In this study, RAW264.7 cells were employed to investigate the effects of honey-processed Astragalus on their energy metabolism and polarization, and explore the scientific connotation of the enhanced efficacy of honey-processed Astragalus on invigorating spleen-stomach and replenishing Qi. The medicated sera were prepared by intragastric administration of rats with water extracts of crude and honey-processed Astragalus, and the composition changes of medicated sera of crude and honey-processed Astragalus were analyzed by using LC-MS technology. The cell survival rates were detected and the concentrations of medicated sera were screened through CCK-8 assay. The differences of cell phagocytic rates, ATP energy metabolism, and NO secretion between crude and honey-processed Astragalus were evaluated by using neutral red phagocytosis assay, ATP detection kit, and NO detection kit. The effects of crude and honey-processed Astragalus on TNF-α secretion of RAW264.7 cells were detected by employing ELISA kit. The effects of crude and honey-processed Astragalus on polarization of RAW264.7 cells were evaluated by utilizing flow cytometry. The differential metabolites related to glycolysis in cell lysates and culture media were screened by using LC-MS technology. The experiment was approved by the experimental animal ethics committee from Shanxi University of Chinese Medicine (No. AWE202407352). The results showed that the contents of betaine, amino acids, and ononin in the prepared medicated sera of rats treated by intragastric administration with water extracts of honey-processed Astragalus increased compared to those in crude one. The results of CCK-8 experiment showed that there were no cytotoxic effects on RAW264.7 cells in the medicated sera of crude and honey-processd Astragalus at different concentration. The phagocytic index and ATP yield both increased to varying degrees after administration of the medicated sera to cells. The secretion of NO in normal and inflammatory cells increased and decreased respectively, and the effect of honey-processed Astragalus was better than that of crude one. The results of ELISA kit showed that the medicated sera of both crude and honey-processed Astragalus could promote the secretion of TNF-α in a concentration-dependent manner, and the promoting effect of honey-processed Astragalus was stronger than that of crude one. The results of flow cytometry showed that the medicated sera of both crude and honey-processed Astragalus could promote M1-type polarization and inhibit M2-type polarization of RAW264.7 cells, and the effect of honey-processed Astragalus was better than that of crude one. Compared to crude Astragalus, the metabolites related to glycolysis in the cell lysates and culture media of the medicated sera of honey-processed Astragalus were generally on the rise, indicating that the effect on promoting glycolysis of honey-processed Astragalus was better than that of crude one. In summary, honey-processed Astragalus can promote the polarization and energy metabolism of RAW264.7 cells, and participate in positive immune regulation, which is correlated with its enhanced effect of invigorating spleen-stomach and replenishing Qi.

OBJECTIVE To investigate the effects of Setaria italica extract on improving insomnia model mice and to explore its potential mechanisms. METHODS The mice were randomly assigned into blank group， model group， positive control group （diazepam， 2.6 mg/kg）， and S. italica extract low-dose， medium-dose and high-dose groups （1.2， 2.4， 4.8 g/kg）， with 10 mice in each group. Except for the blank group， all other groups received intraperitoneal injection of para-chlorophenylalanine （PCPA） to establish the insomnia model. After modeling， the blank group and model group were given a constant volume of normal saline intragastrically， and administration groups were given relevant medicine intragastrically， with a volume of 0.01 mL/g， once a day， for 7 consecutive days. After the administration， the open-field test was conducted to observe the praxiological changes of mice， and to determine the levels of 5-hydroxytryptamine （5-HT） and 5-hydroxyindoleacetic acid （5-HTAA） in the hippocampal tissue， as well as the contents of 5-HT， brain-derived neurotrophic factor （BDNF）， interleukin-2 （IL-2）， IL-6， B-cell lymphoma-2 （Bcl- 2）， and Bcl-2-associated X protein （Bax） in the serum. The expression of phosphoinositide 3-kinase/protein kinase B/nuclear factor- κB （PI3K/Akt/NF-κB） signaling pathway related protein was determined in the hippocampus of mice. RESULTS Compared with the model group， the total exercise time of mice in S. italica extract high-dose group was significantly prolonged， but the total rest time was significantly shortened （P＜0.01）； the number of standing times and modification times were significantly reduced （P＜ 0.01）. The contents of 5-HT， BDNF， and Bcl-2 in serum， and Bcl-2/Bax were significantly increased， while the contents of IL-2， IL-6， and Bax were significantly reduced （P＜0.05 or P＜ 0.01）. The content of 5-HTAA in the hippocampal tissue and 202104010910029）；the phosphorylation levels of PI3K and Akt proteins were increased significantly， while the phosphorylation level of NF-κB p65 protein was decreased significantly （P＜0.05）.CONCLUSIONS High-dose of S. italica extract demonstrates significant therapeutic effects on insomnia in mice， and the mechanism of which may be associated with the regulation of PI3K/Akt/NF-κB signaling pathway.

