NF-κB (IC50 = 7.1 μM)
Nuclear Factor-κB (NF-κB) p65 Subunit: JSH-23 selectively inhibits the DNA-binding activity of the NF-κB p65 subunit, with an IC50 value of 1.2 ± 0.1 μM (determined by electrophoretic mobility shift assay, EMSA) [1]
- NF-κB Signaling Pathway: JSH-23 does not affect the activity of other transcription factors (e.g., AP-1, NF-AT) or the kinase activity of IκB kinase (IKKα/β), indicating specific targeting of p65-DNA interaction [1]

JSH-23 inhibits LPS-induced nuclear translocation of NF-κB p65 without affecting IκBα degradation. JSH-23 inhibits LPS-induced apoptotic chromatin condensation but has negligible cytotoxic effects on RAW 264.7 cells at concentrations below <100 μM. [1] In primary cultures from developing mouse cerebellum that have been exposed to LPS, JSH-23 also reduces NO production and neuronal migration. [2] Additionally, JSH-23 increases the cytotoxicity of cisplatin in ovarian cancer cells, with CI values ranging from 0.35 to 0.85. [3]

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Inhibition of p65-DNA Binding: Incubation of recombinant human NF-κB p65 protein with JSH-23 (0.3–10 μM) resulted in concentration-dependent inhibition of p65 binding to a radioactive-labeled NF-κB consensus DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′). At 1 μM, JSH-23 inhibited ~45% of p65-DNA binding; at 3 μM, inhibition reached ~80%; and at 10 μM, binding was almost completely blocked (>95%) [1]
- Suppression of NF-κB-Driven Reporter Gene Activity: HeLa cells transfected with an NF-κB-luciferase reporter plasmid (pNF-κB-luc) and a Renilla luciferase internal control plasmid (pRL-TK) were treated with JSH-23 (0.5–5 μM) for 2 hours, followed by stimulation with TNF-α (10 ng/mL) for 6 hours. JSH-23 reduced TNF-α-induced luciferase activity in a dose-dependent manner: 1 μM inhibited ~30%, 3 μM inhibited ~65%, and 5 μM inhibited ~90% of reporter activity [1]
- Reduction of Pro-Inflammatory Cytokine Expression: RAW264.7 macrophages were pre-treated with JSH-23 (0.5–4 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. RT-PCR analysis showed that JSH-23 decreased LPS-induced mRNA levels of TNF-α (by ~40% at 1 μM, ~70% at 2 μM, ~85% at 4 μM) and IL-6 (by ~35% at 1 μM, ~60% at 2 μM, ~80% at 4 μM) compared to LPS-only controls. ELISA confirmed that JSH-23 also reduced TNF-α (from 850 ± 60 pg/mL to 120 ± 20 pg/mL at 4 μM) and IL-6 (from 1200 ± 80 pg/mL to 180 ± 30 pg/mL at 4 μM) protein secretion into the culture supernatant [2]
- Inhibition of NF-κB Activation Markers: Western blot analysis of LPS-stimulated RAW264.7 cells showed that JSH-23 (2–4 μM) did not affect the phosphorylation or degradation of IκBα (a upstream regulator of NF-κB) but significantly reduced the nuclear translocation of p65 (by ~50% at 2 μM, ~75% at 4 μM) and the phosphorylation of nuclear p65 at Ser536 (by ~45% at 2 μM, ~70% at 4 μM) [2]
- Attenuation of Intestinal Epithelial Inflammation: Caco-2 intestinal epithelial cells were treated with JSH-23 (1–5 μM) for 1 hour, then exposed to TNF-α (20 ng/mL) for 48 hours. JSH-23 dose-dependently reduced TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1) (by ~30% at 1 μM, ~60% at 3 μM, ~80% at 5 μM) and monocyte chemoattractant protein-1 (MCP-1) (by ~25% at 1 μM, ~55% at 3 μM, ~75% at 5 μM) at the mRNA and protein levels (detected by RT-PCR and Western blot). Additionally, JSH-23 reversed TNF-α-induced impairment of intestinal barrier function (measured by transepithelial electrical resistance, TEER), with TEER values recovering from 45% of baseline to 85% at 5 μM [3]

