TNF-α (cat. no. AF7014), Keap1 (cat. no. AF5266), Nrf2 (cat. no. AF0639) and phosphorylated (p-)Nrf2 (p-Nrf2; cat. no. DF7519) were obtained from Affinity Biosciences. GCLM (cat. no. 14241-1-AP) and GCLC (cat. no. 12601-1-AP) were obtained from ProteinTech Group, Inc. NQO1 (cat. no. SC-376023) was obtained from Santa Cruz Biotechnology, Inc. Bax (cat. no. ab32503), Bcl-2 (cat. no. ab59348) and caspase-3 (cat. no. ab13847) were obtained from Abcam. Small interfering (si)RNA and fluorescent siRNA transfection kits (cat. nos. R10043.8 and R11077.2, respectively) and HiPerFect transfection reagent (cat. no. 301702) were obtained from Ruibo Bio-Technology Co., Ltd. ELISA kits for glutathione (GSH; cat. no. CEA294Ge), malondialdehyde (MDA; cat. no. CEA294Ge), peroxidation catalase (CAT; cat. no. SEC418Ra) and angiotensin (Ang)II (cat. no. CEA005Ge) were obtained from Cloud-Clone Corp. Total antioxidation capacity (T-AOC) ELISA kit (cat. no. SU-BN31087) was obtained from Jiangsu Jingmei Biotechnology Co., Ltd. Reverse transcription-quantitative (RT-q)PCR Primer was obtained from Sangon Biotech Co., Ltd. eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (cat. no. 88-8824-00) was obtained from Thermo Fisher Scientific, Inc. LBP was obtained from Ningxia Qiyuan Pharmaceutical Co., Ltd. One-step TUNEL apoptosis in situ detection (cat. no. KGA7061) and BCA protein assay kits (cat. no. KGPBCA) were obtained from Nanjing KeyGen Biotech Co., Ltd. Human AngII (cat. no. 4474-91-3) was obtained from Merck KGaA.

When the body is stimulated by the external environment (exercise), it activates the renin-Ang system in the body. The expression of AngII and oxidative stress rise, which is manifested by increased levels of oxidase and decrease levels of antioxidant enzymes. SOD and CAT regulate oxidative stress and free radicals. When the level of oxidative stress increases, MDA can be used as an indicator of lipid peroxidation. T-AOC reflects the degree of oxidative stress and total levels of all antioxidants. Therefore, the oxidative stress state can be reflected by measuring the levels of AngII, oxidase and antioxidant enzymes in serum (25,26). ELISA kits were used according to the manufacturer's protocols and used to detect the levels of AngII in rat serum, as well of those in GSH, MDA, CAT and T-AOC in rat serum and cell culture medium using a microplate reader to measure the optical density value and plot a standard curve to calculate the concentration and reflect oxidative stress level.

Animal experiments were approved by the Animal Ethics and Use Committee of Ningxia Medical University (Yinchuan, China) in accordance with the guidelines of the Council of the Chinese Physiological Society. A total of 60 healthy male Sprague-Dawley rats (6–8 weeks, 180–220 g; Experimental Animal Center of Ningxia Medical University) are kept at room temperature (18–22°C), humidity 45–65%, with a 12 h light/dark cycle and had free access to food and water. Rat thoracic aortic endothelial cells (RTAECs) were purchased from Shanghai Saiqi Biological Engineering Co., Ltd.

Rats were divided into normal control (Control), aerobic exercise (AE), exhaustive exercise (EX) and EX + LBP groups (n=15/group). Control rats did not do any exercise, while the other three groups swam for 1 h. The period of swimming training was 5 weeks; adaptive swimming training was performed in the first week (10 min on day 1, increasing by 10 min/day to 60 min on day 6, followed by rest on day 7). For the remaining 4 weeks, rats swam for 60 min/day, 5 days/week (rest on Tuesdays and Saturdays). After the final swimming session, rats in the EX and EX + LBP groups continued to swim to exhaustion, as previously described (27). Rats in the EX + LBP group were fed with LBP at a dose of 200 mg/kg/day (Ningxia Qiyuan Pharmaceutical Co., Ltd.) after exercise. Animals were anesthetized with 10% chloral hydrate (300 mg/kg). Hemostatic forceps were used to clamp the limbs; lack of resistance and pain reflex indicated that anesthesia was successful (absence of peritonitis following the use of 10% chloral hydrate). Blood (4–5 ml), thoracic aorta and myocardium samples were collected from rats under anesthesia with chloral hydrate. Following blood centrifugation (1,798 × g at 4°C for 3 min), serum was collected and placed in a refrigerator at −80°C. Part of the myocardium and blood vessels were fixed in 4% paraformaldehyde at room temperature for 24 h; the other part of the heart and blood vessels were placed in a refrigerator at −80°C. The specimens were used for subsequent experiments.

