通讯机构:
[Zeng, JG; Yang, ZH ] H;Hunan Agr Univ, Hunan Key Lab Tradit Chinese Vet Med, Changsha 410128, Peoples R China.;Hunan Agr Univ, Coll Vet Med, Changsha 410128, Peoples R China.
关键词:
sanguinarine;UPLC-Q-TOF-MS/MS;metabolites;rats;in vivo
摘要:
Sanguinarine (SAN), as the main active component of a traditional Chinese veterinary medicine, has been widely used in the animal husbandry and breeding industry. However, the metabolites of SA are still uncertain. Therefore, this research aimed to investigate the metabolites of SA based on rats in vivo. The blood, feces, and urine of rats were collected after the oral administration of 40 mg/kg SAN. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was employed to identify the metabolites of SAN. The elemental composition of sanguinarine metabolites was inferred by analyzing their exact molecular weight, and the structures of the metabolites were predicted based on their fragment ions and cleavage pathways. A total of 12 metabolites were identified, including three metabolites in the plasma, four in the urine, and nine in the feces. According to the possible metabolic pathways deduced in this study, SAN was mainly metabolized through reduction, oxidation, demethylation, hydroxylation, and glucuronidation. This present research has summarized the metabolism of SAN in rats, which is helpful for further studying the metabolic mechanism of SAN in vivo and in vitro.
通讯机构:
[Wan, FC ; Liu, L ] H;Hunan Agr Univ, Coll Anim Sci & Technol, Changsha, Hunan, Peoples R China.;Hunan Agr Univ, Coll Vet Med, Changsha, Hunan, Peoples R China.
关键词:
L-leucine;beef cattle;in vitro technique;rumen protection rate;rumen fermentation;bacterial community
摘要:
This study was conducted to compare the effects of rumen-protected (RP-Leu) and unprotected L-leucine (RU-Leu) on the fermentation parameters, bacterial composition, and amino acid metabolism in vitro rumen batch incubation. The 5.00 g RP-Leu or RU-Leu products were incubated in situ in the rumen of four beef cattle (Bos taurus) and removed after 0, 2, 4, 6, 12, 16, and 24 h to determine the rumen protection rate. In in vitro incubation, both RP-Leu and RU-Leu were supplemented 1.5 mmol/bottle (L-leucine HCl), and incubated after 0, 2, 4, 6, 8, 12, and 16 h to measure gas production (GP), nutrient degradability, fermentation parameters, bacterial composition, and amino acids metabolism. Results from both in vitro and in situ experiments confirmed that the rumen protection rate was greater (p < 0.01) in RP-Leu than in RU-Leu, whereas the latter was slow (p < 0.05) degraded within incubation 8 h. Free leucine from RP-Leu and RU-Leu reached a peak at incubation 6 h (p < 0.01). RU-Leu supplementation increased (p < 0.05) gas production, microbial crude protein, branched-chain AAs, propionate and branched-chain VFAs concentrations, and Shannon and Sobs index in comparison to the control and RP-Leu supplementation. RU-Leu and RP-Leu supplementation decreased (p < 0.05) the relative abundance of Bacteroidota, which Firmicutes increased (p < 0.05). Correlation analysis indicated that there are 5 bacteria at the genus level that may be positively correlated with MCP and propionate (p < 0.05). Based on the result, we found that RP-Leu was more stable than RU-Leu in rumen fluid, but RU-Leu also does not exhibit rapid degradation by ruminal microbes for a short time. The RU-Leu was more beneficial in terms of regulating rumen fermentation pattern, microbial crude protein synthesis, and branched-chain VFAs production than RP-Leu in vitro rumen conditions.
作者机构:
[Ye, Mengke; Wang, Ji; Liu, Xiangyan; Li, Xiaowen; Wang, Xianglin; Liu, Sha; Qu, Jianyu] Hunan Agr Univ, Coll Vet Med, Lab Anim Clin Toxicol, Changsha, Peoples R China.;[Wu, Jing; Li, Rongfang; Yi, Jine; Wen, Lixin; Yuan, Zhihang] Hunan Agr Univ, Coll Vet Med, Hunan Engn Res Ctr Livestock & Poultry Hlth Care, Changsha, Peoples R China.