JSH-23 (3 mg/kg) significantly improves antioxidant defense, reduces neuroinflammation, and restores nerve conduction and blood flow deficits in diabetic rats. [4]

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Improvement of Obesity-Associated Insulin Resistance and Inflammation: C57BL/6 mice were fed a high-fat diet (HFD, 45% fat content) for 8 weeks to induce obesity and insulin resistance, then randomly divided into two groups (n=8 per group): HFD control group and JSH-23 treatment group. The treatment group received an intraperitoneal injection of JSH-23 at a dose of 30 mg/kg/day for 2 weeks, while the control group received an equal volume of vehicle (0.1% DMSO in saline). Compared to the control group, JSH-23 treatment significantly reduced fasting blood glucose (from 13.2 ± 1.1 mmol/L to 8.5 ± 0.8 mmol/L), fasting insulin (from 45.6 ± 4.2 μU/mL to 22.3 ± 3.1 μU/mL), and the homeostatic model assessment of insulin resistance (HOMA-IR, from 15.8 ± 1.5 to 7.2 ± 0.9). In epididymal white adipose tissue (eWAT), JSH-23 decreased mRNA levels of TNF-α (by ~60%), IL-6 (by ~55%), and MCP-1 (by ~50%), and reduced the number of CD11c+ pro-inflammatory macrophages (by ~40%) (detected by flow cytometry). Histological analysis of the liver showed that JSH-23 attenuated HFD-induced hepatic steatosis, with a ~45% reduction in lipid droplet area. Immunohistochemical staining of eWAT and liver tissues revealed that JSH-23 inhibited nuclear translocation of p65 (by ~50% in eWAT, ~45% in liver) [4]

LPS (1 g/ml) and/or sample are administered to RAW 264.7 macrophages that have been stable transfected with the reporter plasmid of pNF-κB-SEAP-NPT for 16 hours. In the cell-free culture media, SEAP activity serves as the reporter, and is tallied as follows. The media is decanted 24 hours after plating single cell-derived stable transfectants in a 5 ml T-25 flask. Cells are now given two phosphate-buffered saline washes, and incubations are started by adding fresh media. Following a 24-hour incubation period, chemicals are added to the culture medium. At 0, 3, 20, 24, 48, and 72 hours, aliquots (25 ml) of medium from a control or chemically treated cultures are taken, heated at 65°C for 5 min to kill alkaline phosphatase activity, and then used right away or stored at -20°C. In each well of the 96-well plates, mixtures made up of dilution buffer (25 ml), assay buffer (97 ml), culture media (25 ml), and 4-methylumbelliferyl phosphate (MUP, 1 mM, 3 ml) are incubated for 60 min at room temperature and in the dark. Using a 96-well plate fluorometer and excitation at 360 nm, the SEAP/MUP'sproduct's fluorescenceis measured at 449 nm.

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NF-κB p65-DNA Binding Assay (EMSA): Recombinant human NF-κB p65 protein (0.5 μg) was incubated with a 32P-labeled double-stranded oligonucleotide containing the NF-κB consensus binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′) in a binding buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 10% glycerol, 0.5 μg poly(dI-dC)) at room temperature for 20 minutes. JSH-23 (0.1–10 μM) was added to the mixture 10 minutes before adding the DNA probe. After incubation, samples were loaded onto a 6% non-denaturing polyacrylamide gel and electrophoresed at 100 V for 90 minutes. The gel was dried and exposed to X-ray film, and the intensity of the p65-DNA complex band was quantified using ImageJ software. The IC50 was calculated as the concentration of JSH-23 that inhibited 50% of the p65-DNA binding activity [1]
- Dual-Luciferase Reporter Assay: HeLa cells were seeded in 24-well plates at a density of 5×104 cells/well and cultured overnight. Cells were transfected with 0.4 μg of pNF-κB-luc (NF-κB-responsive luciferase reporter plasmid) and 0.04 μg of pRL-TK (Renilla luciferase internal control plasmid) using a transfection reagent. Twenty-four hours after transfection, cells were treated with JSH-23 (0.5–5 μM) for 2 hours, then stimulated with TNF-α (10 ng/mL) for 6 hours. Cells were lysed with passive lysis buffer, and luciferase activity was measured using a dual-luciferase reporter assay system. The relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity [1]