RTAECs were cultured in DMEM (Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (Clark Bioscience; cat. no. FB35015). All cells in the experiment were cultured at 37°C, and 5% CO₂ and the subsequent test performed when the cell confluence was 70–80%. A RTAEC oxidative stress model was established to study the mechanism of LBP alleviating oxidative stress injury using the following groups: i) Blank control, cells cultured in fresh culture medium (37°C, 5% CO₂); ii) LBP, cells cultured in medium containing LBP (3,200 µg/ml) for 2 h, then fresh medium for 24 h; iii) AngII, cells cultured in fresh medium for 2 h, then medium containing AngII (1×10⁻⁶ mol/l) for 24 h; iv) AngII + LBP, cells cultured in medium containing LBP for 2 h, then medium containing AngII for 24 h. siRNA (50 nmol) was transfected with endothelial cells with assistance in HiPerfect. Specific steps are as follows: i) A single cell suspension ~1×10⁵/ml was inoculated into a 6-well plate, 1 ml per well, then 4 ml of complete medium added per well, and then placed in the incubator for 24 h. ii) The siRNA stock solution was diluted according to the manufacturer's instructions; 120 µl of the serum-free-free-free medium was added together with 5 µl of 20 µmol siRNA stock solution. iii) HiPerfect (12 µl) was slowly added to the liquid prepared in step ii, and allowed to stand for 15 min, to form a transfection complex. iv) The culture solution was discarded, and 1,863 µl of non-double anti-medium added, and then the composite from step iii slowly added. v) For subsequent related experiments, siRNA transfection was performed using the following groups: i) Blank control, as aforementioned; ii) AngII, as aforementioned; iii) AngII + LBP, as aforementioned; iv) si non-specific control (siNC), siNC was transfected into cells using HiPerFect for 24 h; v) siNC + AngII + LBP, siNC was transfected into cells using HiPerFect for 24 h, then cells were cultured in medium containing LBP for 2 h, then for 24 h in medium containing AngII; vi) siRNA, siRNA was transfected into cells using HiPerFect for 24 h; vii) siRNA + AngII + LBP, siRNA was transfected into cells using HiPerFect for 24 h, then cells were cultured in medium containing LBP for 2 h, then for 24 h in medium containing AngII.

Under anesthesia with chloral hydrate, the rat chest was opened and 3–5 ml blood was removed from the left ventricle with the tip of a 5 ml syringe to prevent hemolysis. Samples were transferred to a 15 ml centrifuge tube. After 30 min, samples were centrifuged at 4°C, 5,035 × g. After 3min, the serum was taken and stored at −80°C for follow-up experiments. Cell supernatant was collected, centrifuged at 4°C, 5,035 × g for 3 min and stored at −80°C for testing. Standards and samples were loaded into the enzyme plate, followed by incubation at 37°C for 1 h. After washing, the substrate solution was loaded into the plate, followed by incubation at 37°C in the dark for 15 min. The absorbance at 450 nm was measured after stop solution was loaded into the plate and the concentration of the sample was calculated.

Rat thoracic aorta and myocardium were fixed in 4% paraformaldehyde at room temperature for 24 h, paraffin-embedded, sectioned at 3 µm, dewaxed at 60°C for 6–8 h, then placed at room temperature and dehydrated (2 min, 95% alcohol 1 min, 90% alcohol 1 min, 80% alcohol 1 min, 75% alcohol 13 min and 50% alcohol 1 min. Sections were stained in hematoxylin for 5 min at room temperature and eosin for 2–3 min at room temperature. The sections were dehydrated with a graded series of alcohol and cleared in xylene, mounted neutral dried at room temperature before being observed under an inverted light microscope (magnification, ×400).

