Article pubs.acs.org/JAFC
Regucalcin Expression as a Diagnostic Tool for the Illicit Use of Steroids in Veal Calves Laura Starvaggi Cucuzza, Bartolomeo Biolatti, Alessandra Sereno, and Francesca T. Cannizzo* Department of Veterinary Sciences, University of Turin, Largo Paolo Braccini 2, 10095 Grugliasco (Turin), Italy ABSTRACT: It has been previously demonstrated that sex steroid hormone treatment down-regulates regucalcin gene expression in the accessory sex glands and testis of prepubertal and adult male bovines. The aim of this study was to investigate whether low doses of sex steroid hormones combined with other drugs significantly affect regucalcin gene expression in the accessory sex glands and testis of veal calves. The regucalcin expression was down-regulated in the bulbo-urethral glands of estrogen-treated calves, whereas it was up-regulated in the prostate of estrogen-treated calves. Only the testis of androgen-treated calves showed a down-regulation of the regucalcin expression. Thus, the administration of sex steroid hormones, even in low doses and combined with other molecules, could affect regucalcin expression in target organs. Particularly, the specific response in the testis suggests regucalcin expression in this organ as a first molecular biomarker of illicit androgen administration in bovine husbandry. KEYWORDS: biomarker, gene expression, regucalcin, sex steroid hormones, veal calves
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regucalcin (RGN) promoter region.6 RGN is a calcium (Ca2+)-binding protein first identified in 1978 in the rat liver7 regulating Ca2+ intracellular homeostasis,8 Ca2+-dependent enzymes activity,9−11 and cell death and proliferation.12−15 The regulation of RGN expression through sex steroids in liver,16 kidney cortex,17 and, more recently, breast, prostate, and testis6,18−20 has been demonstrated. In a previous study, high doses of E2 greatly down-regulated RGN gene expression in the accessory sex glands of veal calves, whereas high doses of testosterone strongly decreased RGN expression only in the testis.20 Because sex steroid hormones have been often administered in low-dosage cocktails, the aim of the present study was to investigate whether low doses of sex steroid hormones combined with other illicit drugs significantly affect RGN gene expression in veal calf target organs, namely, the bulbo-urethral glands, prostate, and testis.
INTRODUCTION Despite the European Union ban on the use of the sex steroid hormones and other growth promoters (GPs) in bovine livestock, these molecules are administered to veal calves to improve live weight gain. The detection of the abuse of these drugs is largely based on the direct identification of specific residues via highly sensitive LC-MS/MS and GC-MS analyses of various matrices, namely, the muscle tissue, blood, and urine.1 Although these methods are highly sensitive, they are expensive and time-consuming. Moreover, the search for a specific chemical residue limits the possibility of investigation only to a few known molecules, whereas hundreds of new drugs are introduced every year on the black market. In addition, GPs are often used in low-dosage cocktails that could elude official controls. Novel techniques allowing indirect detection of illegal GP administration have been developed to circumvent the limits of the official analyses. These indirect methods are based on the detection of GPs’ biological effects in target tissues and organs and could be used for screening purposes. Particularly, the transcriptomic assays are based on the evident changes of gene expression in target tissue or cell population in specific environmental conditions. The identification of a specific transcriptional marker can be used to develop a novel screening method for the meat production chain and national surveillance programs. This approach has recently allowed the identification of molecular biomarkers suitable to detect illegally treated animals. For example, the progesterone receptor (PR) gene expression levels were increased in the bulbo-urethral glands and prostate of 17β-estradiol (E2)-treated calves and beef cattle.2−4 More recently, the change in oxytocin (OXT) gene expression was proved to be an intriguing biomarker to discover estrogen and glucocorticoid abuse directly in meat.5 In addition, androgens affect several genes containing androgen-responsive elements (ARE). Different ARE consensus sequences upstream from the transcription initiation site have been identified in the © 2015 American Chemical Society
