Transcriptome analysis reveals a molecular understanding of nicotinamide and butyrate sodium on meat quality of broilers under high stocking density

Background In recent years, increased attention has been focused on breast muscle yield and meat quality in poultry production. Supplementation with nicotinamide and butyrate sodium can improve the meat quality of broilers. However, the potential molecular mechanism is not clear yet. This study was designed to investigate the effects of supplementation with a combination of nicotinamide and butyrate sodium on breast muscle transcriptome of broilers under high stocking density. A total of 300 21-d-old Cobb broilers were randomly allocated into 3 groups based on stocking density: low stocking density control group (L; 14 birds/m2), high stocking density control group (H; 18 birds/m2), and high stocking density group provided with a combination of 50 mg/kg nicotinamide and 500 mg/kg butyrate sodium (COMB; 18 birds/m2), raised to 42 days of age. Results The H group significantly increased cooking losses, pH decline and activity of lactate dehydrogenase in breast muscle when compared with the L group. COMB showed a significant decrease in these indices by comparison with the H group (P < 0.05). The transcriptome results showed that key genes involved in glycolysis, proteolysis and immune stress were up-regulated whereas those relating to muscle development, cell adhesion, cell matrix and collagen were down-regulated in the H group as compared to the L group. In contrast, genes related to muscle development, hyaluronic acid, mitochondrial function, and redox pathways were up-regulated while those associated with inflammatory response, acid metabolism, lipid metabolism, and glycolysis pathway were down-regulated in the COMB group when compared with the H group. Conclusions The combination of nicotinamide and butyrate sodium may improve muscle quality by enhancing mitochondrial function and antioxidant capacity, inhibiting inflammatory response and glycolysis, and promoting muscle development and hyaluronic acid synthesis.


Background
Intensive stocking in the rapidly developing poultry industry worldwide has become a norm. However, high stocking density causes oxidative stress in broilers [1] and reduces the tenderness and increases the drip loss of breast muscle [2,3]. Oxidation is one of the leading reasons for the deterioration of meat quality [4], and oxidative stress causes protein and lipid peroxidation as well as cellular damage [5,6] which ultimately affects meat quality [7]. Nicotinamide (NAM) reduces oxidative stress and inhibits reactive oxygen species (ROS) production [8,9]. Dietary supplementation with NAM has been observed to minimize the formation of carbonylated proteins in the liver of high-fat fed mice [10]. Butyrate sodium (BA) could also improve antioxidant capacity in a human study [11]. Further, the addition of BA can enhance the activities of superoxide dismutase and catalase and reduce the level of malondialdehyde in serum [12]. Butyrate treatment has been reported to decrease the levels of markers of oxidative stress and apoptosis in mice [13]. As treatment with NAM and BA both can elevate antioxidant capacity and muscle function, it may improve the muscle quality of broilers under high stocking density. Dietary supplementation with 60 mg/kg niacin (NAM precursor) reduces the drip loss of breast muscles in broilers [14]. Dietary supplementation with BA can increase broiler weight, decrease abdominal fat percentage [15], and reduce intramuscular fat content [16].
Mitochondrial biogenesis has previously been associated with preservation of muscle mass and beneficial effects on metabolism [17]. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) is a crucial regulator of mitochondrial biogenesis. Replenishment with nicotinamide adenine dinucleotide (NAD) induces mitochondrial biogenesis by increasing PGC1α expression [18,19]. NAM is the primary source of NAD which is obtained through the salvage pathway. As a precursor of NAD, treatment with NAM also enhances PGC-1α expression [20]. Impaired intramuscular NAD synthesis compromises skeletal muscle mass and strength over time, which can be quickly restored with an oral NAD precursor [21]. Besides, NAD biosynthesis alleviates muscular dystrophy in a zebrafish model [22] and promotes muscle function in Caenorhabditis elegans [23]. Addition of niacin (precursor of NAM) has been reported to increase the number of oxidative type I fibres in skeletal muscles of growing pigs [24] and induce type II to type I muscle fibre transition in sheep [25]. Further, supplementation with butyrate increases mitochondrial function and biogenesis of skeletal muscle in mice and rats [26,27]. Further, the intake of BA increases the percentage of type 1 fibres [26,28] and muscle fibre cross-sectional area in skeletal muscle [13].
Although supplementation with NAM or BA alone can elevate antioxidant capacity and improve the meat quality of broilers, the effect of combined supplementation with NAM and BA on the meat quality of broilers is not clear yet. Therefore, we performed transcriptome sequencing of broiler breast muscles to elucidate the molecular mechanism of the effect of feeding density and nutrient regulation on meat quality.

