Heparin sodium

Primary human umbilical vein endothelial cells (HUVECs) were cultured in M199 Medium (HyClone, South Logan, UT) supplemented with 3.2 ng/ml β-ECGF (Sigma, Billerica, MA), 0.108 mg/ml heparin sodium (Solarbio, Tongzhou, BJ), 2.5 ng/ml thymidine (Solarbio, Beijing), and 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel). The osmolality of M199 medium (control) was about 270 mosmol/kg. To elevate the osmolality of M199 medium, NaCl (Sigma, Billerica, MA) was added into the control medium, with a final osmolality at 290, 310, 330, and 350 mosmol/kg. For all experiments except the long-term proliferation experiments, the logarithmically growing HUVECs between passages 2 and 6 were used. Human THP-1 monocytes were purchased from YiYuan (Guangzhou, China), and grown in RPMI 1640 medium supplemented with 10% FBS (Gibco, Grand Island, NY). Cells were maintained at 37 °C with 5% CO₂ in a humidified atmosphere.
Eight-week male apolipoprotein E-deficient (ApoE^−/−) mice weighed at 20~25 g were purchased from Research Institute of Surgery at Daping Hospital, the Third Military Medical University. The mice were fed with a normal salt diet (0.8% NaCl) for 2 weeks, and then divided into two groups. Group 1 was kept on with a normal salt diet (0.8% NaCl), whereas group 2 was fed with a high-salt diet (8% NaCl). All mice had free access to water.

Aconitine and ALO were provided by Chengdu EFA Biotechnology Co., Ltd. (Chengdu, China). Collagenase type II (batch number: 44N15308B) and bovine serum albumin (BSA, batch number: 8398F) were purchased from Worthington Co. (NJ, United States) and ICN (CA, United States). CsCl (batch number: 101368097), TEA.Cl (batch number: 101394223), taurine (batch number: 101282014T), MgATP (batch number: 1001571838), and HEPES (batch number: 1001621577) were acquired from Sigma-Aldrich (St. Louis, MO, United States). Heparin sodium (batch number: 301A028) and glucose (batch number: 405A096) were obtained from Solarbio Company (Beijing, China). Egtazic acid (EGTA, batch number: 101257840) and tetrodotoxin (TTX, batch number: AF02) were purchased from Sigma-Fluka Company (St. Louis, MO, United States) and AFFIX Company (Kfar Yona, Israel), respectively. All other reagents were of analytical grade.

Doxorubicin (Solarbio, Beijing, China), formaldehyde (Solarbio, Beijing, China), heparin sodium (Solarbio, Beijing, China), acetonitrile (Merck, Germany), formic acid (CNW, Germany), BCA Kit (Solarbio, Shangahi, China), Tris (Sigma, USA), SDS (Bio-Rad, USA), phenylmethylsulfonyl fluoride (Solarbio, Beijing), phosphatase inhibitor (Solarbio, Beijing, China), RIPA buffer (Solarbio, Beijing, China), PTP1B (Abcam, USA), IRS-1, P-IRS, HK2, HIF-1α (Cell Signaling Technology, Inc., USA), Nrf2 (Abcam, USA), NH4HCO3 (Sigma, USA), High pH Reversed-Phase Peptide Fractionation Kit (Pierce, USA), TMT 6/10 plex Isobaric Label Reagent (Thermo, USA) were used.

Human umbilical vein endothelial cells (HUVECs) were cultured with the endothelial growth medium. This medium was prepared by blending the following supplements, 2.5 ng/mL Thymide (Solarbio, Beijing, China), 0.108 mg/mL Heparin sodium (Solarbio, Beijing, China), 3.2 ng/mL β-Endothelial Cell Growth Factor (β-ECGF, Sigma, Billerica, MA) and 10% Fetal Bovine Serum (FBS, Biological Industries, Kibbutz Beit Haemek, Israel) with M199 medium (HyClone, South Logan, UT). Osmolality in normal endothelial growth medium was around 270 mosmol/kg, and then gradually elevated by adding NaCl (Sigma, Billerica, MA). The HUVECs at passages 2 - 6 were used in these experiments. Human THP-1 monocytes were maintained in RPMI 1640 medium (HyClone, South Logan, UT) and split 1:2 - 1:3 every 4 - 5 days at a density of three million per mL culture.
Local government authorities approved these animal experiments, according to internationally accepted laws. The apolipoprotein E-deficient (ApoE^-/-) mice (8-week old) were purchased from Daping Hospital (Chongqing, China), and fed for another two weeks to adapt to the environment. We randomly divided male ApoE^-/- mice into normal-salt chow (0.8% NaCl) and high-salt chow (8% NaCl) groups.

Reference substances salidroside (product no. 181020) and DL-anabasine (product no. 21J064-C2) were obtained from the Beijing Century Aoke Biotechnology Co., Ltd. (Beijing, China). Chlorogenic acid (product no. 110753–201716) was purchased from the China National Institutes for Food and Drug Control (Beijing, China). Protocatechuic acid (product no. MUST-20110310) was procured from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). JGL extract (product no. 190806) and medicinal materials of the CDP group were procured from Guizhou Yibai Pharmaceutical co., LTD (Guiyang, China). Heparin sodium (product no. 125P028) was procured from Beijing Solarbio Science & Technology co., Ltd. (Beijing, China). High-performance liquid chromatography grade acetonitrile and methanol were purchased from Merck & Co., Inc. (Darmstadt, Germany). Formic acid was purchased from Thermo Fisher Scientific Inc. (Shanghai, China). Distilled water was obtained from Guangzhou Watson’s Food & Beverage Co., Ltd. (Guangzhou, China). All other solvents used were of analytical grade and are commercially available.

AGNHP (Batch No.19011426, 3 g/pill) was purchased from Beijing Tongrentang Technologies Co., Ltd. (Beijing, China). Water lapped realgar (Batch No.160319, purity of 92.86%) was obtained from Sanmenxia Yuhuangshan Pharmaceutical Co., Ltd. (Henan, China). Water lapped cinnabar (Batch No. 20181201, purity of 96.23%) was bought from Fenghuang Hongfei cinnabar Pharmaceutical Co., Ltd. (Hunan, China). Nitric acid, sulphuric acid, hydrochloric acid, perchloric acid, potassium borohydride, potassium dichromate, potassium hydroxide, potassium hydroxide, thiourea, ascorbic acid and sodium carboxymethyl cellulose (CMC-Na) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Heparin sodium was supplied by Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Arsenic and mercury standard solutions (1000 mg/L) were obtained from o2si smart solutions Co., Ltd. (Charleston, United States) and Guobiao Testing and Certification Co., Ltd. (Beijing, China), respectively. Deionized water was produced by a PCDX-F10 water purification system purchased from Pincheng Science and Technology Co., Ltd. (Chengdu, China).