Simple Summary: The essential oil of Paederia scandens can remedy non-alcoholic fatty liver disease of chicken, but the mechanisms remain unclear. In this study, proteomics technology was used to declare the anti-non-alcoholic fatty liver disease mechanism of Paederia scandens essential oil. The results show that the essential oil of Paederia scandens significant decreased the oxidative stress of non-alcoholic fatty liver disease in chicken, which was mainly due to the center regulation protein of HSP7C being significantly inhibited. The aim of the study is to determine the underlying pathogenic mechanisms of oxidative stress and detect the anti-oxidative target of essential oil of Paederia scandens in non-alcoholic fatty liver disease (NAFLD). Chicken NAFLD was modeled by feeding with a high-capacity diet and Paederia scandens essential oil was used to treat the disease. The levels of hepatic reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and the differential proteins and network of protein–protein interactions were investigated in model and drug-treated groups. The results showed that essential oil of Paederia scandens down regulated the hepatic ROS and MDA level significantly (p < 0.05 and 0.01, respectively). The heat shock cognate 71 kDa protein (HSP7C) was down regulated significantly, which was in the center of the network and interacted with 22 other proteins. The results showed that oxidative stress played an important role in the pathogenesis of chicken NAFLD. The essential oil of Paederia scandens showed good anti-oxidation activity by down regulating the HSP7C protein, which can be used as a potential therapeutic target in chicken NAFLD.
Keywords: oxidative stress; essential oil of Paederia scandens; non-alcoholic fatty liver disease; HSP7C; chicken
Non-alcoholic fatty liver disease (NAFLD) is the most common worldwide nutritional metabolic disease in the general population due to lifestyle changes, such as increased consumption of high-fat food and lack of exercise, which are also the main reasons for chronic diseases, including dyslipidemia, chronic kidney disease, obesity, and hyperglycemia [[
There is no sufficient explanation of the mechanisms which are associated with the development of NAFLD, including progression to other nosologic units. It is hypothesized that it depends on genetic and environmental factors, and eventual progression is the result of bilateral interactions [[
Some studies have shown that oxidative stress may be the most critical pathogenesis of NAFLD leading to disease progression, and there can be an effective preventive and/or treatment strategy against chicken NAFLD by hepatoprotective effect or anti-oxidant activity. The recent study showed that essential oil of Paederia scandens has the activity of hepatoprotective and anti-oxidant effect [[
Plant materials were harvested from Guizhou (27.7183° N, 109.192° E, southwest China), in June 2015. All solvents and reagents were analytical grade. The harvested samples were washed with tap water and dried at 30 °C for 6 days by oven. A sample of 150 g crushed Paederia scandens was subjected to extraction by hydro distillation for 3 h in 500 mL distilled water using a clevenger type apparatus. The extracted oil was recovered and stored at 4 °C, and its extraction yield was calculated as the ratio of the weight of oil to the weight of fruits using the following equation:
% yield of oil = (weight of oil/weight of dried materials) × 100% (
The main ingredients of linalool, L-α-terpineol, dextro-α-terpineol, methyl salicylate, camphor, borneol, eugenol, and isoeugenol were chosen as the standards to determine the constituent of the essential oil of Paederia scandens. The standards and extraction were analyzed by Agilent 1260 high performance liquid chromatography (HPLC) equipped with an Agilent Zorbax Eclipse XDB-C8 column (4.6 mm × 250 mm, 5 μm) (Agilent Technologies, Santa Clara, CA, USA). The test condition was acetonitrile–water (55:45); flow rate 1.0 mL/min; injection volume 10 μL; wavelength 230 nm; column temperature 30 °C.
Seventy-five, one-day-old, healthy Ross 305 chicks (33−40 g) were selected from the breeding chicken farm of Sichuan Wenjiang China Tai Livestock Co., Ltd. (Chengdu, China). All chickens were housed in wood cages under the recommended environment. Chicks were brooded at 33 °C during the first week; the brooding temperature was reduced 3 °C/week to approximately 24 °C by week four of age. Light was provided continually using incandescent lamps. The environmental humidity was controlled at 60−65%.
