Bawei chenxiang wan

BCW (CAS18017A) used in our experiment was provided by Tibet Ganlu Tibetan Medicine Co.,Ltd. (Lhasa, Tibet, P. R. China), and we adopted the clinical approach of BCW administration (crushing and dissolving with water) for both in vivo and in vitro experiments. BCW was administered to rats via an animal gavage needle.

Chemicals

SU5416(CAS 204005–46-9) and Cell Counting Kit-8 (CCK-8) (CAS C0005) purchased from TargetMol, Trimetazidine (TMZ) was obtained from Servier (Tianjin). 3-TYP (S8628) was purchased from selleck.cn. β-Sitosterol (CAS 83–46–5), luteolin (CAS 491–70–3), Kaempferol (CAS 520–18–3), Costunolide (CAS 553–21–9), Naringenin (CAS 480–41–1),and Quercetin (CAS 117–39–5)were purchased from Chengdu Ruifensi Biotechnology Co., Ltd. (Sichuan, P. R. China).

Antibodies

Target antigen

Vendor or Source

Catalog #

Working concentration

Glut4

Immumoway

YT5523

1:1000

Citrate Synthetase

Abcam

Ab129095

1:1000

CTP1α

Abcam

ab234111

1:1000

SIRT3

Immumoway

YT4304

1:1000

HIF1α

Abcam

Ab179483

1: 500

PDK1

Abcam

ab202468

1:1000

PDK2

Abcam

ab68164

1:1000

PDK4

Abcam

ab214938

1:1000

PDHA1

Abcam

ab110334

1:1000

PDHA1(phosphor S293)

Abcam

ab177461

1:1000

β-actin (Mouse)

Cell SignalingTechnology

#3700

1:1000

α-Tubulin(Rabbit)

proteintech

11,224-1AP

1:1500

GAPDH (Mouse)

BeiJing TDY Biotech CO.,Ltd

TDY042C/F

1:1500

Anti-mouse IgG

Cell Signaling Technology

#7076

1:1500

Anti- rabbit IgG

Cell Signaling Technology

#7074

1:1500

Chromatographic conditions and instrumentation

Methods were performed as previously described [16]. A validation HPLC method was applied onto a Shimadzu (Kyoto, Japan) LC-20AT system. Extraction method of test substance: Weigh 2.0g of sample and put it into volumetric flask, 25ml of methanol and 25ml of water were added separately, and ultrasonicate (power 250W, frequency 33 kHz) for 30 min; stand and filter detection conditions: acetonitrile: 0.2% phosphoric acid water, 0–8 min 9% acetonitrile; 8–20 min 9–60% acetonitrile; 20–55 min 60%; 55–56 min 60%–9%; 56–66 min 9% acetonitrile; detection wavelength: 225nm; flow rate: 1.00 ml/min; and loading volume: 20μL.

Cell culture

H9C2 cells were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences. The cells were cultured in DMEM (SH30022.01B, Hyclone) supplemented with 10% fetal bovine serum (FBS, 10,270–106, Gibco) in an atmosphere containing 5% CO2 and 95% air at 37 °C. The medium was replaced every 24h, and the cells were subcultured or cryopreserved when the confluence reached 80%-90%. Under the conditions of cells to be in good condition cells will be diluted to 1 × 105 cells / mL, 100μl/1mL per well, evenly added into 96-well plates / 6-well plates, and cultured for 24h. After the cells reached logarithmic growth stage, the following experimental procedures were performed and conventional culture for 24h. After that, the cells of each group were detected, and the experiment was repeated three times.

Screening of CoCl2 effective concentration and action time

According to relevant literature [17], different concentrations of CoCl2 were set to screen the conditions for modeling and cell viability. The specific methods were as follows: When the cells reached the logarithmic phase of growth, the cells were washed with PBS, trypsinized and resuspended. The cells were diluted to a density of 1 × 105 cells /mL, and 100μL of the cell solution in each well was inoculated in a 96-well plate and cultured in a CO2 incubator for 24 h. Different concentrations of CoCl2 (400μM, 600μM) solution were added, and the cell survival rate was detected by CCK-8 after 12h and 24h of culture.