The rat thoracic aorta and myocardium were sectioned at 3 µm, deparaffinized in xylene (20 min), and dehydrated with an ethanol gradient. The dehydration sequence and time were absolute ethanol I and II each for 2 min, 95% alcohol immersion for 1 min, 90% alcohol immersion for 1 min, 80% alcohol immersion for 1 min, 75% alcohol immersion for 1 min, and 50% alcohol immersion for 1 min. then add proteinase K working solution, DNaseI reaction solution, TdT enzyme reaction solution and Streptomyces dropwise Avidin-TRITC-labeled working solution were added and the sections placed in a 37°C incubator for 30 min. DAPI (catalog number S2110; Beijing Solarbio Science & Technology Co., Ltd.) was added dropwise and observed under an inverted fluorescence microscope (magnification, ×400).

Rat thoracic aorta and myocardium were paraffin-embedded, dewaxed with xylene, dehydrated with alcohol gradient as aforementioned. Antigens were retrieved at 100°C for 10 min and the sections permeated and blocked with normal goat serum (1:50) at 37°C for 1 h. The sections were incubated with primary antibodies (Bax, 1:100; Bcl-2 and 1:100 and caspase-3, 1:50) at 4°C overnight, followed by incubation with the corresponding horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (cat. no. A21020; Abbkine Scientific Co., Ltd.; 1:50) and horseradish enzyme-labeled goat anti-mouse IgG (H+L) (cat. no. A21010; Abbkine Scientific Co., Ltd.; 1:50) at 37°C for 1 h and DAPI stock solution in the dark for 10 min at room temperature and observed under an inverted fluorescence microscope (magnification, ×400).

RTAEC single-cell suspensions were seeded into 24-well plates. When the cells reached 80% confluence, they were fixed with 4% paraformaldehyde for 30 min, 0.2% Triton X-100 and placed in a 37°C incubator for 30 min (to improve the permeability of the cell membrane) and the serum was blocked with goat serum (OriGene Technologies, Inc.). After incubating with the primary antibody (Nrf2, 1:100 and GCLM, 1:50) at 4°C overnight, the cells were incubated with the corresponding secondary antibody (1:50, 1 h) and DAPI stock solution for 10 min at room temperature in the dark and observed under an inverted fluorescence microscope (magnification, ×400).

Rat thoracic aorta and myocardium tissue were lysed in lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) containing 0.1% protease inhibitor, 1% phosphatase inhibitor and 1% phenylmethylsulfonyl fluoride followed by separation via SDS-PAGE (8%). Protein (50 µg) was loaded in each lane and transferred to PVDF membranes. After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were probed with TNF-α (1:500), Keap1 (1:1,000), Nrf2 (1:700), NQO1 (1:800), GCLM (1:700), GCLC (1:2,000), β-actin (1:1,000) and GAPDH (1:5,000) antibodies at 4°C overnight, washed Tween20 (1:1,000) and followed by incubation (3 times at room temperature, 15 min each time) with the corresponding secondary antibody (Goat anti-rabbit or goat anti-mouse; 1:5,000). The visualisation reagent was Drop ECL (Affinity Biosciences. USA) and the target bands were quantified by ImageJ 180 software (National Institutes of Health).

Tissues were paraffin-embedded, dewaxed in xylene, dehydrated with alcohol gradient, repaired by high temperature antigen and serum-blocked, as aforementioned, then incubated with TNF-α (1:100), Keap1 (1:100), Nrf2 (1:100), NQO1 (1:50), GCLM (1:150) and GCLC (1:100) primary antibodies at 4°C overnight and reheated at 37°C for 1–2 h the next day. Reaction enhancement solution (OriGene Technologies, Inc.) was added to the tissue sections, which were placed in a humidified box for 20 min at 37°C. Universal two-step kit (mouse/rabbit enhanced polymer method detection system) was added and incubated at 37°C for 1 h, followed by DAB color development (1 ml). Sections were washed in deionized water for 3–5 min, hematoxylin staining stock added to stain the nucleus for 2 min (room temperature) and washed 3 times in deionized water for 5 min each. The sections were differentiated in 0.5% 75% alcohol and concentrated hydrochloric acid for 5 sec, washed 3 times in water, each time for 5 min. Following washing, the sections were dehydrated 50, 75, 80, 90 and 95% ethanol, then anhydrous I and anhydrous ethanol II each for 30 sec. After transfer to xylene for 2–3 min, the sections were mounted in neutral resin. The positive results were observed using a light microscope (magnification, ×400) and the results were analyzed using ImageJ v180 (National Institutes of Health).

