Studies have shown that the extract of Macleaya cordata can promote animal growth, improve growth performance (Luo et al. 2021), improve feed conversion rate, and reduce diarrhea rate (Zong et al. 2020). The growth and development of animals are influenced by many genes at the transcriptome level. In this study, transcriptomic techniques were used to investigate the regulatory mechanisms of the genes involved in growth promotion in rats. The experimental results showed that the liver transcriptome sequencing results of rats in the high and low dose group supplemented with Macleaya cordata extract were significantly different from those in the control group.
A GO enrichment analysis was performed on the differentially expressed genes in the three experimental groups (RA vs RB, RA vs RC, and RB vs RC). The results showed that maximum changes occurred in peptide metabolism, cytoamide metabolism, cellular nitrogen compound biosynthesis, purine nucleotide metabolism, ATP metabolism, purine ribose triphosphate metabolism, and purine nucleoside triphosphate metabolism.
A KEGG pathway analysis was performed for the differentially expressed genes in the three experimental groups (RA vs RB, RA vs RC, and RB vs RC), and the first 20 pathways in each group are shown in Fig. 4. The results showed that the splicing body, ribosome, PPAR signaling pathway, oxidative phosphorylation, myocardial contraction, glycolysis/glycogenesis, DNA replication, cell cycle, and other pathways were significantly enriched. At the same time, the high- and low-dose groups and the control group presented different DEGs, indicating that different doses of MCE had different effects on the liver of rats. qRT-PCR was used to verify the eight differentially screened genes, and the expression trends of these eight genes were consistent with the sequencing results, which demonstrated the reliability of this experiment and the accuracy of the sequencing results.
RNA-seq technology can be used to explore the effects of Macleaya cordata extract on the genes of rat liver cells and identify the genes and signaling pathways related to growth and development (Wang 2020). Therefore, in this study, transcriptome sequencing was performed on rat livers. The results showed that 16109, 16332, and 16158 differentially expressed genes were obtained in the RA vs RB, RA vs RC, and RB vs RC groups, respectively. Several common DEGs were also identified, such as COX6C, HDC, MRPL33, NFATC4, RPS25, RPS6, and RAD51D. Through this analysis, we also found genes related to growth and development among the enriched pathways, such as CycD, SCD-1, and EPOR. In addition, five important pathways were identified, thus providing a basis for further understanding the molecular mechanism of Macleaya cordata extract in rats.
BMP7 (bone morphogenetic protein 7) is a protein-encoding gene that plays an important role in calcium regulation and bone homeostasis in vivo by regulating mesenchymal stem cells due to its ability to induce bone and cartilaginous tissue formation (Sun et al. 2005). BMP7 expression also promotes the differentiation of C2C12 cells through the BMP/TGF-β pathway to promote muscle development (Wang et al. 2019). Therefore, earlier encoded primordium is hydrolyzed to form homodimeric disulfide bonds, and each of these subunits can play an important role in the development of bone, kidney, and fat. This gene also has a very important collateral homologue, BMP5, which plays a regulatory role in various cancers to varying degrees (Korkut et al. 2018). In the validation test of this experiment, the level of BMP7 increased successively from the blank group to the low-dose and high-dose groups, indicating that the effect of Macleaya cordata extract on BMP7 is consistent with its function.
COX6C (cytochrome C oxidase subunit 6 C) is the last enzyme in the mitochondrial electron transport chain that drives oxidative phosphorylation, thus enabling electron transfer from reduced cytochrome C to oxygen molecules, and it plays an indispensable role in cellular oxidative phosphorylation (Tian et al. 2021). COX6C can cooperatively transfer the derived electrons from NADH and succinate to oxygen molecules, thus generating an electrochemical gradient on the intima to drive transmembrane transport and ATP synthase (Hashad et al. 2016). The results of the validation test showed that COX6C expression in the high- and low-dose groups was higher than that in the blank group. To a certain extent, Macleaya cordata extract can promote the expression of COX6C, which is beneficial for the growth of rats. However, the validation test was higher in the low-dose group than in the high-dose group compared with transcriptome sequencing, which requires further discussion and research.