The chickens were randomly divided into control group, model group, and drug treated group, 25 individuals per group. The control group was given a normal diet, and model and drug-treated groups were given a high-capacity diet. Cooked pigs' oil was the main calorie sources of the high-capacity diet. The drug group chickens were treated with essential oil of Paederia scandens by nasal drops, 2 mg/kg per day continuously for 4 weeks. The dose of Paederia scandens essential oil was calculated by previous experiments, and 2 mg/kg was the safe and effective dose to treat NAFLD in chicken [[
A small part of fresh liver was homogenized to prepare 10% tissue homogenate by adding 0.9% saline on ice in each group. The homogenate was centrifuged at 1500× g for 10 min at 0 °C. The supernatant was harvested and adjusted to 1% tissue homogenate by adding 0.9% saline. The levels of MDA, SOD and ROS were measured by assays kits from the NanJing Jiancheng bio-engineering research institute (NanJing, China).
The liver (1.0 g) was suspended in 10 mL buffer consisting of 50 mM Tris and 1.0 mM phenylmethylsulphonyl fluoride for preparation of the total protein extract. The suspension was homogenized with the homogenizer for 1 min, sonicated for 30 s, and centrifuged at 14,000× g for 15 min in the buffer. The homogenate was poured into a glass beaker, which was placed on ice, and the homogenate was stirred gently for 30 min at 4 °C. Cell debris and other particulate matter were removed from the homogenate by centrifugation at 14,000× g for 20 min at 4 °C. After filtering the supernatant, the cells were washed by adding 10 mL phosphate buffer saline to the centrifuge tube. The cell pellet was resuspended by pipetting the mixture up and down. The cells were centrifuged again at 4800× g for 5 min at 4 °C. The supernatant was discarded without disturbing the pellet. A pipette was used to transfer the extraction buffer into the tube and vortex the tube briefly (10 s) to resuspend the pellet. Samples of 1.0 mg protein were applied on immobilized pH 3−10 nonlinear strips. Each sample was analyzed in triplicate. Focusing started at 200 V at 3 V/min and was kept constant for a further 24 h. The second-dimensional separation was performed in 12% SDS–polyacrylamide gels. The gels (18 × 18 × 0.15 cm) were run at 40 mA per gel. After protein fixation for 12 h in 40% methanol containing 5% phosphoric acid, the gels were stained with Coomassie blue R250 (0.5 g/L) for 24 h. The gels were scanned as tiff files and analyzed by PDQuest 8.01 (Bio-Rad, Berkeley, CA, USA). The significantly different pots were selected for MALDI-TOF/MS analysis. These proteins were identified by GPM-XE software (The Global Proteome Machine Organisation, USA,
The statistical analyses were performed using the SPSS 19.0 software package (ICM, Armonk, NY, USA) and the data were presented as means and standard deviations (means ± SD), and group comparison was done by independent-sample t-tests.
The extraction rate of essential oil was about 0.5% in Paederia scandens. Eight chemical ingredients were tested by HPLC, and the contents were shown in Table 2. The main ingredients were linalool and borneol, the contents were 261.142 and 118.784 mg/mL in the oil, the results showed in Table 2. These eight components account for 54.14% of essential oil. The HPLC chromatogram is shown in Figure 1.
The clinical manifestations were weight gain, unstable standing, mouth breathing, lethargy, unilateral lying, increased body temperature, and feces rot in most of the chickens treated with high-calorie diets. Even a small number of chickens died. There was no chicken death in the drug treatment group, no obvious clinical features, and the chickens grew well.
In the model group, most chickens showed a large amount of yellow–brown fat deposition in the abdominal cavity and mesentery. The liver was swollen (data shown in Table 3), deep yellow, soft, and brittle. Some livers lost their original shape. In the drug treatment group, the hepatic hemorrhage band was reduced or even disappeared, and some showed small bleeding spots. The overall liver appearance was similar to that of the normal group.