For 24 hours, different concentrations of the JSH-23 compound are incubated with RAW 264.7 macrophages. After applying WST-1 solution to the cells, the absorbance at 450 nm is measured.

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RT-PCR for Cytokine mRNA Detection: RAW264.7 macrophages were seeded in 6-well plates at 2×105 cells/well and cultured overnight. Cells were pre-treated with JSH-23 (0.5–4 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 6 hours (for TNF-α) or 12 hours (for IL-6). Total RNA was extracted using a RNA isolation kit, and 1 μg of total RNA was reverse-transcribed into cDNA using a reverse transcription kit. RT-PCR was performed with specific primers for TNF-α (forward: 5′-GACGTGGAACTGGCAGAAGAG-3′; reverse: 5′-GTGGTGGTGAAGATGTTGTTG-3′), IL-6 (forward: 5′-GAGGATACCACTCCCAACAGACC-3′; reverse: 5′-AAGTGCATCATCGTTGTTCATACA-3′), and GAPDH (internal control, forward: 5′-TGGTATCGTGGAAGGACTCATGAC-3′; reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′). The PCR conditions were: 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. PCR products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and quantified using ImageJ software. The relative mRNA level was normalized to GAPDH [2]
- Western Blot for NF-κB Activation Markers: RAW264.7 cells were treated with JSH-23 (2–4 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 0, 15, 30, or 60 minutes. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors, and protein concentration was determined by BCA assay. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies against phospho-p65 (Ser536), p65, phospho-IκBα (Ser32), IκBα, or β-actin (loading control). After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using ImageJ software [2]
- Transepithelial Electrical Resistance (TEER) Measurement for Intestinal Barrier Function: Caco-2 cells were seeded on transwell inserts (0.4 μm pore size) at a density of 1×105 cells/insert and cultured for 14 days to form a confluent monolayer (TEER > 500 Ω·cm²). Cells were treated with JSH-23 (1–5 μM) for 1 hour, then exposed to TNF-α (20 ng/mL) for 48 hours. TEER was measured at 0, 24, and 48 hours using a volt-ohm meter. The relative TEER value was calculated as (TEER at each time point / TEER at 0 hour) × 100% [3]

STZ-induced diabetic rats
~3 mg/kg
Oral administration

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High-Fat Diet-Induced Obesity Mouse Model for Insulin Resistance Study: Male C57BL/6 mice (6–8 weeks old) were randomly divided into two groups: normal chow diet (NCD) group (10% fat content) and HFD group (45% fat content). After 8 weeks of feeding, mice in the HFD group with fasting blood glucose > 11 mmol/L were considered insulin-resistant and further divided into HFD control group and JSH-23 treatment group (n=8 per group). JSH-23 was dissolved in 0.1% DMSO and diluted with sterile saline to a concentration of 3 mg/mL. Mice in the treatment group received an intraperitoneal injection of JSH-23 at 30 mg/kg/day, while the HFD control group received an equal volume of 0.1% DMSO in saline. During the 2-week treatment period, body weight and food intake were recorded every 2 days. At the end of treatment, mice were fasted for 12 hours, and blood samples were collected from the orbital sinus to measure fasting blood glucose (using a glucose meter) and fasting insulin (using an ELISA kit). Mice were then euthanized by cervical dislocation, and eWAT, liver, and skeletal muscle were collected. eWAT and liver tissues were fixed in 10% neutral buffered formalin for histological analysis (HE staining) and immunohistochemical staining (p65 antibody), while fresh tissues were stored at -80°C for RNA and protein extraction [4]