HDC (histidine decarboxylase) can convert histidine to histamine, which acts as a neurotransmitter and plays an important role in the function of a variety of brain activities, and astrocytes can transport it (Yoshikawa et al. 2016). Stimulation by activating the H1 receptor causes the hypothalamus to release more histamine, resulting in reduced food intake and weight loss (Díaz et al. 2019). The above findings show that the liver is the main metabolic site, which can affect the amount of food intake through neural reflection. The expression of HDC increased successively from the blank group to the low-dose group and finally to the high-dose group, indicating that the extract of Macleaya cordata can cause more significant enrichment of this gene, thus further promoting the growth of rats.
MRPL33 (mitochondrial ribosomal protein L33) can participate in the translation process in mitochondria and plays a certain role in the protein synthesis process in mitochondria, which is necessary for the normal operation of organelles (Cheong et al. 2020). Bioinformatics analysis showed that the overexpression of MRPL33-L and MRPL33-S played a key role in transcription, signal transduction, and apoptosis, and these signaling pathways showed an opposite effect in the chemical reaction of gastric cancer to epirubicin, which was conducive to the resolution of drug resistance in gastric cancer patients (Li et al. 2019). On the one hand, a growth promotion effect was observed for the extract of Macleaya cordata, while on the other hand, increases in the dose did not improve the effect. Moreover, an optimal peak value was observed.
NFATC4 (nuclear factor of activated T cell 4) is a regulated transcription factor involved in multiple biological processes, and aberrantly activated NFATC4 participates in modulating the initiation, proliferation, invasion, and metastasis of various cancers (Zhong et al. 2022). It can also be involved with NFATC3 in the development of the embryonic heart and in the energy metabolism of mitochondria required for its function, which can change the shape of the heart. It can bind to and simultaneously activate the BACE1 / β-secretase 1 promoter, thereby participating in the proteolytic process of amyloid precursor protein regulation (Mei et al. 2015). p38 MAP increases the expression of PPARγ by phosphorylating several residues in the NFAT homology domain of NFATC4, which contributes to adipocyte formation and plays a direct role in adipocyte differentiation (Yang et al. 2002). After drug treatment, the expression of the NFATC4 gene was significantly increased in the blank, high-dose, and low-dose groups, and it could be seen from the KEGG pathway diagram that the decrease in the adrenal ketone hormone content in the body of the rats could lower blood pressure and form fat cells to promote healthy growth of the rats.
RAd51D is a member of the RAd51 family. This protein-coding gene translates into a protein complex that promotes homologous pairing between single- and double-stranded DNA, homologous recombinant repair (HRR) pathways that can participate in the DNA replication process, and double-stranded DNA breaks occur during the overrun or are induced by DNA damage agents (Baldock et al. 2019). Because RAD51D is an important part of homologous recombination, if this gene is defective, it will have a huge negative impact on cell mitosis; therefore, this gene plays an indispensable role in various biological processes of DNA (Wyatt et al. 2018). Through transcriptome high-throughput sequencing and quantitative fluorescence PCR experiments, we found that the expression level of RAD51D in the low-dose and high-dose groups was significantly higher than that in the blank group. These results indicated that Macleaya cordata extracts promoted DNA mitosis, homologous pairing, and other biological processes in the cells, which ensured the growth and development of rats.
RPS6 (ribosomal protein S6) is one of the main substrates for the lipid kinase reaction. Owing to its special structure, RPS6 has five C-terminal serine residue subsets that are phosphorylated by different protein kinases and selectively recognize different mRNAs to control cell proliferation (Meyuhas. 2015). It was found that the expression level in the high-dose group was the highest, followed by the low-dose group and the blank group. According to the KEGG analysis, this gene is mainly involved in the HIF-1 signaling pathway, and inhibition of HIF-1 can improve the severity of osteoporosis (Zhong et al. 2017). As the expression level of the gene is downregulated, it can be inferred that the extract of Macleaya cordata can increase the inhibitory effect on the HIF-1 signaling pathway through the influence of the RPS6 gene. Promote bone development and growth in rats.
