Cmc na

Thirty-six male rats were allowed to acclimatize for seven days in a controlled environment (12 h light/dark cycle, 22 ± 2 °C, relative humidity of 50% ± 5%) with unrestricted access to water and food. Rats were randomly assigned to six groups: control group, IND group, 0.3, 1.5, and 3 g/kg MD-4 and ranitidine groups (a clinical antiulcer drug). They were then given a 21-day pretreatment as follows: sodium carboxymethylcellulose water solution (CMC-Na, Sigma; 0.5%, St. Louis, MO, USA) was given to the control and IND groups throughout the trial, while MD-4 groups at 0.3, 1.5, or 3 g/kg doses (provided by the Affiliated Hospital of Inner Mongolia Minzu University, Tongliao, China) and ranitidine (30 mg/kg) were suspended in 0.5% CMC-Na solution and provided once daily through gastric administration.

The endoglucanase activity and filter paper activity (FPA) were measured using CMC-Na (Sigma, USA) and filter paper (Whatman No.1) as substrates, following the method described by Xue et al. [56 (link)]. Xylanase activity was assayed with oat spelts xylan (Sigma, USA) as the substrates [57 (link)]. The assays for these three enzymes were conducted at 50 °C for 10 min in a 0.1 M acetate buffer (pH 4.8). Subsequently, the released reducing sugars were measured using the DNS method [58 ]. The cellobiohydrolase activity was determined in 0.1 M acetate buffer at 50 °C for 30 min with pNPC (Sigma, USA) as substrates according to Liu et al. [59 (link)]. One enzyme activity unit was defined as the number of enzymes required to liberate one μmol glucose or pNP per minute under the assayed conditions. The respiratory rate of T. guizhouense under SSF was evaluated by tracking the release of CO₂. Briefly, the triangular flasks for SSF were sealed with a parafilm for 2 h before sampling, and then 20 mL of gas was extracted with a syringe. The collected gas was injected into the gas sampling bag (E-SWITCH, China), and the contents of CO₂ were determined by gas chromatography (Agilent 7890A) equipped with a Porapak Q column and a flame ionization detector (FID) according to Zhang et al. [60 (link)].

The activity of cellobiohydrolase was quantified using p-nitrophenyl-β-d-cellobioside (pNPC) (Sigma, USA) as the substrate as described previously [48 (link)]. Enzymes were incubated in 50 mM citrate buffer (pH 4.8) with 2 mM pNPC at 50 °C for 30 min. The reaction was stopped by the addition of 10% sodium carbonate and p-nitrophenol (pNP) released from pNPC was determined at 405 nm. Endoglucanase activity was measured using carboxymethylcellulose sodium salt (CMC-Na; Sigma, USA) as the substrate [49 (link)]. Reducing sugars from hydrolyzed CMC-Na were boiled with dinitrosalicylate (DNS) for 10 min and then detected at 540 nm. β-Glucosidase activity was determined as described previously using p-nitrophenyl-β-d-glucopyranoside (pNPG; Sigma, USA) as the substrate [50 (link)]. Enzymes were mixed in 50 mM citrate buffer (pH 5.0) with 5 mM pNPG at 50 °C for 30 min. 10% sodium carbonate was added to stop the reaction and pNP released from pNPG was detected at 405 nm. One unit of the enzyme activity was defined as the amount of enzyme that released 1 μmol of product (p-nitrophenol or glucose) from the substrate at 50 °C in 1 min.

Pyrrolidine dithiocarbamate (PDTC) and ST2825 were purchased from Sigma- Aldrich (St. Louis, MO, USA). Alanine aminotransferase (ALT) and aspartate amino -transferase (AST) kits were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). A tissue protein extraction kit was obtained from Keygen Biotech. Co., Ltd. (Nanjing, China). Bicinchoninic acid (BCA) protein assay kit was purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). A 3, 3′-diaminobenzidine (DAB) substrate kit was purchased from Zhong- shan Golden Bridge Biotechnology (Beijing, China); 4’, 6′-diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), sodium dodecyl sulphate (SDS) and CMC-Na were purchased from Sigma. RNAiso Plus, a PrimeScript® RT Reagent Kit with gDNA Eraser (Perfect Real Time) and SYBR® Premix Ex Taq™ II (Tli RNase H Plus) were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China).

The primary antibodies VEGF-D, LYVE-1, β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); VEGFR-3/phospho-VEGFR-3, JNK phospho-JNK, Bcl-2 and Bax antibodies were purchased from Cell Signaling Laboratories (Beverly, MA). The terminal deoxynucleotidyl transferase mediated nick-end labeling (TUNEL) assay kit was purchased from Promaga Company (Madison, WI). DMSO, Tween-20, Gelatin and CMC-Na were purchased from Sigma Chemical Co, (St. Louis, MO); Matrigel was purchased from BD Pharmingen (La Jolla, CA).

Equally harvested biomass samples of different treatments were transferred to the medium with rice straw as the sole carbon source and incubated at 37 °C to determine the growth rate. For enzyme activity assays, 1 mL of fresh spore suspension (1.0 × 10⁷ spores·mL⁻¹) of different strains was inoculated for SSF. All samples of different treatments were collected on the 4th day, and three biological replicates were collected at each sampling point. Filter paper activity (FPA) and endoglucanase activity (EG) were measured according to the method described by Xue et al. [27 (link)] with filter paper (Whatman NO.1) and CMC-Na (Sigma, St. Louis, MO, USA) as the substrates. Xylanase activity (XYL) was assayed with oat spelts xylan (Sigma, St. Louis, MO, USA) as the substrate [28 (link)]. The reaction system was executed in 0.1 M acetate buffer (pH 4.8) at 50 °C for 10 min, after which the DNS method was used to measure the released reducing sugars. The cellobiohydrolase activity (CBH) was determined in 0.1 M acetate buffer at 50 °C for 30 min with pNPC (Sigma, St. Louis, MO, USA) as the substrate according to Liu et al. [29 (link)]. One enzyme activity unit was defined as the amount of enzyme required to liberate 1 μmol glucose or pNP per minute under the assayed conditions.