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.

ObjectiveTo observe the effect of Yulin Hukun decoction on autophagy mediated by phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway in the mouse model of cyclophosphamide-induced diminished ovarian reserve and explore the follicular development-improving mechanism of this decoction. MethodsSixty female ICR mice with normal estrous cycle were assigned into a blank group （n=10） and a modeling group （n=50）. The model was established by intraperitoneal injection of cyclophosphamide （60 mg·kg^-1） for 5 days. The successfully modeled mice were randomly grouped as follows： model， estradiol （0.26 mg·kg^-1）， and high-， medium-， and low-dose （56.42， 28.21， 14.105 g·kg^-1， respectively） Yulin Hukun decoction， with 10 mice in each group. The blank group and the model group received normal saline （10 mL·kg^-1）. The intervention was performed once a day for 21 days. The general conditions， estrous cycle， body weight， and ovary index were observed and recorded for each group. Serum levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， estradiol （E₂）， and anti-Müllerian hormone （AMH） were measured by enzyme-linked immunosorbent assay. Histopathological changes in the ovarian tissue were observed by hematoxylin-eosin staining. Western blot was employed to determine the protein levels of PI3K， Akt， mTOR， autophagy-related protein 7 （Atg7）， beclin1， microtubule-associated protein 1 light chain 3Ⅱ （LC3Ⅱ）， ubiquitin-binding adaptor protein （p62）， forkhead box protein O1 （FoxO1）， and acetylated forkhead box protein O1 （Ac-FoxO1） in mouse ovaries. Real-time PCR was adopted to determine the mRNA levels of PI3K， Akt， mTOR， Atg7， beclin1， and LC3Ⅱ in the mouse ovarian tissue. ResultsCompared with the blank group， the model group had disturbed estrous cycle， decreased body weight （P<0.05）， loose ovarian structure with increased atretic follicles， increased serum FSH level （P<0.05）， and decreased AMH and estradiol levels （P<0.05）. Compared with the model group， the treatment groups showed recovered estrous cycles and body weight. The estradiol group and high- and medium-dose Yulin Hukun decoction groups showed declined FSH level （P<0.05） and elevated AMH levels （P<0.05）. In addition， the treatment groups showed downregulated protein levels of Atg7， LC3Ⅱ， beclin1， FoxO1， and Ac-FoxO1 （P<0.01）， upregulated protein levels of PI3K， Akt， mTOR， and p62 （P<0.01） in the ovarian tissue， gradual repair of the ovarian structure， with more intact and numerous follicles of various stages. ConclusionYulin Hukun decoction can inhibit autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR signaling pathway and inhibiting the expression of autophagy-related proteins and transcription factors， thereby improving follicular development and ovarian reserve.

Plasma metabolomics based on UHPLC-Q-TOF-MS/MS technique was developed for profiling the mechanism on attenuating hepatic fibrosis of Bupleuri Radix (BR) and Paeoniae Radix Alba (PRA) before and after vinegar-processing and compatibility, and to screen potential pharmacodynamic substances by spectrum-effect correlation method in this study. Firstly, SD rats with CCl₄-induced hepatic fibrosis were used as an in vivo model. The blood and tissue samples were collected for the analyses of pharmacodynamic indexes and plasma metabolomics after six weeks’ administration of BR, vinegar-processed BR (VPBR), PRA, vinegar-processed PRA (VPPRA), BR-PRA herb-pair, and VPBR-VPPRA herb-pair. The experiment was approved by the experimental animal ethics committee from Nanjing University of Chinese Medicine (No.202103A002). The results of pharmacodynamics indicated that the levels of alanine aminotransferase (ALT, P < 0.01), aspartate aminotransferase (AST, P < 0.01), and hydroxyproline (HYP, P < 0.01) were decreased significantly, while the level of glutathione peroxidase (GSH-Px, P < 0.05) was increased obviously after administration of all treatment groups. Next, UHPLC-Q-TOF-MS/MS was performed to characterize the endogenous metabolites. A total of 20 differential endogenous metabolites related to the pathogenesis of hepatic fibrosis were identified in positive and negative ion modes, mainly involving five metabolic pathways of retinol metabolism, glycerol phospholipid metabolism, glyceride metabolism, fatty acid biosynthesis, and arachidonic acid metabolism. Meanwhile, a concept named correction rate was introduced to evaluate the back-regulation effects of all treatment groups on differential metabolites, and 10 differential metabolites were corrected by all treatment groups. The correction effects of the vinegar-processed herb groups were better than those of the crude ones, and the correction effects of the herb-pair groups were better than those of the single ones. Interestingly, the best correction effect was found in the VPBR-VPPRA herb-pair group, which further verified the efficacy improvement through vinegar-processing and compatibility. Partial least square method and VIP analysis combined with spectrum-effect correlation were applied for screening pharmacodynamic markers, and 38 ingredients with higher correlation with four classical pharmacodynamic indexes (ALT, AST, HYP, and GSH-Px) were identified as pharmacodynamic markers of the anti-hepatic fibrosis effects of BR and PRA before and after vinegar-processing and compatibility. The results of the investigation could not only lay a foundation for clarifying the pharmacodynamic materials and mechanism of vinegar-processing and compatibility of BR and PRA in the treatment of hepatic fibrosis as well as provide a theoretical basis for demonstrating the scientific connotation of processing and compatibility, but also provide a reference for further drug design and development of BR and PRA in clinic.