Production performance and meat quality
There is no significant difference among the H, L and COMB group in corresponding to FI, BW, BWG and FCR (P > 0.05) ( Table 1). Compared with the L group, the H group showed significantly increased cooking loss of breast muscle (P < 0.05). The COMB group showed decreased cooking loss compared with the H group (P < 0.05). Besides, the drip loss in the COMB group was lower than that in the L group, as well (P < 0.05) (Fig. 1). The 45-min pH value in the H group was higher than that in the other 2 groups (P < 0.05) while there was no significant difference in 24-h pH values among the groups. Thus, the pH decline during 45 min to 24 h in the H group was significantly higher than that in the other 2 groups, indicating that the H group had rapid pH drop rate, which was attenuated in the COMB group under high stocking density (Fig. 2).

Anti-oxidant capacity
The stocking density significantly altered the activity of LDH (P = 0.022). The activity of LDH in the H group was higher (P < 0.05) than that in the L group. The COMB group had significantly decreased (P < 0.05) activity of LDH when compared with the H group. However, stocking density had no significant effect on the activities of CK, T-AOC, MDH, anti-superoxide anion and the content of hydroxyproline (Table 2).

RNA sequencing data and differentially expressed genes (DEGs)
In the principal component analysis (PCA), there was a clear divergence among the H, L and COMB groups. In the Venn diagram, the number of identified genes in the H, L and COMB were 11,777, 12,554 and 11,633, respectively (Fig. 3). Compared with the H group, the number of DEGs in the L group and COMB group were 3752 and 773, respectively (Fig. 4).
The gene sets were produced by DEGS. From Venn analysis of genes sets, we found that there were 1310 genes shared in common between the COMB group and the L group. Nevertheless, there were only 6 genes owed by both the COMB group and the H group. Similarly, from the iPath map of metabolic pathways, there were a total of 830 pathways annotated in common. In contrast, there was only 1 pathway owed by both the COMB group and the H group (Fig. 5).

Up-regulated genes in the H group
Compared with those in the L group, a total of 1894 genes were up-regulated in the H group (Fig. 4), which were mainly involved in muscle contraction, cell localization, ion transport, lipid metabolism, glycolysis, proteolysis, and immune stress (Fig. 6).
Muscle contraction-related pathways were enriched in the H group. They involved vital genes including MYLK2, NOS1, TMOD4, and Six1 (Table 3). The H group was enriched for cell-localization-related genes Fig. 2 The pH values of breast muscle. Data are shown as the means ± SEM. Different letters a, b indicate that there are significant differences (P < 0.05) among these groups. L, low stocking density (14 birds/m 2 ); H, high stocking density (18 birds/m 2 ); COMB, combination of NAM and BA (18 birds/m 2 )  For the PCA graph, the distance between each sample point represents the distance of the sample. The closer the distance means higher the similarity between samples; for the VEEN graph, the numbers inside the circle represents the sum of the number of expressed genes in the group. The crossover region represents the number of consensus expressed genes for each group Fig. 4 Volcanic map of differential expression genes. The abscissa is the fold change of the gene expression difference between the two samples and the ordinate is the statistical test value of the gene expression. Each dot in the figure represents a specific gene, the red dot indicates a significantly up-regulated gene, the green dot indicates a significantly down-regulated gene, and the grey dot is a non-significant differential gene such as KEAP1, CDKN1A, ERBB4, and TMOD4 (Table 3). Additionally, high-density up-regulated ion and amino acid transport-related genes included KCNJ12, KCNA7, SLC38A3 and SLC38A4, which are involved in ion transmembrane transport and transporter activity ( Table 4). High-density enriched glycolysis-related pathways included fructose metabolism, fructose-2,6-diphosphate 2-phosphatase activity, and fructose 2,6diphosphate metabolism ( Table 5). The lipid metabolismrelated genes such as MID1IP1, ACACB and Lpin1 were up-regulated in H group, which are involved in lipid synthesis and lipid oxidation ( Table 5). Stress response pathways including non-biologically stimulated cellular responses, extracellular stimuli response and nutritional level response were also enriched in the H group. Furthermore, high-density up-regulated proteolysis-related genes include TINAG, USP24, OTUD1, KEAP1, KLHL34, and SMCR8. Also, high-density enriched immune pathways include the regulation of host defence responses to viruses and prostaglandin receptor-like binding ( Table 6).
In Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, genes involved in calcium signalling pathway (RYR), inflammatory mediator regulation of RTP channels (PLA2) and chemokine signalling pathway (SOS) (Fig. S1, S2 and S3) were enriched in the H group.