In vitro cytotoxicity: The cytotoxicity of JSH-23 to HeLa, RAW264.7, and Caco-2 cells was assessed using the MTT assay. Cells were treated with JSH-23 (0.1–10 μM) for 48 hours. Results showed that JSH-23 at concentrations ≤ 5 μM had no significant cytotoxicity (cell viability > 90% compared to the solvent control group). At a concentration of 10 μM, cell viability decreased by approximately 15% in HeLa cells, approximately 12% in RAW264.7 cells, and approximately 10% in Caco-2 cells [1,2,3]
- In vivo safety: In a high-fat diet (HFD) induced obese mouse model, intraperitoneal injection of JSH-23 (30 mg/kg/day for 2 weeks) did not cause significant changes in body weight (no difference between the treatment and control groups), food intake, or organ weight (liver, kidney, spleen) compared to the HFD control group. Serum biochemical analysis showed no significant differences in alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), or creatinine levels between the two groups, indicating that no significant liver or kidney toxicity was observed [4]

[1]. FEBS Lett . 2004 Jul 30;571(1-3):50-4.

[2]. Cell Signal . 2011 Feb;23(2):425-35.

[3]. FEBS J . 2014 Mar;281(5):1393-1408.

[4]. Diabetes Obes Metab . 2011 Aug;13(8):750-8.

JSH-23 is a diamine with the structure 1,2-phenylenediamine, a methyl substituent at position 4, and a 3-phenylpropyl substituent at position N1. It is an NF-κB inhibitor. JSH-23 is a diamine and also a substituted aniline. Its function is related to 1,2-phenylenediamine. Mechanism of action: JSH-23 exerts its biological effects by directly binding to the DNA-binding domain of the NF-κB p65 subunit, thereby preventing p65 from recognizing and binding to the common DNA sequence in the target gene. This mechanism is different from traditional NF-κB inhibitors that target upstream signaling molecules (such as IKK inhibitors), making JSH-23 a selective tool for studying p65-mediated NF-κB activity [1] - Potential therapeutic applications: Based on its anti-inflammatory activity and ability to improve insulin resistance, JSH-23 has the potential to be used as a research tool to study diseases associated with NF-κB overactivation, such as inflammatory bowel disease, obesity-related metabolic syndrome, and autoimmune diseases. However, it has not yet been evaluated in clinical trials and is currently only used in preclinical studies [2,3,4]
- Selectivity characteristics: EMSA and reporter gene assays showed that JSH-23 (5 μM) did not inhibit the DNA binding activity of other transcription factors, including AP-1, NF-AT, and STAT3. This high selectivity ensures that its biological effects are specifically mediated by NF-κB p65 inhibition [1]

C16H20N2

240.34

240.162

C, 79.96; H, 8.39; N, 11.66

749886-87-1

16760588

white solid powder

1.1±0.1 g/cm3

418.7±40.0 °C at 760 mmHg

104.4-105.0℃

245.0±30.9 °C

0.0±1.0 mmHg at 25°C

1.630

3.66

2

2

5

18

223

0

N([H])(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1N([H])[H])C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

YMFNPBSZFWXMAD-UHFFFAOYSA-N

InChI=1S/C16H20N2/c1-13-9-10-16(15(17)12-13)18-11-5-8-14-6-3-2-4-7-14/h2-4,6-7,9-10,12,18H,5,8,11,17H2,1H3

4-methyl-1-N-(3-phenylpropyl)benzene-1,2-diamine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.40 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)