A quantitative analysis method for six principal active constituents (acubin, geniposidic acid, chlorogenic acid, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside) of crude Eucommiae Cortex (EC) and its salt-processed product extracts was developed to investigate and compare their pharmacokinetic behaviors in adenine-induced renal fibrotic rats in vivo. UHPLC-QqQ-MS/MS technology was employed. Scan was conducted in negative ion mode and quantitative determination was carried out by MRM paired ion. The established method was fully validated by specificity, linearity, precision, accuracy, stability, recovery, and matrix effect, and the results of methodological investigation met the requirements of biological sample analysis. Then, a quick, sensitive, and accurate method was successfully established, which could simultaneously measure the contents of six active constituents of crude and salt-processed EC extracts in rat plasma. After a single administration to renal fibrotic rats of crude EC and its salt-processed product extracts, the plasma concentration of each constituent at different time points was measured, the pharmacokinetic parameters were calculated and the concentration time curves were structured. The experiment was approved by the experimental animal ethics committee from Nanjing University of Chinese Medicine (No. 202103A008). The results showed that compared to the crude Eucommiae Cortex group, the t_max of aucubin, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside in the salt-processed Eucommiae Cortex group rat plasma were significantly lower than those in the crude group (P < 0.05, P < 0.01); the C_max and AUC_{0-48 h} of chlorogenic acid, the C_max, AUC_{0-48 h} and AUC_0-∞ of pinoresinol di-O-glucopyranoside, and the C_max of geniposide and pinoresinol 4-O-glucopyranoside were significantly higher than those in the crude group (P < 0.05, P < 0.01). Our investigation found that compared to crude Eucommiae Cortex, a variety of active ingredients could play a role of quick effect with higher peak blood concentration and bioavailability after oral administration of salt-processed Eucommiae Cortex, which were consistent with the traditional Chinese medicine theory of "salt-processing enhancing drug into kidney meridian", providing an experimental basis for the selection of quality control indexes and the in-depth study of processing mechanisms and metabolic rules in vivo of Eucommiae Cortex and its salt-processed product.

OBJECTIVES: To provide a guideline for genealogy inference and family lineage investigation through a study of the mismatch tolerance distribution of Y-STR loci in Chinese Han male lineage. METHODS: Three Han lineages with clear genetic relationships were selected. YFiler Platinum PCR amplification Kit was used to obtain the typing data of 35 Y-STR loci in male samples. The variation of Y-STR haplotypes in generation inheritance and the mismatch tolerance at 1-7 kinship levels were statistically analyzed. RESULTS: Mutations in Y-STR were family-specific with different mutation loci and numbers of mutation in different lineages. Among all the mutations, 66.03% were observed on rapidly and fast mutating loci. At 1-7 kinship levels, the number of mismatch tolerance ranged from 0 to 5 on all 35 Y-STR loci, with a maximum step size of 6. On medium and slow mutant loci, the number of mismatch tolerance ranged from 0 to 2, with a maximum step size of 3; on rapidly and fast mutant loci, the number of mismatch tolerance ranged from 0 to 3, with a maximum step size of 6. CONCLUSIONS Combined use of SNP genealogy inference and Y-STR lineage investigation, both 0 and multiple mismatch tolerance need to be considered. Family lineage with 0-3 mismatch tolerance on all 35 Y-STR loci and 0-1 mismatch tolerance on medium and slow loci can be prioritized for screening. When the number of mismatch tolerance is eligible, family lineages with long steps should be carefully excluded. Meanwhile, adding fast mutant loci should also be handled with caution.
Male ; Humans ; Haplotypes ; Chromosomes, Human, Y/genetics* ; Microsatellite Repeats ; Mutation ; Asian People/genetics* ; China ; Genetics, Population