Down-regulated genes in the H group
Compared with those in the L group, a total of 1858 genes were down-regulated in the H group (Fig. 4), which were involved in cell adhesion, cell matrix, and cell migration, etc. (Fig. 7).
The genes involved in muscle development include muscle fibre assembly and binding (LMOD2, MYOZ2 and ACTN1, etc.) and muscle fibre development (DSG2, LMOD2 and FSCN1, etc.), which were down-regulated the sum of all the numbers inside the circle represents the total gene of the set. The number, circle intersection area represents the number of shared genes among the gene sets. For the map of KEGG metabolic pathway, the red represents the pathway of the common annotation of the genes in the gene sets of two groups. We thank Kanehisa Laboratories for providing the copyright permission of KEGG pathway maps [29] in H group (Table 7). High-density also down-regulated genes related to cell-matrix pathways such as MMP9, FBLN1, THBS4, and VCAN. High-density also downregulated collagen synthesis and collagen binding related genes including ADAMTS3, ADAMTS14, COL1A2, and LUM (Table 8). Besides, the adhesion-associated genes including DSG2, CSTA, THY1, TGFBI, NOV, CDH11 and FN1 were diminished. Additionally, antioxidant genes including MGST2, PTGS2, NCF1, SOD3, and CYBB were also down-regulated (Table 9).

Up-regulated genes in the COMB group
Compared with those in the H group, up-regulated genes in the COMB group were involved in muscle development, hyaluronic acid synthesis, mitochondrial function, and redox pathway (Fig. 8).
The muscle development-related pathways enriched in the COMB group included positive regulation of muscle tissue development and muscle cell decision processes, which involved key genes such as MYF6, LMCD1 and TRPC3. Besides, the COMB group was enriched for mitochondria-associated pathways such as electron transport chains, mitochondrial respiratory chain complex I and mitochondrial protein complex pathways, which involved genes including TOMM6, NDUFV1, NDUFS5, NDUFB2, NDUFA2, LMCD1, ZNF593 and COASY (Table 10). The hyaluronic acid-related genes upregulated in the COMB group included HYAL1 and HYAL3. Besides, the redox-related genes including LDHD, CPOX, SUOX, NDUFV1, GRHPR, DOHH and NDUFA2 were up-regulated in the COMB group, which were involved in the pathways such as redox process, NAD binding, NADPH binding and NADH dehydrogenase complex (Table 11). In KEGG enrichment analysis, up-regulated genes in the COMB group were involved in The abscissa indicates the GO term, and the ordinate indicates the enrichment ratio. "*"means P < 0.05, "**"means P < 0.01 and "***" means P < 0.001    oxidative phosphorylation (NDUFS5, NDUFV1, NDUFA2, NDUFA13, NDUFB2, NDUFB7 and NDUFC2) (Fig. S7).

Down-regulated genes in the COMB group
Compared with those in the H group, down-regulated genes in the COMB group were involved in the inflammatory response, acid metabolism, fatty acid metabolism, and glycolysis-related pathways (Fig. 9). The inflammatory response-related genes downregulated in the COMB group included CCR5 and ALOX5 while the immune response-related genes included C1S, BLK, CCR5 and MARCH1 (Table 12). The acid metabolism-related pathways include organic acid synthesis process, oxoacid metabolism process and carboxylic acid synthesis process, which involved genes such as PSAT1, SCD, MAT1A, ALOX5, ST3GAL1 and ALDOB. The genes involved in fatty acid metabolism pathways include SCD and ALOX5. In addition, downregulated genes in the COMB group were involved in glycolytic and carbohydrate metabolism, which included GALNT16, ST3GAL1, ALDOB and MAT1A (Table 13).
In KEGG enrichment analysis, genes involved in the regulation of lipolysis in adipocytes (PLIN), glycolysis/ gluconeogenesis (ALDO) and arachidonic acid metabolism (ALOX5) were down-regulated in the COMB group (Fig. S8, S9 and S10).