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MATERIALS AND METHODS
Animals and Experimental Design. Thirty Friesian male veal calves of approximately 4 months of age were used. The calves were housed in 10 × 15 m boxes, with concrete floors lacking litter or lateral partitions. The calves were tethered and fed liquid milk replacer twice a day (providing, per kg, 950 g of dry matter (DM), 230 g of crude protein (CP), 210 g of ether extract (EE), 60 g of ash, 1 g of cellulose, 75 mg of retinol, 50 mg of ascorbic acid, 5 mg of Cu, 0.125 mg of cholecalciferol, and 80 mg of α-tocopherol). The amount of feed was gradually increased to 8 L/calf/day and then gradually increased to 16 L/calf/day. After 1 month, 0.5 kg of barley straw (per kg, 900 g of DM, 20 g of CP, 10 g of EE, 60 g of ash, and 410 g of crude fiber) was added to the diet, according to the recommendations of the European Commission (97/182/EC). At approximately 5 months of age the calves were randomly assigned to four experimental groups: group A (n = 8) was weekly administered 5 mg/animal of estradiol benzoate Received: Revised: Accepted: Published: 5702
March 16, 2015 May 27, 2015 May 27, 2015 May 27, 2015 DOI: 10.1021/acs.jafc.5b01337 J. Agric. Food Chem. 2015, 63, 5702−5706
Article
Journal of Agricultural and Food Chemistry
Figure 1. Treatment schedule of veal calves. Group A was weekly administered 5 mg/animal of estradiol benzoate (E) intramuscularly for 6 weeks in combination with 0.25 mg/animal/day of brotizolam (BR) per os for the last 31 days of treatment (a); group B was weekly administered 5 mg/ animal of estradiol benzoate intramuscularly for 6 weeks in combination with 0.40 mg/animal/day of dexamethasone (DEX) per os for the last 31 days of treatment (b); group C was administered 150 mg/animal of Nandrosol (NA) every 2 weeks four times in combination with 80 mg/animal/ day of ractopamine (RA) per os for the last 31 days of treatment (c). The calves were slaughtered 3 days after the last treatment. intramuscularly for 6 weeks in combination with 0.25 mg/animal/day of brotizolam per os for the last 31 days of treatment; group B (n = 6) was weekly administered 5 mg/animal of estradiol benzoate intramuscularly for 6 weeks in combination with 0.40 mg/animal/ day of dexamethasone (DEX) per os for the last 31 days of treatment; group C (n = 8) was administered 150 mg/animal of Nandrosol (17β19-nortestosterone phenylpropionate) every 2 weeks for four times in combination with 80 mg/animal/day of ractopamine per os for the last 31 days of treatment; group K (n = 8) served as control (Figure 1). The calves were slaughtered 3 days after the last treatment. The hormone dosages were selected according to previous studies.2,21,22 The animals were healthy upon intravitam and post-mortem examinations. The experiments were authorized through the Italian Ministry of Health and the Ethical Committee of the University of Turin. The carcasses of the treated animals were appropriately destroyed (2003/74/CE-DL 16 March 2006, No. 158). Tissue Sampling and Processing. Samples of bulbo-urethral glands, prostate and testis tissues were obtained from each animal at slaughter. The samples were immediately frozen in liquid nitrogen and then stored at −80 °C for molecular and Western blot (WB) analyses. RNA Extraction, Reverse Transcription, and qPCR. Several milligrams of each tissue sample were disrupted using a TissueLyser II (Qiagen, Hilden, Germany) using stainless steel beads in 1 mL of TRIzol reagent (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA concentration was spectrophotometrically determined, and the RNA integrity was evaluated using an automated electrophoresis station (Experion Instrument, Bio-Rad, Hercules, CA, USA). cDNA was synthesized from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (Qiagen). The effect of sex steroid hormones on RGN mRNA expression in the bulbo-urethral glands, prostate, and testis of veal calves was evaluated through quantitative polymerase chain reaction (qPCR). To determine the relative amounts of specific RGN transcripts, the cDNA obtained from retrotranscription was subjected to qPCR23 using the