Screening of the effective concentration of 3-TYP

Different concentrations of 3-TYP (40μM, 50μM, 60μM) were added to the cell culture medium, and after 12 h of culture, cell RNA and protein were extracted for SIRT3 detection.

Safety range screening of BCW

After 600μM CoCl2 was added into the cell culture medium, different concentrations of BCW (800mg/L, 400 mg/L, 200 mg/L, 100 mg/L) were added to the cell culture medium for 12 h, and the cell survival rate was detected by CCK-8.

Group of cells

The cell experimental groups were categorized as follows: control group, CoCl2 group, CoCl2 + BCW group, CoCl2 + 3TYP group, CoCl2 + 3TYP + BCW group, and CoCl2 + 3TYP + TMZ group.

Animals

Male specific pathogen-free Sprague Dawley (SD) rats aged 2–3 months were obtained from the Xian Jiaotong University Animal Center (SCXK (shaan) 2018–003, Xian, China). Rats were housed in the Xizang Minzu University Laboratory Animal Center with a 12h–12h light–dark cycle. They were fed regular chow and purified water ad libitum. The animal experiment was conducted following the internationally accepted laboratory animal use and care principles. It is reviewed by the Ethics Committee of Xizang Minzu University (Ethics Approval No. 20200–7). The total of 50 SD male rats with an average weight of 180-220g were randomly divided to the following groups (n = 10/group): Control, RVH group, RVH + BCW0.8 g kg−1d−1 group, RVH + BCW0.4 g kg−1 d−1 group, RVH + TMZ group.

Vascular endothelial growth factor inhibitor SU5416 was injected subcutaneously at a one-time rate of 20mg/kg + chronic hypoxia (SuHx) simulated oxygen chamber (5000m altitude) for 4 weeks, 23h a day, and the remaining 1h was taken out, fed with water and replaced with bedding material.Animals in the control group were raised in an environment outside the cabin (300m local altitude).

Pulmonary artery pressure, right ventricular pressure was measured by floating catheter

The polyethylene (PE) catheter with transducer at the end was filled with heparin saline. After the air bubble was drained, the transducer was connected to the Power Lab multi-channel electrophysiological recorder, and the pressure waveform curve was simultaneously recorded by Lab Chart 8.0 software. After the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (60 mg/kg), the rats were fixed on the anatomical plate in the supine position. Preserved skin, separated the right subcutaneous tissue, expose the right external jugular vein and dissociated.The PE tube was inserted through the incision after cutting out the V-shaped incision, and the waveform of venous pressure was observed on the recorder. After that, mRVP and mPAP were measured in each group.

Measurement of right ventricular hypertrophy index in rats

Following hemodynamic parameter measurements, the weight of the whole heart was obtained. The right ventricle was then separated from the left ventricle plus septum (LV + Septum). RV hypertrophy was assessed by the Fulton index (FI; ratio of RV to LV + septum weights). All collected tissues were either flash frozen in liquid nitrogen or fixed with 4% paraformaldehyde at room temperature for further analysis.

Pathological HE staining of myocardial tissue

The maximal cross sections of the hearts were perfused with normal saline and then fixed in 4% paraformaldehyde for 24 h. Serial paraffin sections about 0.4μm thick were prepared by rinsing with running water, dehydration, transparency, and embedding in paraffin. Paraffin sections were routinely dehydrated. HE staining was performed with hematoxylin for 10 min at room temperature, followed by a rapid rinse with tap water for 30-60s. The cells were differentiated with 1% alcohol hydrochloride for 1s, and then rinsed with tap water for 60s. The cells were stained with eosin for 5–10 min at room temperature. Gradient ethanol dehydration; Xylene transparent; Seal with neutral gum. Finally, the sealed paraffin sections were observed under a light microscope and photographed.

