Protein Extraction Kits were obtained from KEYGEN Biotech. indicated that TKL markedly inhibits the binding of p65 to the gene in HG-treated HK-2 cells, subsequently NSC-207895 (XI-006) suppressing transcription of the gene. In the dual-luciferase reporter assay, TKL significantly inhibited luciferase activity in cells co-transfected with p65 and a wild-type capase-9 construct instead of mutated caspase-9 constructs. Taken together, our results show that TKL helps to protect against DN by inhibiting the LOX1/NF-B/caspase-9 signaling pathway, suggesting TKL as a promising agent for treating DN. gene, is an initiator involved in the NSC-207895 (XI-006) mitochondrial apoptosis pathway . Hyperglycemia, proteinuria, and angiotensin II all help to activate nuclear factor-B (NF-B), which is a ubiquitous transcription factor capable of controlling DNA transcription, cytokine production, and cell survival [12C14]. Evidence suggests that NF-B is responsible for regulating the expression of genes involved in apoptosis . Blockading the nuclear translocation of NF-B might attenuate the expression of NF-B-related genes such as and Maxim (TK), also referred to as Tian-Hua Fen, is traditionally used for treating diabetes and its complications in Eastern Asia . Recent pharmacological studies have shown that pre-treatment with a TK extract can attenuate histopathological changes in the kidney and reduce the numbers of apoptotic cells . Evidence indicates that the ability of TK to inhibit tumor growth is likely associated with an inhibition of NF-B activity . Lectin compounds comprise the main ingredients responsible for the hypoglycemic activity of TK . lectin (TKL) is usually a RAC1 galactose-specific herb thrombin that not only has the ability to agglutinate blood cells and sperm cells, but also participates in a series of important physiological and pathological processes . In a recent, study, TKL displayed hypoglycemic effects in alloxan-induced diabetic NSC-207895 (XI-006) mice ; however, no publication has reported the protective effects of TKL against DN. We used a high-dose glucose (HG)-induced HK2 cell model and a STZ-induced DM rat DN model to investigate how TKL affects the NF-B p65/caspase-9 signaling pathways. We also discuss the possibility of developing TKL as a novel agent for treating DN. Materials and methods Chemicals and materials Cell Counting Kit-8 (CCK-8), Bicinchoninic acid (BCA) Protein Assay Kits, Annexin V-FITC Apoptosis Detection Kits, and Cell Cycle Analysis Kits were all purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Protein Extraction Kits were obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Diaminobenzidine (DAB) substrate kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) kits were provided by Roche Diagnostics (Germany). 4,6-Diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), and sodium dodecyl sulphate (SDS) were purchased from Sigma (St. Louis, MO, U.S.A.). Anti-LOX1, anti-caspase-9, anti-p65, anti-p-IKK, anti-IKK, anti-p-IkB, anti-IkB, anti-GAPDH, anti–tubulin, and anti-Lamin B primary antibodies, and horseradish peroxidase-conjugated antibody were obtained from Abcam (Cambridge, U.K.). Plasmids harboring the wild-type caspase-9 response element (WT-luciferase-caspase-9) and the corresponding mutant (MUT-luciferase-caspase-9) were purchased from Vipotion Biotechnology (Guangzhou, China). Pierce Agarose Chip Kits were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.). Extraction of TKL TKL was extracted with phosphate-buffered saline (PBS) and purified by dialysis. Briefly, Tian-Hua Fen was added to PBS at a weight to volume ratio of 1 1:30, and the TKL was extracted in a 4C refrigerator for 24 h. The mixture was then centrifuged at 4000 rpm for 10 min, and supernatant was collected. Next, the supernatant was added to a 70% ammonium sulfate answer and let sit for 24 h; after which, the lower sediment was harvested by centrifugation at 10,000 rpm. Finally, the ammonium salt in the TKL answer was removed by dialysis, and the TKL extract was dried in a vacuum freeze dryer. Cell culture HK-2 human kidney tubular epithelial cells were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China) and maintained in low-glucose DMEM supplemented with 10% FBS and antibiotics (100 IU/ml penicillin and 100 mg/ml.