IQ5 detection system (Bio-Rad) and respective gene primers in an IQ SYBR Green Supermix (Bio-Rad). Primer sequences of RGN were designed using Primer3 (vers. 0.4.0), as previously described.20 The cyclophilin A (PPIA) gene was used as a housekeeping gene, as previously described.20 The levels of gene expression were calculated using a relative quantification assay based on the comparative Cq method (ΔΔCq method),24 previously verifying that efficiencies of target and housekeeping gene amplification were similar. Subsequently, the relative abundances of each transcript, normalized to the endogenous housekeeping gene (PPIA) and relative to the control sample, were calculated as 2−ΔΔCq (fold increase).25 Western Blot Analysis. The effect of sex steroid hormones on RGN protein expression in the bulbo-urethral glands, prostate, and testis of veal calves was evaluated through WB analysis. Total protein was isolated from veal calves’ bulbo-urethral glands, prostate, and testis using RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with protease inhibitor cocktail (Sigma). Protein concentration was determined using the Bio-Rad DC Protein Assay. Twenty micrograms of total protein were resolved through 12.5% SDS-PAGE. The proteins were blotted onto Trans-Blot Turbo Mini Nitrocellulose Transfer membrane (Bio-Rad) using a Trans-Blot Turbo Blotting System (Bio-Rad). The blotted membranes were blocked with 5% BSA in TBS-0.1% Tween for 1 h at room temperature, followed by overnight incubation with an anti-RGN rabbit polyclonal antibody (1:200; Sigma, St. Louis, MO, USA). The membranes were subsequently incubated with secondary anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (1:1000), developed using the SuperSignal West Pico IgG Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA), and recorded on CL-XPosure X-ray film (Thermo Fisher Scientific). α-tubulin (1:10000; clone B-5-1-2; Sigma) was used as a total protein loading control. 5703
DOI: 10.1021/acs.jafc.5b01337 J. Agric. Food Chem. 2015, 63, 5702−5706
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effects of estradiol benzoate combined with brotizolam (group A), estradiol benzoate combined with dexamethasone (group B), and Nandrosol combined with ractopamine (group C) on RGN gene expression compared with the control group K in the bulbo-urethral glands (a), prostate (b), and testis (c) of veal calves. The results are presented as the means ± SEM. The y-axes show arbitrary units representing mRNA relative expression levels. A representative Western blot showing RGN protein expression in the bulbo-urethral glands, prostate, and testis (d) of veal calves using an anti-RGN polyclonal antibody (1:200). α-Tubulin (1:10000) was used as loading control. The relative expression levels of RGN protein in the bulbo-urethral glands, prostate, and testis (e) of veal calves were determined and normalized using α-tubulin. The results are presented as the means ± SEM. (∗) P < 0.05 and (∗∗) P < 0.01 versus the control group K.
2.94 × 10−4) compared with the control group K (3.57 × 10−3 ± 4.45 × 10−4) (P < 0.01) (Figure 2a). The RGN expression increased in the prostate of group A (7.82 × 10−3 ± 9.88 × 10−4) (P < 0.01) and group B (6.51 × 10−3 ± 9.05 × 10−4) (P < 0.05) compared with the control group K (3.73 × 10−3 ± 4.84 × 10−4) (Figure 2b). The RGN expression decreased in the testis of group C (4.73 × 10−3 ± 8.88 × 10−4) (P < 0.01) compared with the control group K (1.72 × 10−2 ± 3.80 × 10−3) (Figure 2c). Western Blot Analysis. RGN protein expression was down-regulated in the bulbo-urethral glands of group A compared with control group K (P < 0.01) (Figure 2d,e) and up-regulated in the prostate of groups A and B compared with control group K (P < 0.05 and P < 0.01, respectively) (Figure 2d,e). RGN protein expression decreased only in the testis of
Band densities were obtained according to standard methods using ImageJ software (U.S. National Institutes of Health) and normalized by division with the respective α-tubulin band density. Statistical Analysis. Statistical analyses were performed using Graph-Pad InStat (vers. 3.05) statistical software (GraphPad Inc., San Diego, CA, USA). The analysis of RGN expression was performed using one-way analysis of variance (ANOVA), followed by Dunnett’s post-test versus the control group K. The Grubbs test was used to reveal potential outliers. A P value of