通讯机构:
[Rongfang Li] H;Hunan Engineering Research Center of Livestock and Poultry Health Care, College of Veterinary Medicine, Hunan Agricultural University, Changsha City, People’s Republic of China
摘要:
The aim of this study was to evaluate the effects of anti-stress agents on the growth performance and immune function of broilers under immune stress conditions induced by vaccination. A total of 128, 1-day-old Arbor Acres broilers were randomly divided into four groups. Group normal control (NC) was the control group. Group vaccination control (VC), T 0.5%, and T 1% were the treatment groups, which were nasally vaccinated with two doses of the Newcastle disease virus (NDV) vaccine. The chicks in groups T 0.5% and T 1% were fed conventional diets containing 0.5% and 1% anti-stress agents. Thereafter, these broilers were slaughtered on 1, 7, 14, and 21 days post-vaccination. The results indicated that anti-stress agents could significantly reduce serum adrenocorticotropic hormone (ACTH) (P < 0.01) and cortisol (CORT) (P < 0.05) levels, and improve the growth performance (P < 0.05) and immune function of broilers (P < 0.05); However, the levels of malondialdehyde (MDA) (P < 0.05) were decreased, and the decreased total antioxidant capacity (T-AOC) (P < 0.01) levels mediated by vaccination were markedly improved. In addition, anti-stress agents could attenuate apoptosis in spleen lymphocytes (P < 0.01) by upregulating the ratio of Bcl-2 to BAX (P < 0.01) and downregulating the expression of caspase-3 and -9 (P < 0.01), which might be attributed to the inhibition of the enzymatic activities of caspase-3 and -9 (P < 0.05). In conclusion, anti-stress agents may improve growth performance and immune function in broilers under immune-stress conditions.RESEARCH HIGHLIGHTS Investigation of effects and mechanism of immune stress induced by vaccination.Beneficial effect of anti-stress agents on growth performance, immune function, oxidative stress, and regulation of lymphocyte apoptosis.Demonstration of the effects of apoptosis on immune function in the organism.
摘要:
Ketosis is often accompanied by a reduction in milk production in dairy cows, but the molecular mechanism has not been fully elucidated. Ketotic cows possess systemic oxidative stress (OS), which may implicate apoptosis in mammary glands. Sirtuin 3 (SIRT3) is a vital regulator of cellular redox homeostasis and is under the control of AMP-activated protein kinase (AMPK) signaling in nonruminants. Thus, we aimed to investigate (1) the AMPK-SIRT3 and apoptosis status of mammary glands from ketotic cows, (2) the effect of SIRT3 on OS-induced apoptosis in bovine mammary epithelial cells (BMEC), and (3) the role of AMPK signaling on SIRT3-mediated effects on apoptosis. Mammary gland samples were reused from a previous study, which contained healthy and ketotic cows (both n = 15). BMEC were incubated with 0, 0.3, 0.6, or 0.9 mM H(2)O(2) for 6 h with/without a 30 min incubation of an antioxidant MitoQ (1 μM). Then BMEC were incubated with SIRT3 overexpression adenovirus (Ad-SIRT3) for 6 h followed by a 6 h incubation with 0.6 mM H(2)O(2). Finally, BMEC were treated with the AMPK inhibitor Compound C (Cd C,10 μM) for 30 min before the H(2)O(2) challenge, or cells were initially treated with the AMPK agonist MK8722 (10 μM) for 30 min followed by a 30-h culture with/without si-SIRT3 and eventually the H(2)O(2) exposure. Ketotic cows displayed higher levels of Bax, Caspase-3 and Bax/Bcl-2 but lower levels of Bcl-2 in mammary glands. H(2)O(2) incubation displayed similar results, exhibiting a dose-dependent manner between the H(2)O(2) concentration and the apoptosis degree. Mito Q pretreatment reduced cellular reactive oxygen species and rescued cells from apoptosis. Ketotic cows had a lower mammary protein abundance of SIRT3. Similarly, H(2)O(2) incubation downregulated both mRNA and protein levels of SIRT3 in a dose- and time-dependent manner. Ad-SIRT3 infection lowered levels of cellular reactive oxygen species, Bax, Caspase-3 and Bax/Bcl-2 but increased levels of Bcl-2. TUNEL assays confirmed that Ad-SIRT3 infection mitigated H(2)O(2)-induced apoptosis. Both ketotic cows and H(2)O(2)-induced BMEC had lower levels of p-AMPK and p-AMPK/AMPK. Additionally, Cd C pretreatment decreased SIRT3 and Bcl-2 expression but increased levels of Bax and Caspase-3. Contrary to the inhibitor, MK8722 had opposite effects and reduced the percentage of apoptotic cells. However, these effects of MK8722 were reversed upon SIRT3 silencing. In conclusion, in vivo data confirmed that ketosis is associated with greater apoptosis and restricted AMPK-SIRT3 signaling in mammary glands; in vitro data indicated that SIRT3 mitigates OS-induced apoptosis via AMPK signaling. As such, there may be potential benefits for targeting the AMPK-SIRT3 axis to help counteract the negative effects of mammary glands during ketosis.