ELISA protein analysis

Rat ELISA kits (BNP, CK-MB, LDH, PFK, acetyl-CoA and pyruvate) from mlbio (Shanghai, China) was used accordingly to the manufacturer’s recommendation. Briefly, 50μL of each standard and sample was added into each well of a 96-well plate, followed by 100μL of 1X HRP–streptavidin solution and incubated for 1h at 37℃ while gently shaking. After 1 h of shaking at 37℃, washed 5 times, and 50μL A, B substrate Reagent added. After 15 min of 37℃ incubation in the dark and the addition of 50μL of Stop Solution to each well, the plate was immediately read at 450nm.

Determination of lactic acid and free fatty acids

The arterial blood samples harvested from all groups were centrifuged at 3,000 rpm for 10 min at 4 °C. The plasma supernatant was obtained and stored at − 80°C. Subsequently, operational testing was performed according to the relevant kit instructions. Free fatty acids (FFA), Lactic acid (LD) obtained from Nanjing jiancheng Bioengineering Institute, Nanjing, China.

Real-time quantitative polymerase chain reaction

According to the manufacturer’s instructions, total RNA from rats heart tissue was extracted with Magzol reagent (MGBio, Shanghai, China)(RNA extraction methods are described in the Supplementary Material). Reverse transcription was performed using the two-step RT kit (Vazmye, China). PCRs were performed with the SYBR Green Quantitative PCR kit using the following primers: PDHA1, PDK1, PDK2, PDK3, PDK4, SIRT1-7 (sangon Biotch, Shanghai, China), Quantification was performed using the efficiency-corrected − 2ΔΔCT method with the housekeeping gene GAPDH as endogenous control. Data were presented as folding change over the control group.The sequences of target genes and internal reference primers are as follows:

Target Gene

Sequence

PDHA1-Rat

FORWARD: CTGAGGGTAGATGGAATGGA

REVERSE: TGGTAGCGGTAAGTCTGTAG

GAPDH-Rat

FORWARD: AGTTCAACGGCACAGTCAAGGC

REVERSE: CGACATACTCAGCACCAGCATCAC

PDK1-Rat

FORWARD: CGAGACGGCTTTGTGATTT

REVERSE: GAGATGGGACGGAACATAAAC

PDK2-Rat

FORWARD: GACTTGCAGCTCTTCTCTATG

REVERSE: CAGGCAGACTTGTTGTAGAC

PDK3-Rat

FORWARD: CCGCTCATCCGAAACATATAG

REVERSE: TTCTGGAGCTACCAGGTAATA

PDK4-Rat

FORWARD: GAACCAGCACATCCTCATATT

REVERSE: CTCAAAGGCATCTTCGACTAC

SIRT3-Rat

FORWARD: CTGCGGCTCTACACACAGAA

REVERSE: CATCACGTCAGCCCGTATGT

β-actin-Rat

FORWARD: TCGTGCGTGACATTAAAGAG

REVERSE: ATTGCCGATAGTGATGACCT

Western blotting

Western blotting assay was performed as previously described [18]. Protein extraction methods are described in the Supplementary Material. Equal amount of proteins (30mg of total proteins) from rats heart tissue was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubating with appropriate horseradish peroxidase–conjugated secondary antibodies at room temperature for1h. The blots were developed with super signal chemiluminescent substrate (Diyibio, Shanghai, China) and detected by the ChemiDocTM XRS + (Bio-Rad, CA, United States). The intensities of the blots were quantified by Quantity One software and α-Tubulin,β-actin or GADPH served as a loading control.

Data analysis

The statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software). The data were presented as mean ± SE. Statistical analysis among various groups was performed by one-way analysis of variance (ANOVA) with the Bonferroni post hoc test. In all cases, difference between groups was statistically significant when p < 0.05.