Intranasal administration of INT777 (MedChemExpress,USA) was performed as previously described [20 (link)], with some modifications: rats were administered either saline, INT777 (0.16 mg/kg), INT777 (0.48 mg/kg) or INT777 (1.44 mg/kg) intranasally (5 μL/drop) over a period of 20 mins, alternating drops every 2 min between left and right nares. The total volume delivered was 50 μL at 1 h following MCAO.
Int 777
Manufactured by MedChemExpress
Sourced in United States, China
INT-777 is a synthetic bile acid receptor agonist. It selectively activates the farnesoid X receptor (FXR), a nuclear hormone receptor that plays a key role in bile acid, lipid, and glucose homeostasis. INT-777 has been used in research to study the pharmacological effects of FXR activation.
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Lab products found in correlation
Int 747, by MedChemExpress (4 mentions)
Kt5720, by Merck Group (2 mentions)
Ab76252, by Abcam (2 mentions)
Ab134181, by Abcam (2 mentions)
Ab72608, by Abcam (2 mentions)
Ab205270, by Abcam (2 mentions)
Ab16663, by Abcam (2 mentions)
Sc 376830, by Santa Cruz Biotechnology (2 mentions)
Pb9611, by Boster Bio (2 mentions)
Ursodeoxycholic acid, by Targetmol (2 mentions)
Sbi 115, by Selleck Chemicals (2 mentions)
Sq22536, by Selleck Chemicals (2 mentions)
Sc 47724, by Santa Cruz Biotechnology (2 mentions)
Ab32072, by Abcam (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Lps from escbricbia coli 0111 b4, by Merck Group (1 mentions)
Piresneo3, by Takara Bio (1 mentions)
Human cd14 microbeads, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Mouse cd4 microbeads, by Miltenyi Biotec (1 mentions)
15 protocols using int 777
1
Intranasal Delivery of INT777 in Rats
2
TGR5 Receptor Agonist Evaluation
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LPS (from Escbricbia coli 0111:B4) and S3I-201 was purchased from Sigma Chemical (St Louis, MO). INT-777 was from MedChem Express. IL-6 was purchased from PeproTech. 23(S)-mCDCA was provided by Dr. Wendong Huang and Dr. Donna Yu (City of Hope, Duarte, CA). GPBARA (TGR5 Receptor Agonist, 3-(2-Chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethylisoxazole-4-carboxamide) was purchased from BioVision (Milpitas, CA). The pmTGR5 expression vectors were created in our laboratory. The mouse TGR5 gene was cloned into pIRESneo3 (Clontech) plasmid to generate pmTGR5 overexpression plasmid. The p65 expression vector and the phRL-TK vector were kindly provided by Xufeng Chen and Akio Kruoda (both City of Hope, Duarte, CA), respectively. The NF-κB-dependent reporter (NF-κBx3-LUC) was provided by Dr. Peter Tontonoz (UCLA, Los Angeles, CA) and Dr. Bruce Blumberg (UCLA, Los Angeles, CA).
3
Modulation of BMDC and T-cell responses by bile acids
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BMDCs from the mice were extracted and cultured as previously described 26 (link). Briefly, 5×105 BMDCs were stimulated using lipopolysaccharides (LPS) (1 μg/mL, Sigma-Aldrich) and bile acids (10 µM, Sigma-Aldrich) or INT-747(100 µM, MedChem Express, USA) or INT-777 (100 µM, MedChem Express) for 24 h.
Mice CD4+ T cells were isolated using mouse CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cocultured with BMDCs pretreated with LCA or INT-777 at a ratio of 5:1 (CD4+ T cells: BMDCs) for 5 days.
Human CD14+ monocytes in PBMCs were isolated using Human CD14 microbeads (Miltenyi Biotec). CD14+ monocytes were incubated with 50 ng/mL IL-4 (AcroBiosystems, Newark, NJ, USA) and 100 ng/mL GM-CSF (AcroBiosystems) to induce DC maturation. 7 days later, 5×105 MD-DCs were treated with 100 ng/ml LPS and 10 µM LCA or 100 µM INT-777 for 24 h.
Mice CD4+ T cells were isolated using mouse CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cocultured with BMDCs pretreated with LCA or INT-777 at a ratio of 5:1 (CD4+ T cells: BMDCs) for 5 days.
Human CD14+ monocytes in PBMCs were isolated using Human CD14 microbeads (Miltenyi Biotec). CD14+ monocytes were incubated with 50 ng/mL IL-4 (AcroBiosystems, Newark, NJ, USA) and 100 ng/mL GM-CSF (AcroBiosystems) to induce DC maturation. 7 days later, 5×105 MD-DCs were treated with 100 ng/ml LPS and 10 µM LCA or 100 µM INT-777 for 24 h.