摘要:
In goats, most follicles in the ovaries will be atresia and only a few dominant follicles (DFs) may eventually mature and ovulate at a follicular wave. To investigate the potential microRNAs (miRNAs) that regulate the expression of genes associated with follicular dominance or atresia, small RNA sequencing was performed on granulosa cells of DF and subordinate follicle at the first follicular wave in goats. A total of 108 differentially expressed miRNAs were detected in the two types of follicle granulosa cells: 16 upregulated miRNAs and 92 downregulated miRNAs. Kyoto Encyclopedia of Genes and Genomes analysis of the target genes showed that TKTL1, LOC102187810, LOC102184409 and ALDOA are closely associated with follicle dominance and are involved in the pentose phosphate pathway. Furthermore, a coexpression network of miRNAs and follicular dominance-related genes was constructed. The qPCR results well correlated with the small RNA sequencing data. Our findings provide new insight for exploring the molecular mechanism of miRNAs in regulating follicular development in goats.
摘要:
Branched-long-chain monomethyl fatty acids (BLCFA) are consumed daily in significant amounts by humans in all stages of life. BLCFA are absorbed and metabolized in human intestinal epithelial cells and are not only oxidized for energy. Thus far, BLCFA have been revealed to possess versatile beneficial bioactivities, including cytotoxicity to cancer cells, anti-inflammation, lipid-lowering, reducing the risk of metabolic disorders, maintaining normal β cell function and insulin sensitivity, regulation of development, and mitigating cerebral ischemia/reperfusion injury. However, compared to other well-studied dietary fatty acids like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), BLCFA has received disproportionate attention despite their potential importance. Here we outlined the major food sources, estimated intake, absorption, and metabolism in human cells, and bioactive properties of BLCFA with a focus on the bioactive mechanisms to advocate for an increased commitment to BLCFA investigations. Humans were estimated to absorb 6-5000 mg of dietary BLCFA daily from fetus to adult. Notably, iso-15:0 inhibited the growth of prostate cancer, liver cancer and T-cell non-Hodgkin lymphomas in rodent models at the effective doses of 35-105 mg/kg/day, 70 mg/kg/day, and 70 mg/kg/day, respectively. Feeding formula prepared with 20% w/w BLCFA mixture to neonatal rats with enterocolitis mitigated the intestine inflammation. Iso-15:0 at doses of 10, 40, and 80 mg/kg relieved brain ischemia/reperfusion injury in rats. In the future, it is crucial to conduct research to establish the epidemiology of BLCFA intake and their impacts on health outcomes in humans as well as to fully uncover the underlying mechanisms for their bioactivities.
