Edited by: Xiu-Wei Yang, Peking University, China
Reviewed by: Haolong Liu, Peking University Health Science Centre, China; Dan Yan, Capital Medical University, China
*Correspondence: Hong Nie,
This article was submitted to Ethnopharmacology, a section of the journal Frontiers in Pharmacology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine which is used for hundreds of years for treating the central nervous system diseases. Atherosclerosis is a poly-aetiological chronic inflammatory vascular disease. Preventing inflammation is fundamental for treating atherosclerosis in early stages. In this study, we investigated the protective effects and possible mechanisms of ANP action on a high-fat diet induced early and mid-term atherosclerosis ApoE-/- mice. The effects of ANP were compared with accepted drug simvastatin. Twelve male C57BL/6J mice were used as the control group, and 60 male ApoE-/- mice were randomly divided into five groups: Model group, Simvastatin group, Low-, Medium-, and High-dose ANP group these groups received, respectively, saline, simvastatin (3.0mg/kg), low-dose ANP (0.25 g/kg), medium-dose ANP (0.50 g/kg), and high-dose ANP (1.0 g/kg), once every other day for 10 weeks. After administration, serum biochemical indices were detected by the automatic biochemical analyzer, the concentrations of IL-6 and IL-10 in the serum were assayed by ELISA, expression levels of IL-1β, TNF-α, MMP-2, MMP-9, CCL2, and its receptor CCR2 in the full-length aorta, and expression levels of transcription factors Foxp3, RORγt in the spleen were assayed
Cardiovascular diseases (CVD) and related chronic diseases are the number one cause of death. About 80% of CVD-related deaths occur in low-income and middle-income countries (
Pathogenesis of AS includes lipid infiltration, damage response, mononuclear macrophage invasion, and inflammation response. At present, it is generally accepted that AS is a chronic inflammatory disease (
Chronic inflammation represents the most basic pathogenetic factor of AS (
Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine, used to treat stroke, encephalitis, and meningitis. Its main components include
ANP was provided by Guangzhou Baiyunshan Zhongyi pharmaceutical co., Ltd (Guangzhou, China). It contains the following components:
Main reagents used in the study were as follows: 1) ANP (Guangzhou Baiyunshan Zhongyi Pharmaceutical Co, Ltd, Lot: WA0076); 2) Simvastatin (Shangdong Xinqi Pharmaceutical Co., Ltd, Lot: 20170101); 3) High fat feed composed of 3% cholesterol, 0.5% sodium cholate, 0.2% propylthiouracil, 5% sugar, 10% lard, and 81.3% basic feed (Medical Science Experimental Animal Center, Guangdong, China, Lot: 201814); 4) Kits for IL-6, IL-10 (Wuhan Huamei Biological Co, Ltd, Lot: U23013173, U24018179), kit for reverse transcription, fluorescent dyes of SYBR (TAKARA, Lot: AI12361A, AK9304), kit for Masson staining (Sinopharm Chemical Reagent Co, Ltd, Lot: G1006), kit for BCA (Shanghai Beyotime biotechnology, Lot: 051018180514), and kit for ECL plus (Applygen, Lot: P1010); 5) Anti-αSMA, anti-CD11c, anti-CD68, fluorescent secondary antibody with FITC marker, and fluorescent secondary antibody with CY3 marker (Servicebio, Lot: GB130441, GB11058, GB11067, GB223011, GB21303); 6) β-actin, anti-MCP-1 (CST, Lot: 15,2), anti-CCR2, anti-Foxp3, anti-IL-1β, anti-TNF-α (Abcam, Lot: GR286626-10, GR239541-14, GR309542-2, GR235155-19), anti-RORγ (Santa, Lot: F3017), anti-MMP-2, and anti-MMP-9 (Wanleibio, Lot: WL03334, WL02141); 7) Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody, horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Shanghai Beyotime biotechnology, Lot: 040818180510, 040818184521); 8) PE anti-mouse CD25, APC anti-mouse CD127, PE/Cy7 anti-mouse CD4, Alexa Fluor® 488 anti-mouse IL-17A, APC Rat IgG2a κ Isotype Ctrl, PE Rat IgG2b κ Isotype Ctrl, Alexa Fluor® 488 Rat IgG1 κ Isotype Ctrl, PE/Cy7 Rat IgG2a κ Isotype Ctrl, Cell Activation Cocktail with Brefeldin A, 10×RBC Lysis Buffer, Fixation Buffer, 10×Intracellular Staining Permeabilization Wash Buffer (Biolegad, Lot: B247733, B232775, B249463, B259048, B238057, B255375, B236504, B236195, B248910, B250015, B252968, B252856).