ObjectiveTo analyze the differential components in water extract of Chuanxiong Rhizoma before and after processing with wine， and to explore the molecular mechanism of Chuanxiong Rhizoma processed with wine in enhancing anti-cerebral ischemia injury. MethodUltra high performance liquid chromatography tandem quadrupole orbitrap high resolution mass spectrometry （UHPLC-Q-Orbitrap HRMS） was used to qualitatively analyze the main chemical components in water extract of Chuanxiong Rhizoma based on the spectral information of compound， comparison of reference substance and references. The chemical pattern recognition method was used to screen the differential components of Chuanxiong Rhizoma before and after processing. Based on these differential components， the potential targets of differential components were predicted by online databases， and the related targets of cerebral ischemia were searched. Cytoscape 3.6.0 was used to establish the network diagram of differential components-action targets-diseases of Chuanxiong Rhizoma processed with wine. The protein-protein interaction （PPI） network of intersection targets was constructed by STRING 11.5. The potential targets of differential components against cerebral ischemia were analyzed by Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis through DAVID 6.8. At the same time， the chemical compounds with high relative content and increased peak area after wine processing were docked with their corresponding targets to verify the mechanism of enhanced effect after wine processing. ResultA total of 71 chemical components were identified from Chuanxiong Rhizoma， 34 differential components and 603 potential targets were screened out. At the same time， a total of 769 disease targets and 60 intersection targets were obtained. Seven key targets were identified through PPI network analysis， including JUN， signal transducer and activator of transcription 3 （STAT3）， mitogen-activated protein kinase 3 （MAPK3）， interleukin-1β （IL-1β）， vascular endothelial growth factor A （VEGFA）， Caspase-3 （CASP3） and mtrix metalloproteinase 9 （MMP9）. Tumor necrosis factor （TNF） signaling pathway was the main differential signaling pathway. The results of molecular docking showed that differential components （senkyunolide K， senkyunolide F， 3-n-butylphthalide， Z，Z′-6，8′，7，3′-diligustilide， ferulic acid and Z-ligustilide） and corresponding targets had good binding activities. ConclusionThe synergistic mechanism of Chuanxiong Rhizoma processed with wine may be related to the enhanced inhibitory effect of inflammatory reaction.

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

Metabonomics technology was employed to investigate and identify the mechanisms and metabolic pathways of the crude and wine-processed Fructus Corni extracts on anti-hepatic fibrosis effects in rats, and to compare and analyze the potential mechanism of enhanced interference of the wine-processed Fructus Corni on hepatic fibrosis effects in rats. The rats were randomly divided into the blank control group, the model group, the colchicine group, the crude Fructus Corni groups with low, medium, and high-doses, and the wine-processed Fructus Corni groups with low, medium, and high-doses, and there were six rats in each group. The hepatic fibrosis model was established by subcutaneous injection of 40% carbon tetrachloride, and the intragastric administration was performed at the third week of modeling. The blood and liver samples of rats were taken and carried out for pharmacodynamic index detection and UHPLC-Q-TOF-MS/MS analysis after intragastric administration for six weeks. The results of pharmacodynamic investigation showed that both the crude and wine-processed Fructus Corni had the effects of anti-hepatic fibrosis in rats. Metabonomics analysis indicated that, compared to the blank control group, the twenty-four potential biomarkers related to hepatic fibrosis were screened and identified in the model group, which mainly involved in primary bile acid metabolism, glycerol phospholipid metabolism, pentose and glucuronide metabolism, retinol metabolism, and arachidonic acid metabolism. The crude and wine-processed Fructus Corni extracts had different degrees of callback effects on the ten of the above potential biomarkers, and the effect of wine-processed Fructus Corni was better than that of crude one. The present study clarifies the mechanism of enhanced efficiency of wine-processed Fructus Corni from the perspective of plasma metabolism, and provides the theoretical foundation for further development and clinical application of Fructus Corni.

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.