摘要:
ETHNOPHARMACOLOGICAL RELEVANCE: Koumine, an indole alkaloid extracted from Gelsemium elegans Benth, exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H(2)O(2) and its potential molecular mechanisms need larger studies. AIM OF THE STUDY: We established an IPEC-J2 cell damage model induced by H(2)O(2) to explore the protective mechanism of koumine on intestinal injury. MATERIALS AND METHODS: In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H(2)O(2)-induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, transmission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H(2)O(2)-trigged IPEC-J2 cell damage. RESULTS: The results of CCK-8 and LDH implied that koumine has a mitigative effect on H(2)O(2)-induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1β, IL-6, IL-8, TNF-α and TGF-β), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H(2)O(2). Moreover, koumine dramatically attenuated H(2)O(2)-triggered IPEC-J2 cell apoptosis and autophagy. Subsequently, Western blot analysis identified NF-ΚB, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H(2)O(2)-stimulated IPEC-J2 cell inflammation. CONCLUSIONS: This in vitro experimental study suggests that koumine suppresses the H(2)O(2)-induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy, which provide a rationale for therapeutically use in major intestinal diseases.
期刊:
Lipids in Health and Disease,2023年22(1):1-14 ISSN:1476-511X
通讯作者:
Xiujun Fan<&wdkj&>Qing Yang
作者机构:
[Tan, Lunbo; Yang, Qing; Chen, Zhilong] Hunan Agr Univ, Coll Vet Med, Changsha 410128, Peoples R China.;[Sun, Fen; Tan, Lunbo; Zhang, Jian V.; Fan, Xiujun; Chen, Zhilong] Chinese Acad Sci, Inst Biomed & Biotechnol, Shenzhen Inst Adv Technol, Ctr Energy Metab & Reprod, Shenzhen 518055, Peoples R China.;[Danser, A. H. Jan; Tan, Lunbo; Verdonk, Koen; Mulder, Monique] Erasmus MC, Dept Internal Med, Div Vasc Med & Pharmacol, Rotterdam, Netherlands.;[Ouyang, Zijun] Shenzhen Polytech, Inst Marine Biomed, Sch Food & Drug, Shenzhen 518055, Peoples R China.;[Guo, Haichun] Changsha Hosp Maternal & Child Hlth Care, Changsha 410007, Peoples R China.
通讯机构:
[Xiujun Fan; Qing Yang] C;Center for Energy Metabolism and Reproduction, Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China<&wdkj&>College of Veterinary Medicine, Hunan Agricultural University, Changsha, China
摘要:
The adipokine chemerin regulates adipogenesis and the metabolic function of both adipocytes and liver. Chemerin is elevated in preeclamptic women, and overexpression of chemerin in placental trophoblasts induces preeclampsia-like symptoms in mice. Preeclampsia is known to be accompanied by dyslipidemia, albeit via unknown mechanisms. Here, we hypothesized that chemerin might be a contributor to dyslipidemia. Serum lipid fractions as well as lipid-related genes and proteins were determined in pregnant mice with chemerin overexpression in placental trophoblasts and chemerin-overexpressing human trophoblasts. In addition, a phospholipidomics analysis was performed in chemerin-overexpressing trophoblasts. Overexpression of chemerin in trophoblasts increased the circulating and placental levels of cholesterol rather than triglycerides. It also increased the serum levels of lysophosphatidic acid, high-density lipoprotein cholesterol (HDL-C), and and low-density lipoprotein cholesterol (LDL-C), and induced placental lipid accumulation. Mechanistically, chemerin upregulated the levels of peroxisome proliferator-activated receptor g, fatty acid-binding protein 4, adiponectin, sterol regulatory element-binding protein 1 and 2, and the ratio of phosphorylated extracellular signal-regulated protein kinase (ERK)1/2 / total ERK1/2 in the placenta of mice and human trophoblasts. Furthermore, chemerin overexpression in human trophoblasts increased the production of lysophospholipids and phospholipids, particularly lysophosphatidylethanolamine. Overexpression of placental chemerin production disrupts trophoblast lipid metabolism, thereby potentially contributing to dyslipidemia in preeclampsia.