Twelve 4-week old Specific Pathogen Free (SPF) male C57BL/6J mice (weight:18 ± 2.0g) and sixty 4-week old SPF male ApoE-/- mice (weight:18 ± 2.0g), were provided by the department of Laboratory Animal Science of the department of medicine, Peking University [Certificate No. SCXK (jing) 2011-0012]. Mice were housed in the Jinan University Medical School Laboratory Animal Management Center [Certificate No. SCXK (Guangdong) 2012-0117] and were maintained at 24°C and 65% humidity. Mice were maintained on a 12-h light/dark cycle, C57BL/6J mice were given free access to standard laboratory mouse chow and tap water, ApoE-/- mice were given free access to high fat feed and tap water. The experiments were approved by the Laboratory Animal Ethics Committee of Jinan University (No. 201812374), and were performed according to the instructions of the National Institute of Health (OLAW/NIH Revised 2015) (
After ten days of adaptive feeding, 60 SPF male ApoE-/- mice were randomly divided into five groups, i) model group, ii) simvastatin group (3.0 mg/kg), iii–v) low-, ledium-, and high-dose ANP groups (0.25 g/kg, 0.50 g/kg, 1.0g/kg, respectively) and given free access to high fat feed and tap water for 10 weeks. Each group contained twelve mice. Twelve SPF male C57BL/6J mice were used as controls and were given free access to standard laboratory mouse chow and tap water for 10 weeks. On the first day of high-fat diet administration, all experimental drug treatments (simvastatin and ANP) were administered intragastrically once every other day for 10 weeks. Drug dosages were titrated according to animal weight. Saline was given intragastrically in control group and model group. All drug treatments were prepared immediately before administration as a suspension by dissolving an appropriate amount of the drug in distilled water.
After administration, serum was collected for biochemical analysis. The aortic root was separated for immunofluorescence staining and Masson staining. The full-length aorta was separated for Real-time quantitative polymerase chain reaction (RT-qPCR) and Western Blotting analysis. The liver, kidney, spleen, and thymus were separated for organ coefficient or organ index. Primary splenocytes were separated for flow cytometric analysis from the spleen.
The cholesterol (CHOL), triglyceride (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) were measured using automatic biochemical analyzer. All protocols were followed in accordance to the manufacturer recommendations.
Liver coefficient, kidney coefficient, thymus index, and spleen index were calculated according to corresponded calculation formula separately. The formula of liver and kidney coefficient: weight of liver and kidney (mg)/weight of animal (g). The formula of thymus and spleen index: weight of thymus and spleen (mg)/weight of animal (g)*10.
The ratio of Th17 to Treg cells in splenocyte suspensions was analyzed by flow cytometry. The splenocyte suspensions were stimulated with 10 μl of Cell Activation Cocktail (with Brefeldin A) and mixed. After incubation at 37°C for 6 h, the sample was centrifuged, and the precipitate was suspended in 1 ml RBC. The suspensions were then centrifuged and the precipitate re-suspended in PBS twice. After antibody labeling of surface proteins, the sample was incubated in the dark for 20 min followed by centrifugation (1,500 rpm). The cells in the precipitate were then fixed with fixation buffer, followed by incubation in the dark for 20 min. The sample was again centrifuged and suspended in 1 mL Permeabilization Wash Buffer. The cells were then washed twice and labeled with an immunofluorescent antibody. After incubation in the dark for 20 min, the sample was washed using 1 ml Permeabilization Wash Buffer twice and suspended in PBS prior to flow cytometric detection.
Masson staining was used to detect collagen fibers in the plaque of aortic root. According to kit for Masson staining manufacturer instructions, the aortic root fixed in 4% paraformaldehyde were dehydrated in alcohol, paraffin-embedded, sectioned and subjected to Masson staining. The stained sections were observed under the light microscope. Masson staining of the whole aorta was measured by Image Pro Plus 6.0. The area proportion of collagen fiber was calculated as the ratio of stained area to area of interest calculated by Image Pro Plus 6.0.