Regucalcin Expression as a Diagnostic Tool for the Illicit Use of Steroids in Veal Calves Laura Starvaggi Cucuzza, Bartolomeo Biolatti, Alessandra Sereno, and Francesca T. Cannizzo* Department of Veterinary Sciences, University of Turin, Largo Paolo Braccini 2, 10095 Grugliasco (Turin), Italy ABSTRACT: It has been previously demonstrated that sex steroid hormone treatment down-regulates regucalcin gene expression in the accessory sex glands and testis of prepubertal and adult male bovines. The aim of this study was to investigate whether low doses of sex steroid hormones combined with other drugs significantly affect regucalcin gene expression in the accessory sex glands and testis of veal calves. The regucalcin expression was down-regulated in the bulbo-urethral glands of estrogen-treated calves, whereas it was up-regulated in the prostate of estrogen-treated calves. Only the testis of androgen-treated calves showed a down-regulation of the regucalcin expression. Thus, the administration of sex steroid hormones, even in low doses and combined with other molecules, could affect regucalcin expression in target organs. Particularly, the specific response in the testis suggests regucalcin expression in this organ as a first molecular biomarker of illicit androgen administration in bovine husbandry. KEYWORDS: biomarker, gene expression, regucalcin, sex steroid hormones, veal calves
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regucalcin (RGN) promoter region.6 RGN is a calcium (Ca2+)-binding protein first identified in 1978 in the rat liver7 regulating Ca2+ intracellular homeostasis,8 Ca2+-dependent enzymes activity,9−11 and cell death and proliferation.12−15 The regulation of RGN expression through sex steroids in liver,16 kidney cortex,17 and, more recently, breast, prostate, and testis6,18−20 has been demonstrated. In a previous study, high doses of E2 greatly down-regulated RGN gene expression in the accessory sex glands of veal calves, whereas high doses of testosterone strongly decreased RGN expression only in the testis.20 Because sex steroid hormones have been often administered in low-dosage cocktails, the aim of the present study was to investigate whether low doses of sex steroid hormones combined with other illicit drugs significantly affect RGN gene expression in veal calf target organs, namely, the bulbo-urethral glands, prostate, and testis.
INTRODUCTION Despite the European Union ban on the use of the sex steroid hormones and other growth promoters (GPs) in bovine livestock, these molecules are administered to veal calves to improve live weight gain. The detection of the abuse of these drugs is largely based on the direct identification of specific residues via highly sensitive LC-MS/MS and GC-MS analyses of various matrices, namely, the muscle tissue, blood, and urine.1 Although these methods are highly sensitive, they are expensive and time-consuming. Moreover, the search for a specific chemical residue limits the possibility of investigation only to a few known molecules, whereas hundreds of new drugs are introduced every year on the black market. In addition, GPs are often used in low-dosage cocktails that could elude official controls. Novel techniques allowing indirect detection of illegal GP administration have been developed to circumvent the limits of the official analyses. These indirect methods are based on the detection of GPs’ biological effects in target tissues and organs and could be used for screening purposes. Particularly, the transcriptomic assays are based on the evident changes of gene expression in target tissue or cell population in specific environmental conditions. The identification of a specific transcriptional marker can be used to develop a novel screening method for the meat production chain and national surveillance programs. This approach has recently allowed the identification of molecular biomarkers suitable to detect illegally treated animals. For example, the progesterone receptor (PR) gene expression levels were increased in the bulbo-urethral glands and prostate of 17β-estradiol (E2)-treated calves and beef cattle.2−4 More recently, the change in oxytocin (OXT) gene expression was proved to be an intriguing biomarker to discover estrogen and glucocorticoid abuse directly in meat.5 In addition, androgens affect several genes containing androgen-responsive elements (ARE). Different ARE consensus sequences upstream from the transcription initiation site have been identified in the © 2015 American Chemical Society