作者机构:
[Xie, Shi-Chen; Mei, Jin-Jin; Xie, SC; Zheng, Wen-Bin; Liu, Ya-Ya; Liu, Qing; Zhu, Xing-Quan; Liang, Yao; Gao, Wen-Wei] Shanxi Agr Univ, Coll Vet Med, Lab Parasit Dis, Jinzhong 030801, Peoples R China.;[Xie, Shi-Chen; Xie, SC; Zhu, Xing-Quan] Hunan Agr Univ, Coll Vet Med, Res Ctr Parasites & Vectors, Changsha 410128, Peoples R China.;[Zhu, Xing-Quan] Yunnan Agr Univ, Coll Vet Med, Key Lab Vet Publ Hlth Higher Educ Yunnan Prov, Kunming 650201, Peoples R China.
通讯机构:
[Xie, SC ; Zhu, XQ] S;[Zhu, XQ ] Y;Shanxi Agr Univ, Coll Vet Med, Lab Parasit Dis, Jinzhong 030801, Peoples R China.;Hunan Agr Univ, Coll Vet Med, Res Ctr Parasites & Vectors, Changsha 410128, Peoples R China.;Yunnan Agr Univ, Coll Vet Med, Key Lab Vet Publ Hlth Higher Educ Yunnan Prov, Kunming 650201, Peoples R China.
关键词:
Cryptosporidium spp.;Blastocystis sp.;genotypes;subtypes;prevalence;cattle;Shanxi Province
摘要:
Both Cryptosporidium spp. and Blastocystis sp. are common intestinal protozoa, which can cause zoonotic diseases and economic losses to livestock industry. To evaluate the prevalence and genetic population structure of Cryptosporidium spp. and Blastocystis sp. in beef and dairy cattle in Shanxi Province, north China, a total of 795 fecal samples were collected from beef and dairy cattle in three representative counties in Shanxi Province, and these fecal samples were examined using molecular approaches based on 18S small-subunit ribosomal RNA (SSU rRNA) of Cryptosporidium spp. and Blastocystis sp., respectively. Among 795 cattle fecal samples, 23 were detected as Cryptosporidium-positive and 103 were detected as Blastocystis-positive, and the overall prevalence of Cryptosporidium spp. and Blastocystis sp. in cattle in Shanxi Province was 2.9% and 13.0%, respectively. For Cryptosporidium spp., DNA sequence analysis indicated that all 23 positive samples were identified as C. andersoni. Furthermore, five known subtypes (ST1, ST10, ST14, ST21 and ST26) and three unknown subtypes of Blastocystis sp. were detected among 103 positive samples using DNA sequence analysis. This study reported the occurrence and prevalence of Cryptosporidium spp. and Blastocystis sp. in cattle in Shanxi Province for the first time, which extends the geographical distribution of these two zoonotic parasites and provides baseline data for the prevention and control of these two important zoonotic parasites in cattle in Shanxi Province.
摘要:
Tick saliva is a reservoir of bioactive proteins. Saliva protein compositions change dynamically during blood-feeding. Decipherment of protein profiles in different blood-feeding stages may bring deeper insight into tick feeding physiology and provide targets for immunologic control alternatives. However, having the infancy of tick genome sequencing, assembly, annotation, and limited knowledge of tick salivary proteins restrain the data interpretation. Here, we aimed to depict the saliva protein profile in partially- (PE) and fully-engorged (FE) Haemaphysalis flava ticks, with a special focus on the analysis of those uncharacterized proteins. Saliva was collected from PE and FE adult female H. flava ticks. Saliva proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS). MS data were searched against an in-house salivary gland transcriptome library for identification of tick-derived proteins. Abundances of proteins were compared between PE and FE ticks. The uncharacterized proteins detected in saliva were further bioinformatically analyzed. In total, 614 proteins were identified including 94 host proteins and 520 tick-derived proteins. The 226 tick-derived high-confidence proteins were classified into 10 categories: transporters, enzymes, protease inhibitors, immunity-related proteins, lipocalins, glycine-rich proteins, muscle proteins, secreted proteins, uncharacterized proteins and others. A total of 98 proteins were shared in both PE and FE with 74 only in PE and 54 only in FE. Abundances of 24 shared proteins were significantly higher in PE. The profile of top 15 most abundant proteins was also different between PE and FE ticks. The 65 uncharacterized proteins detected in tick saliva were branched into subclusters 1A, 1B, 2, 3A, 3B and 3C based on particular motifs like RGD, LRR, indicating their diverse predicted functions like anti-coagulation, regulation of innate immune, or other functions. This study provides and compares saliva proteomes of H. flava ticks in two feeding stages with special cluster analysis on the uncharacterized proteins. Further investigations are needed to confirm the roles of these uncharacterized proteins in ticks.