Immunofluorescence staining was used to detect inflammatory cells infiltration such as macrophages, DCs, and VSMCs in the plaque of aortic root. Anti-CD68, anti-CD11c, and anti-αSMA were respectively used as special marker in macrophages, DCs, and VSMCs. Frozen slices of aortic root were blocked in blocking buffer for 60 min, aspirated blocking solution, applied diluted primary antibody, incubated overnight at 4°C, rinsed three times in PBS for 5 min each, and then incubated specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 h at room temperature in the dark, rinsed in PBS, thereafter, coverslips stained with DAPI. The stained sections were observed under fluorescence microscope, and Image J was used to semi-quantitatively analyze positive cells.
The serum concentrations of IL-6 and IL-10 were measured using the IL-6 and IL-10 ELISA kits. In line with the manufacturer’s instructions, standard, or sample (100 µl) was added to each well and incubated for 2 h at 37°C. Then, the medium in each well was discarded, and biotin antibody (100 µl) was added to each well, followed by incubation for 1 h at 37°C. The medium in each well was aspirated and the wells were washed three times. Thereafter, HRP-avidin (100 µl) was added to each well and incubated for 1 h at 37°C. The medium in each well was aspirated and the wells were washed five times, followed by addition of tetramethylbenzidine substrate (90 μl) and incubation for 20 min at 37°C in the dark. Finally, the stop solution (50 ul) was added to each well, and the absorbance of the wells was read at 450 nm within 5 min.
The mRNA levels of
Primers used for RT-qPCR.
Gene | Primer | Sequence 5′-3′ |
---|---|---|
Foxp3 | Forward primer | 5’- TGGAACCACGGGCACTATCACA-3’ |
Foxp3 | Reverse primer | 5’- GAGGCTGCGTATGATCAGTTATGC-3’ |
RORγt RORγt | Forward primer Reverse primer | 5’- TGCAAGACTCATCGACAAGG-3’ 5’- AGGGGATTCAACATCAGTGC-3’ |
CCR2 | Forward primer | 5’- AGAGAGCTGCAGCAAAAAGG-3’ |
CCR2 | Reverse primer | 5’- GGAAAGAGGCAGTTGCAAAG-3’ |
CCL2 | Forward primer | 5’- AGGTCCCTGTCATGCTTCTG-3’ |
CCL2 | Reverse primer | 5’- TCTGGACCCATTCCTTCTTG-3’ |
IL-1β | Forward primer | 5’- ACTCATTGTGGCTGTGGAGA-3’ |
IL-1β | Reverse primer | 5’- TTGTTCATCTCGGAGCCTGT-3’ |
TNF-α | Forward primer | 5’- TCTACTGAACTTCGGGGTGATCG-3’ |
TNF-α | Reverse primer | 5’- ACGTGGGCTACAGGCTTGTCA-3’ |
MMP-2 | Forward primer | 5’-CACACCAGGTGAAGGATGTG-3’ |
MMP-2 | Reverse primer | 5’-AGGGCTGCATTGCAAATATC-3’ |
MMP-9 | Forward primer | 5’-TGAATCAGCTGGCTTTTGTG-3’ |
MMP-9 | Reverse primer | 5’-GTGGATAGCTCGGTGGTGTT-3’ |
GAPDH | Forward primer | 5’-CATCCATGACAACTTTGGCA-3’ |
GAPDH | Reverse primer | 5’-CCTGCTTCACCACCTTCTTG-3’ |
Protein levels of Foxp3, RORγt in the spleen, as well as CCR2, CCL2, IL-1β, TNF -α, MMP-9, and MMP-2 in the full-length aorta were detected by western blotting analysis. Total proteins were extracted from full-length aorta and spleen. The concentrations of protein were determined by BCA protein assay. Each sample containing 30 µg protein was separated, respectively, on 8%, 10%, and 15% SDS-PAGE gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. Thereafter, membranes were incubated in a 5% (weight in volume) milk solution for 2 h and then incubated at 4°C overnight with one of the following primary antibodies: β-actin, IL-1β, TNF-α, CCL2, CCR2, Foxp3, RORγt, MMP-2 (1:1000 dilution each), and MMP-9 (1:500). After primary antibody incubation, membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000, 374°C, 2h) or horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:2000, 374°C, 2h). The blots were visualized using enhanced chemiluminescence reagent, and UVP BioSpectrum Imaging System was used to expose immune-positive bands. The bands were semi-quantitatively analyzed with Image J, and the results are expressed as ratios of IL-1β, TNF-α, CCL2, CCR2, Foxp3, RORγt, MMP-2, MMP-9 to β-actin densitometry readings.