RPS25 (ribosomal protein S25) encodes a cytoplasmic ribosomal protein that is part of the S25E family and is present in the cytoplasm, similar to other ribosomal genes. Rps25 is directly involved in the translation control of proteins, and after RPS25 knockout, the target ribosome is altered, showing virus and toxin resistance phenotypes, and these phenotypes cannot be saved by functional cDNA expression (Johnson et al. 2020). Transcriptome sequencing and qRT-PCR validation tests showed that the expression level of the blank group was the lowest, followed by the low-dose group and the highest high-dose group, which indicated that the influence of the RPS25 gene could enhance protein translation and thus promote the growth of rats.
In addition to the eight common DEGs mentioned above, we identified eight DEGs related to growth and development in two pathways. Animal growth, whether it is the development of each organ or system, cannot be separated from the basic biological processes of cell division and differentiation. According to the KEGG enrichment pathway, it can be seen that after the intragastric administration of the extract, many pathways conducive to growth were enriched. One such pathway is the JAK-STAT signaling pathway, which is a cytokine-stimulatory signaling pathway (Fig. 6a). The JAK-STAT signaling pathway is involved in the pathogenesis of inflammation and immune diseases.
In this signaling pathway, the CyCD gene is upregulated, which belongs to one of the cyclins, plays the role of a CDK kinase regulator, and contributes to the time coordination during mitosis of each cell. The RBF/E2F pathway enables mitosis to proceed in an orderly and regular manner. It also plays an important role in cell proliferation (Romero-Pozuelo et al. 2020; Frei et al. 2005). EPOR (erythropoietin receptor) is also an upregulated gene that enhances the activation of JAK2 tyrosinase. In mice, erythropoietin can also inhibit the NLRP3 inflammasome through EPOR to control inflammation (Cao et al. 2020). In other words, the upregulation of this gene can also effectively prevent the occurrence of inflammation, promote mitosis, and make cells proliferate and differentiate, thus promoting the growth and development of rats.
The PPAR (peroxisome proliferator-activated receptor) pathway plays a key role in glucose and lipid metabolism, mainly through peroxisome proliferator-activated receptors acting as ligand-induced transcription factors (Mirza et al. 2019). In the PPAR pathway, the receptor is induced by transcription factors and acts on the target gene. The target gene is upregulated or downregulated to varying degrees in rats after intragastric administration of the extracts, and the biological effect also changes to some extent.
In the pathway obtained in this experiment (Fig. 6b), the SCD-1 (stearyl coenzyme A desaturase) gene is upregulated. This gene can add a double bond to the saturated fat acyl-CoA substrate, catalyze the cis-double bond at Δ9 to insert fatty acyl-CoA substrate, and convert saturated fatty acids to monounsaturated fatty acids. It also plays an important role in lipid biosynthesis, regulating the expression of genes related to fat formation, the oxidation of mitochondrial lipoic acid, and the dynamic energy balance of the body and contributes to the biosynthesis of membrane phospholipids, cholesterol esters, and triglycerides. PPAR-γ is a key regulator of adipocyte differentiation and glucose homeostasis. It activates the nuclear receptors of peroxidase proliferators, binds to the specific PPAR response element (PPRE) of DNA, and regulates the transcription of its target genes( such as acyl-coenzyme A oxidase). Five genes, Apo-A II, Apo-C III, ACBP, FABP1, and AP2, were downregulated, and they were the target genes involved in lipid transport, fatty acid transport, and fatty acid oxidation. Therefore, these significantly upregulated and downregulated genes play an important role in promoting rat growth.