摘要:
The T-2 toxin (T2) poses a major threat to the health and productivity of animals. The present study aimed to investigate the regulatory mechanism of Nrf2 derived from broilers against T2-induced oxidative damage. DF-1 cells, including those with normal characteristics, as well as those overexpressing or with a knockout of specific components, were exposed to a 24 h treatment of 50 nM T2. The primary objective was to evaluate the indicators associated with oxidative stress and the expression of downstream antioxidant factors regulated by the Nrf2-ARE signaling pathway, at both the mRNA and protein levels. The findings of this study demonstrated a noteworthy relationship between the up-regulation of the Nrf2 protein and a considerable reduction in the oxidative stress levels within DF-1 cells (p < 0.05). Furthermore, this up-regulation was associated with a notable increase in the mRNA and protein levels of antioxidant factors downstream of the Nrf2-ARE signaling pathway (p < 0.05). Conversely, the down-regulation of the Nrf2 protein was linked to a marked elevation in oxidative stress levels in DF-1 cells (p < 0.05). Additionally, this down-regulation resulted in a significant decrease in both the mRNA and protein expression of antioxidant factors (p < 0.05). This experiment lays a theoretical foundation for investigating the detrimental impacts of T2 on broiler chickens. It also establishes a research framework for employing the Nrf2 protein in broiler chicken production and breeding. Moreover, it introduces novel insights for the prospective management of oxidative stress-related ailments in the livestock and poultry industry.
摘要:
血管生成素 4(ANG4)是一种新型抗菌肽蛋白, 具有抗菌、抗炎、血管生成、调节机体免疫反应等多种生物学功能, 在机体先天免疫过程中发挥重要的作用。 ANG4在新生小鼠肠道的表达受到多种因素的调节, 如细菌信号和发育过程等。本试验选取细菌细胞壁表面分子的类似物壳寡糖( COS)作为诱导物, 探究其对小鼠肠道 ANG4表达和对肠道形态的影响。实时荧光定量聚合酶链式反应( qRT-PCR)和蛋白质免疫印迹( Western blot)分析表明, COS能增加小鼠肠道 ANG4 mRNA及 ANG4蛋白的表达;形态学观察结果显示, COS使小肠绒毛高度明显增加, 平均隐窝深度均有降低, V/C值增加, 表明 COS可通过诱导 ANG4等抗菌蛋白的表达, 或通过其杀菌和免疫调节作用而不同程度地提高小肠黏膜的完整性。研究结果为 COS以及 ANG4在仔猪肠道疾病防治的应用提供了理 Angiogenin 4 (ANG4), a novel antimicrobial protein with multiple biological functions, such as antimicrobial, anti-inflammation, angiogenesis and regulating the body’s immune response, plays a key role in innate immunity. The expres-sion of ANG4 in neonatal mice intestine is regulated by some factors including microbial signal and developmental stage. Being an analogue of surface molecules of bacterial cell wall, chitosan oligosaccharide (COS) was selected as a inducer to explore the effects on ANG4 expression and mophology in post weaning mice intestine. qRT-PCR and Western blot analyses showed that the mRNA and protein levels of ANG4 increased suggesting that COS can upregulate the expression of ANG4. The results of HE staining of mouse intestinal tract showed that the height of intestinal villi, the depth of crypt and the value of V/C increased significantly. It indicates that COS can improve the integrity of intestinal mucosae in some extent through either inducing the expression of some intestinal antimicrobial proteins, such as ANG4, or its antimicrobial and immunomodulation activities, and provide the theoretical basis for the applications of COS and ANG4 in controlling the intestinal diseases in piglets. Key words: ANG4; COS; AMPs; qRT-PCR; Western blot