■
MATERIALS AND METHODS
Animals and Experimental Design. Thirty Friesian male veal calves of approximately 4 months of age were used. The calves were housed in 10 × 15 m boxes, with concrete floors lacking litter or lateral partitions. The calves were tethered and fed liquid milk replacer twice a day (providing, per kg, 950 g of dry matter (DM), 230 g of crude protein (CP), 210 g of ether extract (EE), 60 g of ash, 1 g of cellulose, 75 mg of retinol, 50 mg of ascorbic acid, 5 mg of Cu, 0.125 mg of cholecalciferol, and 80 mg of α-tocopherol). The amount of feed was gradually increased to 8 L/calf/day and then gradually increased to 16 L/calf/day. After 1 month, 0.5 kg of barley straw (per kg, 900 g of DM, 20 g of CP, 10 g of EE, 60 g of ash, and 410 g of crude fiber) was added to the diet, according to the recommendations of the European Commission (97/182/EC). At approximately 5 months of age the calves were randomly assigned to four experimental groups: group A (n = 8) was weekly administered 5 mg/animal of estradiol benzoate Received: Revised: Accepted: Published: 5702
March 16, 2015 May 27, 2015 May 27, 2015 May 27, 2015 DOI: 10.1021/acs.jafc.5b01337 J. Agric. Food Chem. 2015, 63, 5702−5706
Article
Journal of Agricultural and Food Chemistry
Figure 1. Treatment schedule of veal calves. Group A was weekly administered 5 mg/animal of estradiol benzoate (E) intramuscularly for 6 weeks in combination with 0.25 mg/animal/day of brotizolam (BR) per os for the last 31 days of treatment (a); group B was weekly administered 5 mg/ animal of estradiol benzoate intramuscularly for 6 weeks in combination with 0.40 mg/animal/day of dexamethasone (DEX) per os for the last 31 days of treatment (b); group C was administered 150 mg/animal of Nandrosol (NA) every 2 weeks four times in combination with 80 mg/animal/ day of ractopamine (RA) per os for the last 31 days of treatment (c). The calves were slaughtered 3 days after the last treatment. intramuscularly for 6 weeks in combination with 0.25 mg/animal/day of brotizolam per os for the last 31 days of treatment; group B (n = 6) was weekly administered 5 mg/animal of estradiol benzoate intramuscularly for 6 weeks in combination with 0.40 mg/animal/ day of dexamethasone (DEX) per os for the last 31 days of treatment; group C (n = 8) was administered 150 mg/animal of Nandrosol (17β19-nortestosterone phenylpropionate) every 2 weeks for four times in combination with 80 mg/animal/day of ractopamine per os for the last 31 days of treatment; group K (n = 8) served as control (Figure 1). The calves were slaughtered 3 days after the last treatment. The hormone dosages were selected according to previous studies.2,21,22 The animals were healthy upon intravitam and post-mortem examinations. The experiments were authorized through the Italian Ministry of Health and the Ethical Committee of the University of Turin. The carcasses of the treated animals were appropriately destroyed (2003/74/CE-DL 16 March 2006, No. 158). Tissue Sampling and Processing. Samples of bulbo-urethral glands, prostate and testis tissues were obtained from each animal at slaughter. The samples were immediately frozen in liquid nitrogen and then stored at −80 °C for molecular and Western blot (WB) analyses. RNA Extraction, Reverse Transcription, and qPCR. Several milligrams of each tissue sample were disrupted using a TissueLyser II (Qiagen, Hilden, Germany) using stainless steel beads in 1 mL of TRIzol reagent (Ambion, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA concentration was spectrophotometrically determined, and the RNA integrity was evaluated using an automated electrophoresis station (Experion Instrument, Bio-Rad, Hercules, CA, USA). cDNA was synthesized from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (Qiagen). The effect of sex steroid hormones on RGN mRNA expression in the bulbo-urethral glands, prostate, and testis of veal calves was evaluated through quantitative polymerase chain reaction (qPCR). To determine the relative amounts of specific RGN transcripts, the cDNA obtained from retrotranscription was subjected to qPCR23 using the