All data were performed as mean ± standard error of the mean (mean±SEM) and were analyzed by One-way ANOVA with Dunnett T3 test in SPSS Statistics 18.0.
The body weight of ApoE-/- mice and C57BL/6J mice over a 10-week period was shown in
The body weight, liver coefficient and kidney coefficient of ApoE-/- mice and C57BL/6J mice.
Body weight of C57BL/6 mice and ApoE-/-mice in 10 weeks.
Weeks | Groups | |||||
---|---|---|---|---|---|---|
Control | Model | Low-dose ANP | Medium-dose ANP | High-dose ANP | Simvastatin | |
1 | 20.3 ± 0.4 | 21.1 ± 0.2 | 21.2 ± 0.3 | 21.6 ± 0.4 | 21.2 ± 0.4 | 21.3 ± 0.3 |
2 | 22.8 ± 0.5 | 23.6 ± 0.3 | 22.7 ± 0.4 | 23.3 ± 0.5 | 23.4 ± 0.4 | 23.4 ± 0.4 |
3 | 24.0 ± 0.5 | 25.5 ± 0.4# | 24.7 ± 0.5 | 24.6 ± 0.6 | 24.4 ± 0.4 | 24.6 ± 0.4 |
4 | 25.2 ± 0.6 | 26.4 ± 0.5 | 25.5 ± 0.5 | 25.2 ± 0.6 | 25.5 ± 0.4 | 25.6 ± 0.4 |
5 | 25.7 ± 0.6 | 27.7 ± 0.5## | 26.2 ± 0.6* | 25.8 ± 0.7* | 25.9 ± 0.4 | 26.5 ± 0.3* |
6 | 27.2 ± 0.5 | 28.2 ± 0.4 | 26.9 ± 0.7 | 26.8 ± 0.6 | 26.7 ± 0.5 | 26.8 ± 0.5 |
7 | 27.2 ± 0.5 | 28.4 ± 0.5 | 27.2 ± 0.7 | 27.4 ± 0.5 | 27.3 ± 0.4 | 27.8 ± 0.4 |
8 | 28.2 ± 0.6 | 29.4 ± 0.5 | 28.0 ± 0.6 | 28.0 ± 0.5 | 28.2 ± 0.4 | 28.6 ± 0.4 |
9 | 28.4 ± 0.6 | 29.4 ± 0.5 | 27.8 ± 0.7* | 28.1 ± 0.5* | 27.8 ± 0.5* | 27.9 ± 0.5* |
10 | 28.4 ± 0.7 | 30.3 ± 0.5# | 28.4 ± 0.6* | 28.5 ± 0.5* | 28.4 ± 0.5* | 28.4 ± 0.6* |
Compared with Control, #p < 0.05, ##p < 0.01; Compared with model group, *p < 0.05, (mean ± SEM, n = 12).
LDL-C/HDL-C ratio and concentrations of CHOL, TG, LDL-C, HDL-C in model group were higher than in control group (
Effect of ANP on concentrations of serum CHOL, TG, HDL-C, LDL-C and LDL-C/HDL-C ratio.
Serum concentrations of IL-6 and IL-10 were shown in
Effect of ANP on serum concentrations of pro-inflammation IL-6 and anti-inflammation IL-10.
According to results of thymus index and spleen index (
Effect of ANP on Th17/Treg balance in the spleen.
RORγt and Foxp3 are transcription factors for Th17 cells which regulate Th17/Treg balance. To evaluate effect of ANP on Th17/Treg balance, expression levels of RORγt, and Foxp3 were detected in the spleen. In the model group, mRNA and protein expression levels of RORγt was significantly up-regulated (
Effect of ANP on expression of RORγt and Foxp3 in the spleen.
Cytokines and chemokines play important roles in early and mid-term of AS and have a profound influence on the pathogenesis of AS. When compared to the control group, mRNA and protein expression levels of IL-1β, TNF-α, CCL2, CCR2 in the model group were significantly up-regulated (
Effect of ANP on mRNA expression levels of IL-1β, TNF-α, CCL2, and CCR2 in the aorta.
