In order to clarify the influence of acute hypobaric hypoxia on the bile acids of the rat small intestine, we used ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to identify bile acids in the contents of the small intestine from untreated and acute hypobaric hypoxia-treated rats. Thirty-nine bile acids were detected; PCA and OPLS-DA analysis revealed marked differences in the composition of bile acids between the untreated and the acute hypobaric hypoxia groups. Bile acids were screened with VIP > 1, |log₂FC|≥ 1, P < 0.05, and a total of 7 bile acids with significant differences in content between the two groups were obtained, including 5 conjugated bile acids, 2 unconjugated bile acids; in addition, the content of conjugated bile acids has risen in the treated group. This study demonstrated the influence of high-altitude hypoxic environment on bile acid composition and metabolism in rats. All the animal experiments in this study were approved by the 940th Hospital Ethics Committee (approval No: 2020KYLL012).

We investigated the effect of methyl jasmonate (MeJA) on the content of asperosaponin VI and the expression of genes involved in its synthesis. Dipsacus aspero seedlings were treated with MeJA at different concentrations of 50, 100, 150, 200 and 300 μmol·L^-1, and leaves and roots were sampled following treatment for 1, 3 and 5 days. The content of asperosaponin VI and superoxide anion in the roots, malondialdehyde (MDA) content in leaves and superoxide dismutase were determined. The results show that 150 μmol·L^-1 MeJA significantly increased the accumulation of asperosaponin VI in roots. The content of asperosaponin VI was greatest after treatment for 3 days, and was 2.16 times higher than the control. After MeJA treatment, SOD activity decreased and MDA content increased in leaves. Moreover, superoxide anion content in roots increased. The expression of squalene epoxidase (DaSE1) and geranyl diphosphate synthase (DaGPS), key enzymes in the synthesis of asperosaponin VI, were up-regulated compared with the control group. These results indicate that an optimal concentration of 150 μmol·L^-1 MeJA increases the accumulation of asperosaponin VI by up-regulating the expression of key enzymes involved in the synthesis of asperosaponin VI, which facilitates resistance to adversity stress stimulated by MeJA.

Gentiana crassicaulis Duthie ex Burk. in Gentiana (Sect. Cruciata), Gentianaceae, is one of the original plants of both Gentianae Macrophyllae Radix and Tibetan herb Jie-Ji Na-Bao, which contain such bioactive iridoids as gentiopicroside, loganic acid and others. In this study, based on previous work, the transcriptome of G. crassicaulis was sequenced and analyzed to construct transcriptome databases of roots, stems, leaves and flowers. qRT-PCR verification was conducted for parts of unigenes that may be key enzymes in the pathway of iridoid biosynthesis. The results are as follows: ① a total of 159 534 unigenes were obtained, with an average length of 679 bp. According to the functional classification of GO, unigenes can be divided into 3 categories with 67 branches. The unigenes were aligned in the KOG database and were classified into 25 categories according to function. ② In the KEGG database, 215 unigenes were implicated in 20 standard secondary metabolism pathways. The analysis shows that 305 unigenes encoded 28 key enzymes in the pathway of iridoid biosynthesis, and their expression in different organs is different; and ③ qRT-PCR was approximately consistent with RNA-Seq results. The 7 annotated unigenes identified in this study, HMGS, DXS, MCS, GPPS, G10H, 7-DLNGT and STR, all had higher relative expression levels in the above-ground parts (stem, leaf and flower) than in the underground part (root). Iridoids are common active and index components of such traditional Chinese medicines as Qinjiao, Longdan, Dangyao, and Qingyedan, among others. Therefore, this work provides basic scientific data for further development including obtaining active components or intermediates through biotechnology, exploring the accumulation of effective components, evaluating the quality of different ecotype varieties, and identifying authentic biosynthesis pathways of medicinal materials.

