference (RNAi) very slightly, but the cell survival rate decreased with the increase of siRNA concentrations. It was well concluded that by optimizing the experimental conditions, cationic liposome can transfer low concentration siRNA to silence target gene's activity efficiently.