Effect of ANP on protein expression of IL-1β, TNF-α, CCL2 and CCR2 in the aorta.
MMPs are a group of proteases that degrade extracellular matrix, and they are mainly secreted by macrophages and VSMCs in atherosclerotic plaques. Overexpression of MMPs promotes the degradation of collagen and elastic fibers in plaque, destroying the stability of arterial plaque, which facilitate induction of cardiovascular risk events. As shown in
Effect of ANP on mRNA and protein expression of MMP-2 and MMP-9 in the aorta.
Masson staining was performed in the aortic root. The area of fibrosis which was stained blue covered a much larger area in the model group than in the control group (
Effect of ANP on collagen fibers in the aortic root.
Immunofluorescence staining was performed in the aortic root. In the model group, the positive area of macrophages which were stained red was higher than in the control group (
Effect of ANP on macrophages infiltration in the aortic root.
Effect of ANP on VSMCs and dendritic cells infiltration in the aortic root.
We found that treatment with ANP corrected the immune imbalance of Th17/Treg cells in the early AS ApoE-/- mice model by regulating RORγt and Foxp3 expression; ANP suppressed the release of pro-inflammatory mediators, promoted the release of anti-inflammatory factors, down-regulated the expression of chemokines and their receptors promoting thus an anti- inflammatory effects in the early AS. Furthermore, ANP down-regulated expression of MMP-2, MMP-9, reduce collagen fibers and decreased infiltration of macrophages, dendritic cells, and vascular smooth muscle cells into plaques thus facilitating stability of the latter.
In this study, ANP was a kind of tawny powder, and distilled water was used as the solution medium. Due to the relatively complex composition of ANP, ANP was tawny suspension after dissolution. In order to maintain the stability of drug suspension, the ANP suspension was prepared before administration. In our next study, we will focus on the metabolic components of ANP in mice and the pharmacodynamic substance basis of ANP, and analyze the prescription.
It has been argued that increase in combined ratios (LDL-C/HDL-C) relates to the severity and prevalence of coronary artery disease (
The high-fat diet is a potential risk factor for the liver and kidney. Treatment with ANP significantly reduced the liver coefficient but did not improve the kidney coefficient; thus pointing on a certain hepatoprotective effect of ANP. This agreed with previous findings showing that ANP has a preventive and therapeutic effect on liver damage caused by bacterial toxins (
Chronic inflammation is the key factor in AS pathogenesis, while inhibiting inflammatory response controls AS evolution (
Atherosclerotic plaque, the morphological substrate of AS, is induced by multiple factors, including accumulation of VSMCs, macrophages and T lymphocytes (
In conclusion, ANP protects early and mid-term atherosclerotic ApoE-/- mice by regulating Th17/Treg balance, inhibiting chronic inflammation, reducing plaque collagen fibers, and reducing inflammatory cells infiltration. Moreover, ANP has anti-inflammatory effects in small doses, and ANP has immunoregulation effects on high doses. Possible mechanisms are shown in
Possible mechanism pathway of ANP. Previous research findings found that ANP could down-regulated mRNA expression levels of IFN-γ, CCL5 and CCR5, these results were shown in
All datasets generated for this study are included in the article/
The experiments were approved by the Laboratory Animal Ethics Committee of Jinan University (No. 201812374), and were performed according to the instructions of the National Institute of Health (OLAW/NIH Revised 2015).
HN supported and designed the experiments, and revised the manuscript. QF performed the experiments. YL, JR, ZZ, KY, WX, TZ, XC, NN, ZY, YC, and YX provided reagents/materials. YL and JR analyzed the data. RL participated in checking data statistics and manuscript revision linguistically. AV gave guidance during the experimental design process, and revised the manuscript. QF and YL wrote the manuscript.
This work was supported by the National Natural Science Foundation of China (No. 81673634) and Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd (No. 40116103).
Authors NN, ZY, YC and YX are employed by company Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd. Author RL is a student of International Department, the Affiliated High School of SCNU. Author AV is a professor in the Faculty of Biology, Medicine and Health, The University of Manchester.
The authors declare that this study received funding from Guangzhou Baiyunshan Zhongyi Pharmaceutical Co., Ltd. The funders had no role in study design, data collection and analysis, decision to publish and preparation of the manuscript.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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