Transcriptome differential gene verification
The transcriptome differential genes were verified by real-time PCR, and the gene expression pattern was consistent with the transcriptome results (Fig. 10).

Discussion
In the current study, the H group showed significantly increased cooking loss of breast muscle when compared with the L group. The muscle disease such as PSE (Pale, Soft and Exudative) meat [30] and wooden breast [31] have higher cooking loss than normal meat. The abscissa indicates the GO term, and the ordinate indicates the enrichment ratio. "*"means P < 0.05, "**"means P < 0.01 and "***" means P < 0.001 Stress is an essential cause of the decline in meat quality. In this study, the activity of LDH in the H group was higher than that in the L group. In transcriptome analysis, the enriched genes in the H group were involved in stimuli response pathway. In the H group, genes encoding nitric oxide synthase 1 (NOS1), Kelch-Like ECHassociated protein 1 (KEAP1) and cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) were upregulated. High levels of NO reduce the antioxidant capacity of post-mortem muscles, increasing the accumulation of ROS and reactive nitrogen, resulting in high levels of protein oxidation. Studies have shown that inhibition of nitric oxide synthase can significantly reduce protein carbonyl content and protein oxidation [32]. Inhibition of CDKN1A expression by miRNAs promotes myoblast proliferation [33]. Up-regulation of KEAP1 expression increases the degradation of Nrf2 in cells, making cells more susceptible to free radical damage [34]. Heat stress can reduce the oxidative stability of broiler muscle protein and reduce the strength of the myofibrillar gel, resulting in increased drip loss and cooking loss in broilers [35]. A study has shown that genes involved in the stimulation response pathway are significantly enriched in muscles with high drip loss [36]. Therefore, increased expression of stress pathwayrelated genes such as KEAP1 and CDKN1A may be one of the causes of muscle quality deterioration.
This study found that the H group had the fastest pH decline rate. The rapid decline in pH is usually accompanied by an increase in the rate of glycolysis and the accumulation of lactic acid, resulting in a decrease of muscle function [37]. In this study, high stocking density led to up-regulation of genes involved in glycolysis and fat metabolism pathways. Anaerobic glycolysis is a vital energy metabolism pathway for post-mortem broilers. Under anaerobic conditions, muscle glycogen degradation occurs through glycolysis, which causes pyruvate to synthesize lactic acid, thus leading to a decrease in muscle pH due to the accumulation of lactic acid [38,39]. High stocking density in this study also caused upregulation of striated muscle contraction pathwayrelated genes such as SIX homeobox 1 (Six1). It has been found that white streak muscles have up-regulated expression of striated muscle contraction-related genes compared with normal meat [40]. Six1 converts slow muscle fibres into fast muscle fibres [41,42]. The proportion of fast muscle fibres was negatively correlated with post-mortem pH [43]. Besides, the enriched genes in the H group were involved in calcium transport, sodium transport, and cation transport. Importantly, ion balance is the basis for maintaining normal physiological functions. Abnormal metabolism caused by high concentrations of calcium ions may be associated with the incidence of turkey PSE [44]. Furthermore, changes in muscle cation homeostasis may mark the beginning of muscle degeneration [45] and cause a reduction in meat quality [46]. Dietary supplementation with niacin (nicotinamide precursor) at 60 mg/kg was reported to reduce the drip loss of breast muscles in broilers [14]. In our study, the COMB group showed significantly reduced drip loss and cooking loss compared with the H group. Further, the COMB group showed significantly decreased activity of LDH compared to the H group. Besides, the COMB group showed inhibited expression of glycolytic and inflammation genes [37].