IQ5 detection system (Bio-Rad) and respective gene primers in an IQ SYBR Green Supermix (Bio-Rad). Primer sequences of RGN were designed using Primer3 (vers. 0.4.0), as previously described.20 The cyclophilin A (PPIA) gene was used as a housekeeping gene, as previously described.20 The levels of gene expression were calculated using a relative quantification assay based on the comparative Cq method (ΔΔCq method),24 previously verifying that efficiencies of target and housekeeping gene amplification were similar. Subsequently, the relative abundances of each transcript, normalized to the endogenous housekeeping gene (PPIA) and relative to the control sample, were calculated as 2−ΔΔCq (fold increase).25 Western Blot Analysis. The effect of sex steroid hormones on RGN protein expression in the bulbo-urethral glands, prostate, and testis of veal calves was evaluated through WB analysis. Total protein was isolated from veal calves’ bulbo-urethral glands, prostate, and testis using RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) supplemented with protease inhibitor cocktail (Sigma). Protein concentration was determined using the Bio-Rad DC Protein Assay. Twenty micrograms of total protein were resolved through 12.5% SDS-PAGE. The proteins were blotted onto Trans-Blot Turbo Mini Nitrocellulose Transfer membrane (Bio-Rad) using a Trans-Blot Turbo Blotting System (Bio-Rad). The blotted membranes were blocked with 5% BSA in TBS-0.1% Tween for 1 h at room temperature, followed by overnight incubation with an anti-RGN rabbit polyclonal antibody (1:200; Sigma, St. Louis, MO, USA). The membranes were subsequently incubated with secondary anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (1:1000), developed using the SuperSignal West Pico IgG Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA), and recorded on CL-XPosure X-ray film (Thermo Fisher Scientific). α-tubulin (1:10000; clone B-5-1-2; Sigma) was used as a total protein loading control. 5703
DOI: 10.1021/acs.jafc.5b01337 J. Agric. Food Chem. 2015, 63, 5702−5706
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effects of estradiol benzoate combined with brotizolam (group A), estradiol benzoate combined with dexamethasone (group B), and Nandrosol combined with ractopamine (group C) on RGN gene expression compared with the control group K in the bulbo-urethral glands (a), prostate (b), and testis (c) of veal calves. The results are presented as the means ± SEM. The y-axes show arbitrary units representing mRNA relative expression levels. A representative Western blot showing RGN protein expression in the bulbo-urethral glands, prostate, and testis (d) of veal calves using an anti-RGN polyclonal antibody (1:200). α-Tubulin (1:10000) was used as loading control. The relative expression levels of RGN protein in the bulbo-urethral glands, prostate, and testis (e) of veal calves were determined and normalized using α-tubulin. The results are presented as the means ± SEM. (∗) P < 0.05 and (∗∗) P < 0.01 versus the control group K.
2.94 × 10−4) compared with the control group K (3.57 × 10−3 ± 4.45 × 10−4) (P < 0.01) (Figure 2a). The RGN expression increased in the prostate of group A (7.82 × 10−3 ± 9.88 × 10−4) (P < 0.01) and group B (6.51 × 10−3 ± 9.05 × 10−4) (P < 0.05) compared with the control group K (3.73 × 10−3 ± 4.84 × 10−4) (Figure 2b). The RGN expression decreased in the testis of group C (4.73 × 10−3 ± 8.88 × 10−4) (P < 0.01) compared with the control group K (1.72 × 10−2 ± 3.80 × 10−3) (Figure 2c). Western Blot Analysis. RGN protein expression was down-regulated in the bulbo-urethral glands of group A compared with control group K (P < 0.01) (Figure 2d,e) and up-regulated in the prostate of groups A and B compared with control group K (P < 0.05 and P < 0.01, respectively) (Figure 2d,e). RGN protein expression decreased only in the testis of
Band densities were obtained according to standard methods using ImageJ software (U.S. National Institutes of Health) and normalized by division with the respective α-tubulin band density. Statistical Analysis. Statistical analyses were performed using Graph-Pad InStat (vers. 3.05) statistical software (GraphPad Inc., San Diego, CA, USA). The analysis of RGN expression was performed using one-way analysis of variance (ANOVA), followed by Dunnett’s post-test versus the control group K. The Grubbs test was used to reveal potential outliers. A P value of