Flow cytometry was performed to detect intracellular TNF-α antigen. Cells cultured at 70–80% confluence were trypsinized to make a single cell suspension and centrifuged at 200 × g at 4°C 5 min. Following centrifugation, the cells were washed with 1 ml PBS, transferred to a 1.5ml EP tube, and diluted so that the number of cells per ml was 1×10⁶ cells Permeation solution was added to increase the cell permeability to ensure fluorescent labelled TNF-α antibody entered the cell and combined with the intracellular antigen. Antibody directly coupled to the intracellular antigen was added for 1 h and permeation solution was added to promote binding of antigen and antibody before centrifugation at 200 × g at 4°C 5 min. Supernatant was discarded and expression levels of TNF-α in the cells were detected by flow cytometry with PBS and PE staining. A total of 50,000 cells was collected at an excitation wavelength of 488–561 nm. The number of TNF-α-positive cells was detected at 578 nm. Finally, the percentage of positive cells in the total number of cells was calculated as the expression level of TNF-α. Each experiment was repeated 6 times independently. GraphPad prim6 (GraphPad Software, Inc.) was used for statistical analysis.

Before transfection, the cells need to grow well, and the cells need to pass resuscitation, passage, growth and fusion to the experimental state. siNrf2 was purchased from Guangzhou Ruibo Biotechnology Co., Ltd. HiPerFect was used to transfect siNrf2 (50 nmol) into endothelial cells. A single-cell suspension was prepared according to the method of cell passage, and cells were counted with an automatic cell counter. Cells (1×10⁵ cells/ml) were inoculated into a 6-well plate and incubated at 37°C for 24 h. siRNA stock solution was diluted according to the manufacturer's instructions the next day. A total of 120 µl serum-free and anti-double-antibodies medium (Beijing Solarbio Science & Technology Co., Ltd.) and 5 µl 20 µmol siRNA stock solution were mixed. A total of 12 µl HiPerFect was added and left to stand for 15 min to form a transfection complex. The culture medium was discarded, then 1,863 µl anti-bibial antibody-free medium (Thermo Fisher Scientific, Inc.) and complex were added and cultured at 37°C for 24 h). The protein level and mRNA levels were detected after 24 h. The following groups were used: Blank control, AngII + LBP, siNC, siNC + AngII + LBP, siRNA and siRNA + AngII + LBP.

A total RNA extraction kit (cat. no. DP171221; Tiangen Biotech Co., Ltd.) was used for RTAEC total RNA extraction. PrimeScript RT Master Mix (Perfect Real Time; cat. no. RR036B; Takara Bio, Inc.) was used for cDNA preparation. The reaction conditions were as follows: 37°C for 15 min, 85°C for 5 sec, 4°C. Amplification was performed using TB Green Premix Ex Taq II (Tli RNase H Plus; cat. no. RR820B; Takara Bio, Inc.). The thermocycling conditions were as follows: 95°C for 30 sec, 95°C for 5 sec, 60°C for 34 sec for 40 cycles. The primer sequences are listed in Table I. The 2^−ΔΔCq method (28) was used to calculate the mRNA expression levels of the target gene. β-actin was used as the reference gene.

SPSS22.0 (IBM Corp.) and GraphPad Prism6 software (GraphPad Software, Inc.) were used to analyze the data. Each experiment was repeated six times independently. Data are expressed as the mean ± SD. Data were analyzed using paired Student's t test or one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

ELISA was performed to study the antioxidant stress effect of LBP. Compared with the Control, there was no significant change in expression levels of AngII and T-AOC, while those of CAT increased and those of MDA and GSH decreased in the AE group. Compared with the AE group, expression levels of AngII and MDA in the EX group increased, whereas those of CAT, T-AOC and GSH expression levels decreased. These changes in MDA, T-AOC and GSH were significant. Compared with the EX group, the expression levels of AngII and MDA decreased in the EX + LBP group, whereas those of CAT, T-AOC and GSH increased (Fig. 1).