To compare the pancreatic proteomics and autophagy between Rehmanniae Radix-and Rehmanniae Radix Praeparata-treated mice with type 2 diabetes mellitus(T2DM). The T2DM mouse model was established by high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days). The mice were then randomly assigned into a control group, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) catalpol groups, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) 5-hydroxymethyl furfuraldehyde(5-HMF) groups, and a metformin(250 mg·kg~(-1)) group. In addition, a normal group was also set and each group included 8 mice. The pancreas was collected after four weeks of administration and proteomics tools were employed to study the effects of Rehmanniae Radix and Rehmanniae Radix Praeparata on protein expression in the pancreas of T2DM mice. The expression levels of proteins involved in autophagy, inflammation, and oxidative stress response in the pancreatic tissues of T2DM mice were determined by western blotting, immunohistochemical assay, and transmission electron microscopy. The results showed that the differential proteins between the model group and Rehmanniae Radix/Rehmanniae Radix Prae-parata group were enriched in 7 KEGG pathways, such as autophagy-animal, which indicated that the 7 pathways may be associated with T2DM. Compared with the control group, drug administration significantly up-regulated the expression levels of beclin1 and phosphorylated mammalian target of rapamycin(p-mTOR)/mTOR and down-regulated those of the inflammation indicators, Toll-like receptor-4(TLR4) and Nod-like receptor protein 3(NLRP3), in the pancreas of T2DM mice, and Rehmanniae Radix showed better performance. In addition, the expression levels of inducible nitric oxide synthase(iNOS), nuclear factor erythroid 2-related factor 2(Nrf2), and heine oxygenase-1(HO-1) in the pancreas of T2DM mice were down-regulated after drug administration, and Rehmanniae Radix Praeparata demonstrated better performance. The results indicate that both Rehmanniae Radix and Rehmanniae Radix Praeparata can alleviate the inflammatory symptoms, reduce oxidative stress response, and increase the autophagy level in the pancreas of T2DM mice, while they exert the effect on different autophagy pathways.
Mice ; Animals ; Diabetes Mellitus, Type 2/genetics* ; Streptozocin/pharmacology* ; Diet, High-Fat/adverse effects* ; Proteomics ; Inflammation ; TOR Serine-Threonine Kinases ; Autophagy ; Mammals

OBJECTIVES: To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. METHODS: Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. RESULTS: The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. CONCLUSIONS The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.
Animals ; Humans ; Rats ; Multivariate Analysis ; Postmortem Changes ; Protein Array Analysis ; Technology

Background: It is well-established that serum testosterone in men decreases with age, yet the underlying mechanism of this change remains elusive. Methods: The expression patterns of Fancd2 opposite-strand (Fancd2os) in BALB/c male mice and testicular tissue derived cell lines (GC-1, GC-2, TM3, and TM4) were assessed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence. The Fancd2os-overexpressing or knockdown TM3 cells were constructed by infecting them with lentivirus particles and were used to evaluated the function of Fancd2os. The testosterone production was measured using enzyme linked immunosorbent assay (ELISA) and the steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were analysed using RT-PCR. The apoptosis of TM3 cells induced by ultraviolet light or testicular tissues was detected using flow cytometry, Western blot or dUTP-biotin nick end labeling (TUNEL) assays. Pearson correlation analysis was used to assess the correlation between the Fancd2os expression and TUNEL-positive staining in mouse testicular Leydig cells. Results: The Fancd2os protein was predominantly expressed in mouse testicular Leydig cells and its expression increased with age. Fancd2os overexpression inhibited testosterone levels in TM3 Leydig cells, whereas knockdown of Fancd2os elevated testosterone production. Fancd2os overexpression downregulated the levels of StAR, P450scc and 3β-HSD, while Fancd2os knockdown reversed this effect. Fancd2os overexpression promoted ultraviolet light-induced apoptosis of TM3 cells. In contrast, Fancd2os knockdown restrained apoptosis in TM3 cells. In vivo assays revealed that higher Fancd2os levels and mouse age were associated with increased apoptosis in Leydig cells and decreased serum testosterone levels. Pearson correlation analysis exhibited a strong positive correlation between the expression of Fancd2os and TUNEL-positive staining in mouse testicular Leydig cells. Conclusion Our findings suggest that Fancd2os regulates testosterone synthesis via both steroidogenic enzymes and the apoptotic pathway.