The results showed that hepatic ROS levels were significantly higher in the model and drug groups than in the control group during the whole experiment (p < 0.01 or p < 0.05), and hepatic ROS levels were lower in the drug group than in the model group in the early stage. Hepatic MDA levels were significantly higher in the model group than in the drug group and control group from 2 weeks to 4 weeks (p < 0.01 or p < 0.05). Hepatic SOD activity was significantly lower in the model group and drug group than in the control group from 3 weeks (p < 0.01 or p < 0.05). The essential oil of Paederia scandens significantly down regulated the hepatic ROS and MDA levels in NAFLD chicken. The results are listed in Table 4.
The total proteins were analyzed by 2D electrophoresis. The samples were analyzed on broad pH range immobilized pH gradient (IPG) strips and the spots were visualized following staining with Coomassie brilliant blue. The results showed a representative analysis of total liver proteins separated on a broad pH range 3−10 2D gel. On each gel, 1.0 mg of total protein amount was applied. The analysis of two-dimensional maps in PDQuest showed that there were about 400 spots detected by Commassie Brilliant Blue-stained in each group. There were 73 markedly differential spots in B gel compared with group A, and there were 22 markedly differential spots in C gel compared with B gel. These differential spots are shown on Figure 2.
In total, 22 differential proteins were identified in C gel, eight of which were up-regulation, such as heat shock 70 kDa protein 5, heat shock 70 kDa protein 8, long-chain specific acyl-CoA dehydrogenase, etc. There were four proteins with the function of lipid metabolism and fatty acid beta-oxidation, such as 3-alpha-hydroxysteroid dehydrogenase, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2, Carbonic anhydrase 3, and Catalase. Additionally, there were four proteins with the function of electron transport and cell communication, such as electron transfer flavoprotein subunit alpha, ATP synthase subunit d, Regucalcin, and Guanine nucleotide-binding protein subunit beta-2-like 1. The results are listed in Table 5.
The analysis of the network of protein–protein interactions was done by Cytoscape v2.6.3. The results showed that HSP7C, heat shock cognate 71 kDa protein, was in the center of the network and interaction among 22 other proteins, which showed that essential oil of Paederia scandens had the activity of hepatoprotective effect and anti-oxidant by up-regulation of HSP7C expression and affected other proteins by interaction. The results are listed in Table 6 and Figure 3.
Oxidative stress can be described as a condition resulting from an uncontrolled increase in ROS and MDA or an insufficiency in the anti-oxidant system under certain pathological states [[
The results showed that hepatic ROS and MDA levels were significantly higher in the model group than other groups, but SOD activity was significantly lower at the same time, which indicated that oxidative stress occurred and played a significant role in liver injury and disease progression in chicken NAFLD. Recent studies have shown that high levels of lipid accumulation exceeded the storage capacity of liver in NAFLD [[
The differential proteins were analyzed by two-dimensional (2D) gel electrophoresis and mass spectrometry in chicken NAFLD and drug treated groups, which indicated that chicken NAFLD followed many differential protein expressions, and essential oil of Paederia scandens can up or down regulate some proteins, which is related to the hepatic ROS and MDA level regulation by the network of protein–protein interactions. There were 22 differential proteins in the drug-treated group compared with the model group. Of these, there were four proteins with the function of lipid metabolism and fatty acid beta-oxidation, such as 3-alpha-hydroxysteroid dehydrogenase, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2, Carbonic anhydrase 3, and Catalase. Additionally, there were four proteins with the function of electron transport and cell communication, such as electron transfer flavoprotein subunit alpha, ATP synthase subunit d, Regucalcin, and Guanine nucleotide-binding protein subunit beta-2-like 1. Eight proteins were up-regulated, such as heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa), heat shock 70 kDa protein 8, Long-chain specific acyl-CoA dehydrogenase, Beta-actin, and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2.