4
Bile Acid Administration in Mice
5
TGR5 Signaling Pathway Regulation
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Antibodies plasmids and chemicals used in the study were listed: GAPDH (sc-47724, Santa cruz biotechnology, for western blotting), YAP1 (ab205270, abcam, for western blotting; sc-376830, Santa cruz biotechnology, for Immunofluorescence (IF) and Immunohistochemistry), p-YAP(Ser127) (ab76252, abcam, for western blotting), CYR61 (26689-1-AP, proteintch, for western blotting), c-Myc (ab32072, abcam, for western blotting), CyclinD1 (ab16663, abcam, for western blotting), LaminB1 (PB9611, Boster, for western blotting), RhoA (#2117, CST, for western blotting), ROCK1 (ab134181, abcam, for western blotting), PKA C-ɑ(#4782, CST, for western blotting), TGR5 (ab72608, abcam, for western blotting, for IF and Immunohistochemistry), and Ki67 (#9449, CST, for Immunohistochemistry). Ursodeoxycholic acid was obtained from Target Mol (Shanghai, China), while INT-777 was obtained from Medchemexpress. TGR5 receptor antogonist SBI-115, H89, and SQ22536 were purchased from Selleck, while KT5720 was purchased from Sigma.
6
Intranasal Administration of INT-777 in SAH
7
Intranasal drug delivery after SAH
8
Intrathecal Drug Injection Protocol
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For single intrathecal injection, direct transcutaneous intrathecal injection was performed following previous work.28 , 29 Briefly, a 25‐Ga 3 10 needle connected to a Hamilton syringe was inserted into the tissues between the dorsal aspects of L5 and L6 of mice, perpendicular to the vertebral column. The injection was considered successful if a tail flick was observed immediately. Then, 5 μL INT‐777, INT‐747, bicuculline SBI‐115, or Z‐guggulsterone (all the above drugs were purchased from MedChemExpress) was administered. For repetitive INT‐777 injection, the drug was injected intrathecally via a polyethylene 10 (PE‐10) tube, the tip of which was placed on the spinal lumbar enlargement level 1 week before. Whether the tube was correctly placed in the intervertebral space was confirmed by paralysis in the bilateral hind limb after a 2% lidocaine injection (3 μL) through the catheter within 30 s.
9
Mitochondrial Dynamics Modulation in Diabetic Cardiomyopathy
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Mdivi-1, 3-bromopyruvic acid, INT-777, 3-methyladenine, Rapamycin, BAPTA-AM, and Z-VAD (OMe)-FMK were procured from MedChemExpress (NJ, United States). MitoBright LT Red was obtained from DOJINDO (Kumamoto, Japan). Glucose, streptozocin (STZ) and Evans Blue were obtained from Sigma-Aldrich (St. Louis, United States). Fluo-4 AM, Cell counting kit-8 (CCK8), JC1 assay kit and cell mitochondria isolation kit were obtained from Beyotime (Shanghai, China). The TUNEL assay and haematoxylin-eosin (HE) staining kits were obtained from Roche (Mannheim, Germany) and BOSTER (Wuhan, China), respectively. The primary antibodies used were as follows: PKC δ, Phospho-PKCδ, HK2, LC3B, P62, DRP1 (Abcam, Cambridge, United States); Parkin, PINK1, Phospho-DRP1, Cleaved caspase-3 (Cell Signaling Technology, Danvers United States), Bax, β-actin, COX4 (Proteintech, Wuhan, China); and TOMM20 (Santa, CA, United States). information on reagents and antibodies is shown in Supplementary Tables S1, S2 .
10
LPS-Induced Inflammation Modulation
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LPS (Escherichia coli strain O111:B4) was obtained from Sigma (L2630; Sigma, St. Louis, MO, USA). PZH powder was obtained from Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd (Zhangzhou, China). The powder was dissolved in phosphate buffered solution (PBS) and ultrasonicated at 70 Hz for 40 min. Then, the supernatant was collected for in vitro assays, and the suspension was used for the in vivo assays. Stattic (Cat# HY-13818), INT-777 (Cat# HY-15677), INT-767 (Cat# HY-12434) and GW4064 (Cat# HY-50108) were purchased from MedChemExpress (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO). Cholestyramine resin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Antibodies against phospho-p38 (Cat# 4511s), p38 (Cat# 9218s), phospho-JNK (Cat# 4668s), JNK (Cat# 9252s), phospho-ERK1/2 (Cat# 4377s), ERK1/2 (Cat# 9102s), phospho-p65 (Cat# 3033s), p65 (Cat# 8242s), A20 (Cat# 5630s), and STAT3 (Cat# 9139s, CST) were purchased from Cell Signalling Technology (Boston, MA, USA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat# AC002), β-actin (Cat# AC002) and phospho-STAT3 (Cat# AC004) were obtained from ABclonal Technology (Wuhan, China). Antibodies against TGR5 (Cat# ab72608) were obtained from Abcam (Cambridge, UK).
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