ObjectiveTo investigate the effect and mechanism of Guilu Erxiangao on Alzheimer's disease （AD） model rats induced by hydrocortisone and amyloid β-protein（Aβ） based on the theory of kidney-brain correlation. MethodIntraperitoneal injection of hydrocortisone and intracerebroventricular injection of Aβ were performed to induce AD in rats， and different concentrations of Guilu Erxiangao were used for intervention. The indexes of hippocampus， kidney and adrenal gland were measured， and the spatial learning and memory ability of AD rats was observed by Morris water maze experiment. The levels of testosterone （T） and corticosterone （CORT） in serum samples were determined by enzyme-linked immunosorbent assay （ELISA）. Liquid chromatography-mass spectrometry （LC-MS） was used to collect and analyze the serum metabolic data of model rats. The active components and corresponding targets of Guilu Erxiangao were collected using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）， a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine （BATMAN-TCM） and Traditional Chinese Medicine Integrated Database（TCMID）. GeneCards and Online Mendelian Inheritance in Man （OMIM） were retrieved to obtain AD-related targets， and protein-protein interaction （PPI） network was constructed to perform gene ontology （GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The expression level of interleukin-6 （IL-6） in the hippocampus of rats was detected by Western blot. ResultCompared with the model group， the low-， medium- and high-dose groups of Guilu Erxiangao exhibited significantly increased hippocampus index， kidney index and adrenal gland index， reduced CORT levels in serum and down-regulated IL-6 levels in hippocampal tissues. According to the results of water maze experiment， as compared with the model group， the platform crossing times of rats was significantly increased in the low- and high-dose groups of Guilu Erxiangao， with evidently prolonged distance traveled in quadrant Ⅲ （%） and time in quadrant Ⅲ （%）. A total of 24 serum differential metabolites associated with AD were identified by LC-MS， and 50 high-frequency common compounds and 187 high-frequency common targets for AD treatment were screened by network pharmacology method. Results demonstrated phosphatidylinositol 3-kinases（PI3K）/protein kinase B（Akt） signaling pathway plays an important role in the complex AD pathological mechanism. ConclusionGuilu Erxiangao can significantly improve the cognitive dysfunction of AD model rats induced by hydrocortisone and Aβ， reduce serum CORT levels and IL-6 levels in hippocampal tissues， and regulate the metabolic level， which provides a reference for its clinical application.

OBJECTIVES: To explore the mRNA differential expressions and the sequential change pattern in acute myocardial infarction (AMI) mice. METHODS: The AMI mice relevant dataset GSE4648 was downloaded from Gene Expression Omnibus (GEO). In the dataset, 6 left ventricular myocardial tissue samples were selected at 0.25, 1, 4, 12, 24 and 48 h after operation in AMI group and sham control group, and 6 left ventricular myocardial tissue samples were selected in blank control group, a total of 78 samples were analyzed. Differentially expressed genes (DEGs) were analyzed by R/Bioconductor package limma, functional pathway enrichment analysis was performed by clusterProfiler, protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software, the key genes were identified by Degree topological algorithm, cluster sequential changes on DEGs were analyzed by Mfuzz. RESULTS: A total of 1 320 DEGs were associated with the development of AMI. Functional enrichment results included cellular catabolic process, regulation of inflammatory response, development of muscle system and vasculature system, cell adhesion and signaling pathways mainly enriched in mitogen-activated protein kinase (MAPK) signaling pathway. The key genes of AMI included MYL7, TSC22D2, HSPA1A, BTG2, NR4A1, RYR2 were up-regulated or down-regulated at 0.25-48 h after the occurrence of AMI. CONCLUSIONS The functional signaling pathway of DEGs and the sequential expression of key genes in AMI may provide a reference for the forensic identification of AMI.
Animals ; Computational Biology/methods* ; Gene Expression Profiling/methods* ; Mice ; Mitogen-Activated Protein Kinases/metabolism* ; Myocardial Infarction/metabolism* ; RNA, Messenger ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Transcriptome

OBJECTIVES: To explore the differential expression of messenger RNA (mRNA) in myocardial tissues of rats with sudden coronary death (SCD), and to provide ideas for the forensic identification of SCD. METHODS: The rat SCD model was established, and the transcriptome sequencing was performed by next-generation sequencing technology. Differentially expressed genes (DEGs) in myocardial tissues of SCD rats were screened by using the R package limma. A protein-protein interaction (PPI) network was constructed by using the STRING database and Cytoscape 3.8.2 on DEG, and hub genes were screened based on cytoHubba plug-in. Finally, the R package clusterProfiler was used to analyze the biological function and signal pathway enrichment of the selected DEG. RESULTS: A total of 177 DEGs were associated with SCD and were mainly involved in the renin-angiotensin system and PI3K-Akt signaling pathway. The genes including angiotensinogen (AGT), complement component 4a (C4a), Fos proto-oncogene (FOS) and others played key roles in the development of SCD. CONCLUSIONS Genes such as AGT, C4a, FOS and other genes are expected to be potential biomarkers for forensic identification of SCD. The study based on mRNA expression profile can provide a reference for forensic identification of SCD.
Rats ; Animals ; RNA, Messenger/genetics* ; Gene Regulatory Networks ; Gene Expression Profiling ; Phosphatidylinositol 3-Kinases/genetics* ; Biomarkers