To define the mechanism of anti-oxidation and further determine the targets of essential oil of Paederia scandens in treating chicken NAFLD, the protein network analysis results showed that HSP7C, heat shock cognate 71 kDa protein, was in the center of the network and interaction among the other 22 proteins, which indicated that HSP7C may be a core target for essential oil of Paederia scandens against chicken NAFLD. Some recent studies showed that HSP7C is a member of the heat shock protein 70 (HSP70) family and is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). As this protein interacts with many ER proteins, it may play a key role in monitoring protein transport through the cell [[
In conclusion, excessive production of ROS and MDA and decreased SOD activity overwhelmed antioxidant defenses and further generated highly toxic lipid peroxides, which induced the formation of oxidative stress in chicken NAFLD. The essential oil of Paederia scandens has a hepatoprotective effect and anti-oxidant activity by up-regulation of HSP7C protein expression, which may be a potential therapeutic target in treating NAFLD chicken.
Graph: Figure 1 High performance liquid chromatogram of 100 μg/L mixed standard solution (A) and 500 μg/L in Paederia scandens essential oil (B).
Graph: Figure 2 Two-dimension gel electrophoresis of proteins from liver extract stained with Coomassie Brilliant Blue. pH 3−10 nonlinear first dimension, and 12% SDS–PAGE second dimension. (A) shows that there were 73 differential spots in the drug-treated group compared with model group; (B) shows that there were 22 differential spots in the drug-treated group compared with control group. Differentially expressed proteins are marked in each spot sustained an individual identification number (SSP).
Graph: Figure 3 The network of differential protein expression.
Table 1 Composition and nutrient levels of the diets.
Ingredients (%) High-Capacity Diets Normal Diets Corn 53.00 62.48 Soybean meal - - Fish meal 1.00 3.00 Fried meal 29.30 28.10 Rapeseeds - 2.20 Wheat bran - - Cooked pigs' oil 12.95 - Additives 0.75 0.75 Lys 0.12 0.09 Met 0.12 0.18 CaHPO4 1.11 1.22 Limestone meal 1.52 1.82 Salt 0.13 0.16 ME (MJ/kg) 16.07 13.39 CP (%) 16.00 18.40 Ca 0.90 1.00 AP 0.35 0.45 Lys 1.00 1.10 Met 0.38 0.50
Table 2 Contents of main components of essential oil in Paederia scandens.
No. Ingredients Raw Sample (mg/mL) 1 Linalool 261.142 2 L-α-terpineol 10.126 3 D-α-terpineol 11.233 4 Methyl salicylate 78.902 5 Camphor 15.234 6 Borneol 118.784 7 Eugenol 24.634 8 Isoeugenol 21.346
Table 3 The change of liver index (%) in broilers.
Time (Week) Control Group Model Group Drug Group 2 2.20 ± 0.15 3.02 ± 0.18 ** 3.02 ± 0.18 ** 3 2.22 ± 0.21 3.15 ± 0.11 ** 2.83 ± 0.21 ** 4 2.22 ± 0.22 3.05 ± 0.30 ** 2.84 ± 0.17
Table 4 The level of hepatic ROS, MDA, and SOD.
Item Week Control Group Model Group Drug Group ROS (U/mg protein) 1 88.56 ± 1.44 113.34 ± 4.06 ** 97.64 ± 1.62 * 2 88.56 ± 2.20 106.66 ± 4.48 ** 98.33 ± 1.39 * 3 93.60 ± 3.55 107.49 ± 7.96 ** 105.01 ± 6.20 * 4 100.69 ± 6.33 125.00 ± 5.86 ** 109.08 ± 4.46 MDA (nmol/mg protein) 1 6.57 ± 1.46 10.60 ± 1.34 ** 9.04 ± 1.27 ** 2 8.32 ± 1.63 10.51 ± 1.29 ** 9.47 ± 1.56 ** 3 8.82 ± 1.26 13.47 ± 1.66 ** 10.42 ± 1.79 ** 4 9.57 ± 1.18 13.46 ± 1.42 ** 9.94 ± 1.03 SOD (U/mg protein) 1 498.96 ± 35.83 461.32 ± 37.49 436.11 ± 43.69 * 2 536.60 ± 31.67 501.19 ± 27.13 476.48 ± 26.99 ** 3 564.89 ± 26.22 492.36 ± 30.23 ** 425.99 ± 26.58 ** 4 523.83 ± 29.05 443.22 ± 33.35 ** 417.21 ± 30.79 **
Table 5 Different proteins expression in chicken liver.