H&E staining was performed to study the protective effect of LBP on the thoracic aorta and myocardium of exhausted rats. H&E staining showed that the wall of the thoracic aorta of the Control group was intact and elastic fibers and endothelial cells were aligned. In the AE group, the interstitial wall of the thoracic aorta was slightly widened and the structure remained unchanged. Vascular endothelial cells in the thoracic aorta of the EX group were markedly lost, and the elastic fibers were broken, irregular and exhibited disordered arrangement. Compared with Ex group, the thoracic aorta endothelial cells in the EX + LBP group were more complete, the elastic fibers arranged more regularly and exhibited less injury (Fig. 2A). Myocardial H&E staining showed that in the Control group, the myocardial fibers were slender, arranged neatly and connected to each other in a network, with a small amount of connective tissue between the myocardial fibers. The nucleus was oval and located in the center of the cell. Myocardial fibers of the AE group were arranged neatly and connected in a reticular pattern, and there was a small degree of inflammatory cell infiltration between myocardial fibers. The myocardial fibers of the EX group were disordered, turbid and unclear, the horizontal stripes disappeared and a large number of inflammatory cells infiltrated. The nuclei of myocardial cells in the EX + LBP group were slightly concentrated, myocardial fibers were swollen and there were few inflammatory cells (Fig. 2B).

The anti-apoptotic effect of LBP on the thoracic aorta of exhausted rats was investigated by TUNEL staining. Compared with the Control, the positive expression of the thoracic aorta in the AE group increased slightly. Compared with the AE group, EX notably increased the positive expression of thoracic aorta blood vessels. Compared with the EX group, the positive expression of thoracic aorta blood vessels in the EX + LBP group was notably decreased (Fig. 3A). Compared with the Control, expression levels of Bax and caspase-3 in the myocardial tissue of the AE group decreased and those of Bcl-2 increased. Compared with the AE group, the expression levels of Bax and caspase-3 in myocardial tissue of the EX group increased, whereas those of Bcl-2 decreased. Compared with the EX group, the expression of Bax and caspase-3 in the myocardial tissue of the EX + LBP group decreased, whereas expression of Bcl-2 increased (Fig. 3B-D).

Western blotting was performed to investigate whether LBP enhances the Keap1/Nrf2 antioxidant stress pathway in the thoracic aorta and myocardium of exhausted rats. Compared with the Control, the expression levels of TNF-α and Keap1 in thoracic aorta vascular tissue decreased and those of Nrf2, p-Nrf2 and GCLM increased. Compared with the AE group, expression of TNF-α in the EX group increased, and that of Keap1, Nrf2, GCLC, NQO1 and GCLM decreased. Compared with the EX group, expression of TNF-α and Keap1 in the EX + LBP group decreased, whereas that of Nrf2, p-Nrf2, GCLC, NQO1 and GCLM increased. (Fig. 4A). The rat myocardium western blotting results showed that, compared with the Control, the expression levels of TNF-α, p-Nrf2, GCLC, NQO1 and GCLM in the AE group increased. Compared with the AE group, the expression levels of TNF-α, GCLC and GCLM increased, whereas those of Nrf2, p-Nrf2 and NQO1 decreased in the EX group. Compared with the EX group, the expression levels of TNF-α and Keap1 in the EX + LBP group decreased and those of Nrf2, p-Nrf2, GCLM, GCLC and NQO1 increased (Fig. 4B).

The thoracic aorta vascular IHC results showed that, compared with the Control, positive expression of TNF-α and Keap1 in the nucleus decreased, whereas that of Nrf2 and GCLM increased in the AE group. Compared with the AE group, TNF-α positive expression in the EX group notably increased, while that of Nrf2, GCLC, NQO1 and GCLM decreased. Compared with the EX group, the positive expression of TNF-α in the nucleus decreased, and that of Nrf2, GCLC, NQO1 and GCLM increased in the EX + LBP group (Fig. 5A).

Rat myocardial IHC showed that, compared with the Control group, positive expression of GCLC and NQO1 in the AE group was increased, however no other notable changes were observed. Compared with the AE group, the positive expression of TNF-α in the nucleus of the EX group notably increased, while that of Keap1, GCLC and NQO1 decreased. Compared with the EX group, the positive expression of TNF-α in the nucleus decreased, and that of Nrf2, NQO1 and GCLM increased in the EX + LBP group. (Fig. 5B).

In order to study the antioxidant effect of LBP, ELISA was performed. Compared with the Control, expression levels of CAT and GSH in the LBP group increased, and those of T-AOC exhibited no significant change. Compared with the LBP group, the expression levels of CAT, T-AOC and GSH in the AngII group decreased, and those of MDA, T-AOC and GSH increased. Compared with the AngII group, CAT expression in the AngII + LBP group increased, and that of MDA decreased (Fig. 6).