In KEGG enrichment analysis, the enriched genes in the H group were involved in inflammatory mediator regulation of RTP channels and chemokine signalling pathway. In contrast, the up-regulated genes in the COMB group were involved in the inflammatory response. Macrophage infiltration in the pectoral muscle might cause muscle damage [47]. The muscle disease such as white striped muscle is usually accompanied by elevated expression of immune-related genes [40]. During tissue degeneration, immune cells immediately enter the site of injury, triggering an inflammatory response, and attracting more immune cells to the damaged area. It can cause phagocytosis of cell debris and release of cytokines, prostaglandins and other signalling proteins, resulting in interstitial spaces [48]. We found that key genes down-regulated in the H group, such as MYOZ2, were involved in muscle development, cell adhesion, cell matrix, collagen, and cytoskeleton. MYOZ2 belongs to sarcomeric family and links calcineurin to alpha-actinin at the Z-line of skeletal muscle sarcomere and can play a role in skeletal muscle differentiation and growth [49]. It was suggested that MYOZ2 knockout mice had neuromuscular disease [50].
Also, genes down-regulated in the H group were involved in cell matrix and collagen pathways. Extracellular matrix (ECM) is a major macromolecule in skeletal muscle and has a substantial effect on meat quality. The remodelling of ECM is mainly regulated by matrix metalloproteinases. The expression of matrix metalloproteinase-1 is negatively correlated with cooking loss and positively correlated with hydraulic performance [51]. Collagen is an abundant connective tissue protein that is an important factor in the tenderness and texture of the meat and is well resistant to physical damage during cooking [52]. The addition of collagen increases the ability of pork [53] and poultry [54] to combine with water and reduces cooking losses. Furthermore, high stocking density downregulates cell adhesion, cytoskeletal and integrin binding-related genes such as integrin subunit alpha 8 (ITGA8), integrin subunit beta 8 (ITGB8) and integrin subunit beta like 1 (ITGBL1). Proteolytic degradation of cell adhesion proteins is associated with the production of drip channels [55]. The cytoskeleton is a highly complex network composed of a large number of connections between myofibrils and myofibrillar membranes. Degradation of the cytoskeleton causes extracellular water to flow into the muscle cells, thereby increasing drip loss [56]. Integrins are heterodimeric cell adhesion molecules that bind the extracellular matrix to the cytoskeleton and play an essential role in controlling cell membrane-cytoskeletal attachment and signalling pathways [57]. The β-chain integrin is responsible for the attachment of the cell membrane to the cytoskeleton [58]. Degradation of β1 integrin promotes the formation of water channels between cells and cell membranes, thereby increasing drip loss [59]. In addition, it has been found that integrins are inversely related to pork drip loss [60].
Compared with the H group, the COMB group showed up-regulation of muscle development, hyaluronic acid levels, mitochondrial function, and the redox pathway. Studies have found that hyaluronic acid is a crucial water-holding molecule [61,62]. Furthermore, supplementation with antioxidant isoflavones can be achieved by reducing lipid peroxidation and increasing oxidative stability in the pectoral muscles [63]. Therefore, enhanced hyaluronic acid biosynthesis and antioxidant capacity may improve muscle quality.
Additionally, up-regulated genes in the COMB group involved the complex I-related gene NDUFS5. The mitochondrial respiratory chain (MRC) consists of four membrane-bound electron transport protein complexes (I-IV) and ATP synthase (complex V) that produce ATP for cellular processes. Complex I deficiency, NADH ubiquinone oxidoreductase is the most common form of MRC dysfunction and is associated with a variety of diseases [64,65]. Complex I deficiency leads to various physiological disorders such as ATP depletion, calcium homeostasis, ROS accumulation [66] and induction of apoptosis [67]. A study found that mitochondrial and Fig. 8 GO enrichment analysis of up-regulated genes in the COMB group. The abscissa indicates the GO term, and the ordinate indicates the enrichment ratio. "*"means P < 0.05, "**"means P < 0.01 and "***" means P < 0.001   The abscissa indicates the GO term, and the ordinate indicates the enrichment ratio. "*"means P < 0.05, "**"means P < 0.01 and "***" means P < 0.001 oxidative phosphorylation-related gene expression was negatively correlated with drip loss. A negative correlation with drip loss means that there is a decrease in the number of mitochondria in muscles with high drip loss [68].