Medicinal plant germplasm resources are the foundation of the modern development of traditional Chinese medicine. In-depth study of medicinal plant germplasm resources is a prerequisite for cultivating fine varieties and ensuring the output and standard quality of traditional Chinese medicine（TCM）. Traditional identification methods start with appearance and are greatly affected by natural environment and human factors，with a low efficiency and accuracy of identification are generally low molecularin general. Due to such advantages as easy operation，high sensitivity，accurate results， molecular biology technology has been widely used in the related research of relevant studies for medicinal plant germplasm resources due to its advantages of easy operation，high sensitivity，accurate results，etc. It mainly involving the distinction between wild and cultivated products，researchstudy on substitutes of TCM，identification of Chinese patent medicine，good variety marker breeding，genetic diversity researchstudy，genetic map establishment and omics research，etcstudy. Among them，omics researchstudy is divided into genomics，transcriptomics，metabolomics，and proteomics due toby different analysis purposes. Genomics is divided into three sub-fields namely structural genomics，functional genomics， and comparative genomics. Eukaryotes Because eukaryotes have nuclei and organelles，so omics researchstudy also includes chloroplast genomics，mitochondrial genomics，nuclear genomics，and plastid genomics. Among them，the chloroplast genome has a simple structure，small molecular weight，and good conservation，while the mitochondrial genome has a strong variability and complex structure，the nuclear genome data isfeatures complex， data and the nucleus contains no ribosomes in nucleus，resulting in spatiotemporal differences in the translation process，even if repeated repeatedly test， the result of and the test is alsoresults remained uncertain， even after repeated tests. The molecular biology technology and omics researchstudy involved in theby current medicinal plant researchstudy still hashave shortcomings，and there iswith a large room for development，which needs and need further improvement and supplementation. This articlepaper successively introduces the characteristics and applications of cytology，molecular markers，and omics researchstudy techniques in the identification of medicinal germplasm resources，providingin order to provide a reference for subsequent identification，development and utilization of medicinal plant germplasm resources.

Objective:To develop the specific molecular markers of Codonopsis plants and better identify their germplasm resources considering the significant difference in active ingredients of Codonopsis Radix from various origins and producing areas. Method:Such bioinformatics software as Primer 5.0， NTSYS-pc 2.10e， and PopGene 32 were used for searching the simple sequence repeat （SSR） markers of C. minima chloroplast genome， C. tsinlingensis chloroplast， and C. lanceolata mitochondrial sequences， and 120 pairs of SSR primers were designed by Primer 5.0. Then 16 pairs of cpSSR primers and 10 pairs of mtSSR primers with good screening effect and high polymorphism were selected for analyzing the interspecific versatility of 20 samples. Result:The results showed that 66 cpSSR primer sites and 26 mtSSR sites were identified from the genome sequences， with 86.20% of single nucleotide， 6.9% of dinucleotide， and 6.9% of trinucleotide for C. minima chloroplast， 83.78% of single nucleotide，13.51% of dinucleotide， and 2.71% of trinucleotide for C. tsinlingensis chloroplast， and 46.15% of single nucleotide and 53.85% of dinucleotide for C. lanceolata mitochondria. As demonstrated by polymerase chain reaction （PCR） identification results， the developed 26 pairs of SSR primers had good applicability in the genus Codonopsis. The analysis by NTSYS-pc 2.10e revealed that the genetic similarity coefficients of 20 samples were within the range of 0.38-1.00， and they were divided into two subgroups at a threshold of 0.69. Four pairs of polymorphic primers were screened out in the diversity analysis of 20 samples using PopGene 32. The number of observed alleles （Na） was 12， and the effective number of alleles （Ne） ranged from 1.362 9 to 2.605 9. The percentage of polymorphic loci （PPL） at each site was 100%， and the average values of genetic parameters Ho， He， and I at each site were 0.555 8， 0.444 2， and 0.753 2， respectively， indicating high polymorphism at each site. The screened four pairs of primers were utilized for DNA fingerprinting of the 20 samples， and it was found that the DNA fingerprints enabled the identification of these 20 samples. Conclusion:This study has provided a molecular basis for the study of the genetic relationship between plants in species Codonopsis and the intraspecific genetic differentiation.