Western blotting was performed to investigate whether LBP enhances expression of the Keap1/Nrf2 antioxidant stress pathway in RTAEC. Compared with the Control, expression levels of TNF-α, p-Nrf2, NQO1 and GCLM in the LBP group increased, whereas those of Keap1 decreased. Compared with the LBP group, the expression of TNF-α increased and that of GCLC and GCLM decreased in the AngII group. Compared with the AngII group, the expression levels of TNF-α and Keap1 in the AngII + LBP group decreased, and those of Nrf2, NQO1, p-Nrf2, GCLC and GCLM increased. (Fig. 7).

Compared with the Control, the positive expression of activated Nrf2 in the nucleus of RTAECs in the LBP group increased, while GCLM in the cytoplasm did not notably change. Compared with the LBP group, the Nrf2 positive expression in the nucleus of the AngII group decreased and GCLM positive expression in the cytoplasm increased. Compared with the AngII group, the positive expression of Nrf2 in the cytoplasm and nucleus and GCLM in the cytoplasm increased in the AngII + LBP group (Fig. 8A and B).

RT-qPCR showed that, compared with the Control group, mRNA expression levels of Keap1 in the LBP group decreased; levels of TNF-α, IL-1β, Nrf2 and GCLC did not significantly change. Compared with the LBP group, mRNA expression levels of TNF-α and IL-1β in the AngII group increased and those of Keap1 and Nrf2 decreased. Compared with the AngII group, mRNA expression levels of TNF-α, IL-6, IL-1β and Keap1 in the AngII + LBP group decreased, whereas those of Nrf2, NQO1, GCLC and GCLM increased (Fig. 9).

In order to study whether LBP reduction of oxidative stress-induced cardiovascular damage is related to the enhancement of keap1/Nrf2 anti-oxidative stress signaling pathway in endothelial cells, in vitro experiments were performed for siRNA transfection. Transfection efficiency was determined by transfection of RTAECs with fluorescence-labeled blank siRNA (Fig. 10A). Cell transfection efficiency was highest at a transfection concentration of 50 nmol and 24 h after transfection. The optimal transfection concentration and time were determined (Fig. 10B). The silencing efficiency of siRNA3, siRNA4 and siRNA5 was detected based on the transfection concentration and time; siRNA5 exhibited the best silencing effect and was selected for subsequent experiments. Expression levels of Nrf2 and p-Nrf2 protein in the siRNA5 group were significantly lower than in the Control (P<0.05 and P<0.01). Western blotting showed that, compared with the AngII + LBP group, protein expression levels of Nrf2 and p-Nrf2 in the siRNA + AngII + LBP group decreased (Fig. 10C).

ELISA was performed to study the antioxidant effect of LBP on siNrf2. Compared with the Control, the expression levels of CAT, T-AOC and GSH in the AngII group decreased and those of MDA increased. Compared with the AngII group, the expression levels of CAT, T-AOC and GSH in the AngII + LBP group increased, while those of MDA decreased. Compared with the AngII + LBP group, the expression levels of CAT, T-AOC and GSH in the siRNA + AngII + LBP group decreased, and those of MDA increased. Compared with siNC + AngII + LBP group, the MDA expression levels increased, and those of GSH and T-AOC decreased in the siRNA + AngII + LBP group (Fig. 11).

In order to study the effect of siNrf2 on the anti-inflammatory effect of LBP, RT-qPCR was performed. Compared with the Control, the expression levels of TNF-α, IL-6 and IL-1β increased in the AngII group. Compared with the AngII group, the expression levels of TNF-α, IL-6 and IL-1β decreased. Compared with the AngII + LBP group, expression levels of TNF-α, IL-6 and IL-1β in the siRNA + AngII + LBP group increased. Compared with the siNC + AngII + LBP group, expression of TNF-α was increased in the siRNA + AngII + LBP group. Compared with the siRNA group, expression levels of TNF-α, IL-1β and IL-6 in the siRNA + AngII + LBP group increased (Fig. 12).

Flow cytometry showed that, compared with the Control, the expression of TNF-α in the AngII group increased. Compared with the siNC group, expression of TNF-α in the siNC + AngII + LBP group decreased. Compared with the AngII + LBP group, expression of TNF-α increased in the siRNA + AngII + LBP group (Fig. 13).