SSP Protein Gene MW/PI Trend 1101 Uncharacterized protein - - - 1203 Regucalcin 33/5.3 ↓ 3704 Heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa) 72/5.1 ↑ 4103 ATP synthase subunit d 19/6.2 ↓ 4301 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta 1 35/7.6 ↓ 4401 S-adenosylmethionine synthase isoform type-1 44/5.6 ↓ 4402 Fumarylacetoacetase 46/6.7 ↓ 5201 Carbonic anhydrase 3 29/6.9 ↓ 5301 3-alpha-hydroxysteroid dehydrogenase 37/6.7 ↑ 5603 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 57/8.9 ↑ 5702 Catalase 60/7.1 ↑ 6104 Glutathione S-transferase Mu 1 26/8.3 ↓ 6502 Arginosuccinate synthase 1 46/7.6 ↓ 6503 Long-chain specific acyl-CoA dehydrogenase 48/7.6 ↑ 6702 Heat shock 70 kDa protein 8 71/5.4 ↑ 6901 Uncharacterized protein - - 7003 Beta-actin 15/5.7 ↑ 7303 Uncharacterized protein 39/8.9 8301 Electron transfer flavoprotein subunit alpha 35/8.6 ↓ 8302 Glutathione S-transferase Mu 2 28/8.2 ↓ 9301 Glyceraldehyde-3-phosphate dehydrogenase-like 36/8.4 ↓ 9501 Betaine-homocysteine S-methyltransferase 1 45/8.0 ↓ 8603 Uncharacterized protein - - -
Table 6 The analysis of the network among differential protein expression.
ID Degree Label Gene Name 146490 22 HSP7C Heat shock cognate 71 kDa protein 145948 18 GRP78 78 kDa glucose-regulated protein 93344 16 PABP1 Emerin 71781 16 YBOX1 Nuclease-sensitive element-binding protein 1 57072 16 PRKDC Heterogeneous nuclear ribonucleoproteins A2/B1 51704 16 ACTB Polyadenylate-binding protein 1 50923 16 RO52 E3 ubiquitin-protein ligase TRIM21 50095 16 ROA2 DNA-dependent protein kinase catalytic subunit 15490 16 EMD Actin, cytoplasmic 1 150257 16 G3P Glyceraldehyde-3-phosphate dehydrogenase 89014 12 RS3 40S ribosomal protein S9 88619 12 RS9 40S ribosomal protein S3 5116 8 IF2B Eukaryotic translation initiation factor 2 subunit 2 50028 8 RLA0 60S acidic ribosomal protein P0 4817 8 IF2A Eukaryotic translation initiation factor 2 subunit 1 117387 8 NCBP1 Nuclear cap-binding protein subunit 1 95707 2 CITE1 Cbp/p300-interacting trans-activator 1 15584 2 ERN1 Serine/threonine-protein kinase/endoribonuclease IRE1
Conceptualization, Q.W.; methodology, H.T.; writing—original draft preparation, H.T.; writing—review and editing, Q.W.; supervision, H.W.; funding acquisition, Q.W.
This research was funded by the National Natural Science Foundation of China (Grant No. 31302134) and the National Natural Science Foundation of China's Guizhou (Grant No. 2017-1191).
The authors declare no conflict of interest.
By Qiang Wu; Huaqiao Tang and Hongbin Wang
Reported by Author; Author; Author