Conclusion
High stocking density may cause oxidative stress, abnormal muscle contraction, and abnormal metabolism of glycolipids; destroy ion channels and cell matrix; reduce muscle strength by inhibiting muscle development, and cell adhesion and collagen synthesis, all of which result in reduced muscle function. Supplementation with NAM and BA in combination can improve mitochondrial function and antioxidant capacity, and inhibit inflammatory response and glycolysis by promoting muscle development and hyaluronic acid synthesis, thereby reducing drip loss of the breast muscle and improving muscle quality (Fig. 11).

Experimental birds, diets, and management
Amount of 300 Cobb broilers (21-day-old) were divided into 3 groups: low stocking density (L, 14 birds/m 2 ), high stocking density (H, 18 birds/m 2 ) and combination of NAM and BA (COMB, 18 birds/m 2 ), with 6 replicates  for each group. The stocking densities of this study are referred to Vargas-Galicia et al. [69]. The L and H groups were fed a basal diet. The COMB group was fed basal diet supplemented with 50 mg/kg NAM and 500 mg/kg BA. The dosage 50 mg/kg NAM and 500 mg/kg BA used in this study were based on our previous studies [70,71]. Experimental diets were designed to meet nutrient requirements of National Research Council (1994) [72]. The nutrient levels and composition of basic diet were shown in Table 14. Broilers in this study were raised from 21-day-old to 42-day-old, and feed and water were provided ad libitum.

Production performance determination and sample collection
On 42-day, remove feed for 5 h and record the remaining feed per cage, then weight the body weight (BW) of broilers. Calculate body weight gain (BWG), feed intake (FI) and the feed conversion rate (FCR). For breast collection, one broiler per replicate was randomly selected and euthanized by intravenous injection of pentobarbital sodium (390 mg/ml) at a dose of 300 mg/kg. The breast muscle was collected for meat quality analysis and further study.
Each group had six replicates for the determination of meat quality, enzyme activities and mRNA relative expression; there were three biology replicates in each group for RNA-sequencing.

Meat quality analysis
The meat quality of right side major pectoral muscle was quickly determined after slaughtering. The drip loss was determined according to Liu et al. [73]. Cooking loss was measured according to the protocol described by Cai et al. [74]. The pH values of the pectoral muscle at 45 min and 24 h were measured by a pH meter (testo 205; Germany). Each sample was tested at 3 different locations (top, middle and bottom) and the average of 3 measurements was calculated.

Enzyme activity determination in breast muscle
The total antioxidant capacity (T-AOC, cat#A015), antisuperoxide anion (cat#A052), the activities of creatine kinase (CK, cat#A032), lactate dehydrogenase (LDH, cat#A020-2), malic dehydrogenase (MDH, cat#A021-2), Fig. 11 The graphic description of the normalization effect of nicotinamide and sodium butyrate on breast muscle. This is the original graph drafted by the authors of this article and the content of hydroxyproline (cat# A030-2) in breast muscle were measured by commercial analytical kits (Jian Cheng Bioengineering Institute, Nanjing, China).

RNA extraction, library preparation and Illumina Hiseq X ten sequencing
Total RNA from the breast muscle was extracted by TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA). The RNA quality was then measured by 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA) and quantified using the ND-2000 (Nanodrop Technologies, Wilmington, Delaware).
RNA-seq library was constructed according to Tru-SeqTM RNA sample preparation Kit from Illumina (San Diego, CA, USA), then was sequenced with the Illumina HiSeq X Ten (2 × 150 bp read length).

Read mapping, differential expression analysis and functional enrichment
SeqPrep and Sickle were applied to process raw pairedend reads. Then use TopHat version2.0.0 [75] software to align the clean reads to the reference genome.
FRKM method was applied to identify differentially expressed genes (DEGs). RSEM [76] was used to quantify gene abundances. Differential gene expression was analyzed by R statistical package software EdgeR [77]. Goatools and KOBAS [78] were applied for KEGG pathway enrichment and GO functional analysis.
The mRNA expression of muscle developmental genes Several differentially expressed genes involved muscle development were validated by real-time PCR analysis. The mRNA expression of muscle was determined as we  previously described [71]. The primer sequences of target gene and housekeeping gene beta-actin were shown in Table 15. The results of gene expression were analyzed and compared using 2 -ΔΔCT .

Statistical analysis
The results are expressed as means with their standard error mean (SEM). SPSS 20.0 for Windows (SPSS Inc. Chicago, IL) was applied for One-way ANOVA analysis. Significant difference was considered at P < 0.05.