Tauroursodeoxycholic acid

Aldo, rapamycin (RP), chloroquine (CQ), 3-methyladenine (3-MA), tunicamycin (Tun), tauroursodeoxycholic acid (TAUDC), and anti-β-actin antibody were purchased from Sigma (St Louis, MO). Antibodies against LC3, Akt, p-Akt, mTOR, p- mTOR, S6K1, p-S6K1, 4EBP1, p-4EBP1, GRP78, GRP94, CHOP, FOXO1, p-FOXO1, Rab5, and Rab7 were purchased from Cell Signaling Technology (Beverly, MA). Anti-Podocin, anti-Nephrin, and anti-p62 antibodies were obtained from Abcam (Cambridge, MA). P300, Ac-FOXO1, and nestin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Autophagy, ER stress, and JNK pathway were blocked by pretreatment of cultured cells for 6 h with 3-MA (10 mM), Baf A1 (50 nM), Tauroursodeoxycholic acid (TUDCA, 2.5 mM), SP600125 (20 μM) which purchased from Sigma-Aldrich (MO, USA). Cells were cultured in a 6-well plate, and then CHOP shRNA, Atg5 siRNA, and corresponding scramble siRNA were transfected into cells using Lipofectamine 2000 (Invitrogen, CA, USA) for 48 h, respectively.

Cholic acid (CA), glycoCholic acid (GCA), tauroCholic acid (TCA), chenodeoxyCholic acid (CDCA), glycochenodeoxyCholic acid (GCDCA), taurochenodeoxyCholic acid (TCDCA), ursodeoxyCholic acid (UDCA), glycoursodeoxyCholic acid (GUDCA), tauroursodeoxyCholic acid (TUDCA), deoxyCholic acid (DCA), glycodeoxyCholic acid (GDCA), taurodeoxyCholic acid (TDCA), taurolithoCholic acid (TLCA), and cortisone acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). α-MuriCholic acid (α-MCA) and tauromuriCholic acid (TMCA) were purchased from Steraloids Inc (Newport, RI, USA). All other reagents and solvents were of high performance liquid chromatography (HPLC) grade. Deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). S. baicalensis Georgi (Hebei Province), G. uralensis Fisch (Inner Mongolia of China), P. lactiflora Pall (Anhui Province), and Z. jujuba Mill (Henan Province) were authenticated by Dr. Ehu Liu (State Key Laboratory of Natural Medicine, China Pharmaceutical University, China).

Pharmacodynamic and pharmacokinetic venous blood samples were collected in serum separator and EDTA tubes. Plasma samples were rapidly separated by centrifugation at 4°C. The serum separator tubes were left on the bench at room temperature for the blood to clot before centrifugation. Plasma and serum was stored at −70°C until they were analyzed. Faecal samples were collected while in the unit and stored at −70°C until they were analyzed.
Plasma total and active GLP-1_7–36, total PYY and GIP concentrations and plasma glucose levels were measured by BioAgilytix Labs (Durham, NC) using the assays shown in Table 1.
Venous serum samples, Entero-Test bile samples and faecal samples were analyzed for primary, secondary and conjugated bile acids. Bile acid (BA) standards, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), glycochenodeoxycholic acid (GCDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), lithocholic acid (LCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurolithocolic acid (TLCA), tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA), were purchased from Sigma Aldrich (Gillingham, UK) and were ≥95% purity. The internal standards, cholic acid (24-¹³C) and deoxycholic acid (24-¹³C), were obtained from Cambridge Isotope Laboratories (Massachusetts, USA) and were 99% pure.

Methanol (MeOH), acetonitrile and formic acid were of HPLC grade. Analytical grade of NaOH, propan-1-ol, pyridine, hexane and propylchloroformate (PCF) were purchased as well from Sigma-Aldrich (Saint Quentin Fallavier, France). Deionized water comes from a Milli-Q Elix system fitted with a LC-PaK and a MilliPak filter at 0.22 μm (Merck Millipore, Guyancourt, France). The following bile acid standards: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), lycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), and taurolithocholic acid (TLCA) and ammonium acetate were purchased from Sigma Chemical (St. Louis, MO, USA). All tryptophan reference, isotope labeled metabolites and SCFAs (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, acetate-D3, butyrate-13C2 and valerate-D9) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The stock solutions of bile acids, tryptophan metabolite and SCFAs were prepared separately in methanol at the concentration of 10 mmol/l.