UDP glucosyltransferase (UDPGT) catalyzes the synthesis of secondary metabolites and plant hormones to regulate plant growth and development, pathogen defense and environmental adaptability. In this study 18 members of the RcUDPGT gene family were cloned from Tibetan Rhodiola crenulata and analyzed using bioinformatics. The tissue-specific expression, abiotic stresses and plant hormones (abscisic acid, auxin, methyl jasmonate) induced expression patterns were identified by real-time quantitative PCR. The bait vector of RcUDPGT (JX228125.1) was constructed to select interacting proteins from an Arabidopsis yeast library. The results of the bioinformatics analysis revealed that RcUDPGT nucleotide sequences were about 1 400 bp and encoded 452-498 amino acids. In the primary protein sequences, C-terminal sequences were more conserved compared with N-terminal regions, which held a PSPG (plant secondary product glycosyltransferase) domain. In the tertiary structures, RcUDPGTs contained a UDP sugar donor recognition binding site. In addition, all genes had multiple phosphorylation sites. The results of qRT-PCR showed that RcUDPGTs genes were expressed in root, stem and leaf. The expression levels were regulated by low temperature/ultraviolet light and various plant hormones (ABA, IAA, MeJA), but the expression patterns were quite different among them. For example, RcUDPGT6, RcUDPGT11, and RcUDPGT17 had the highest expression in leaves and were induced by all three hormones, suggesting that the functions of these genes might be to respond to environmental changes. RcUDPGT9, RcUDPGT10, RcUDPGT14 were most abundantly expressed in roots and were significantly induced by ABA and MeJA hormones, indicating that these genes may be involved in the synthesis and accumulation of salidroside. Yeast two-hybrid results showed that RcUDPGT did not exhibit autoactivation and cell toxicity, and two significant interactional genes were identified, AtKCR1 (AT1G67730.1) and AtSNL4 (AT1G70060). The AtKCR1 gene encodes a β-ketoacyl reductase (KCR) involved in synthesis of very long chain fatty acids. The AtSNL4 gene encodes a homolog of the transcriptional repressor SIN3, which could participate in the ABA hormone signaling pathway and enhance the transcriptional repression of AP2/EREBP class factors in Arabidopsis. These results suggest that the accumulation of the secondary metabolite salidroside in Rhodiola crenulata might be affected by several regulatory mechanisms. The above results may lay the foundation for understanding the adaptive mechanism of Rhodiola crenulata in a high altitude environment and stimulate an in-depth study of the synthesis and accumulation of secondary metabolites in this species.

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.

This paper reviews modern science and technology with regard to traditional Chinese medicine (TCM) research methods, quality control, and discusses the establishment and improvement of the evaluation system for TCM, focusing on the following issues: 1) How to incorporate the efficacy of TCM in the overall evaluation of TCM quality; 2) how to quantify the efficacy and properties of TCM; 3) how to understand the correlation between the efficacy and properties of TCM and its application; 4) how to reflect the specificity of the effective components in the overall evaluation of TCM quality; and 5) how to reflect the quality value transfer of effective components in the whole process of production and metabolism in vivo to the overall evaluation of TCM quality. The overall quality evaluation of TCM needs to better reflect its clinically safety and effectiveness. It is suggested to establish a clinical quality assessment method based on the content of the chemical components of TCM to explore and establish an overall evaluation method of TCM quality that reflects the efficacy of TCM and conforms with clinical practice.

As a key signal transduction molecule involved in the innate immune response, stimulator of interferon genes (STING) is triggered by cytosolic DNA from pathogen and host origins, and plays an important role in inducing the secretion of type Ⅰ interferons and proinflammatory cytokines, thereby defending against viral and intracellular bacterial infections and regulating the production of spontaneous antitumor immune responses in vivo. Thus, STING agonists have shown useful therapeutic effects for pathogen infection and cancer. In the past decade research on STING and its agonists has progressed rapidly. Here, we summarize recent advances in the structure and activation of STING and the mechanism of the cGAS-STING pathway. In particular, we review research advances of STING agonists, analyze the crystal structure of STING in complex with its agonists and the structure-activity relationship of STING agonists, and summarize the strong challenges of developing STING agonists.

Nanocrystal drugs have many advantages, such as no carrier materials, easy industrialization, diversified dosage forms, and can significantly improve the solubility and bioavailability of insoluble drugs, so many drugs have been on the market. The traditional nanocrystal preparation technology has the problems of low preparation efficiency and process limitation of the smallest achievable particle size. With the progress of pharmaceutical preparation technology, the preparation technology of nanocrystal drugs is constantly improving, and new preparation technologies are constantly emerging. The emergence of new technologies has greatly shortened the process time and makes it possible to prepare nanocrystal drugs with smaller particle diameters. In this paper, the preparation technologies of nanocrystal drugs, especially the new preparation technologies such as high gravity controlled precipitation, microfluidic reaction technology and various combination technologies, are reviewed from three aspects: "Top-down" technology, "Bottom-up" technology and combination technology. This article also prospects the development of new preparation technologies, hoping to provide reference for the related research of nano-preparations.

In this study, a rat morphine drug discrimination model with a fixed ratio (FR) of 10 (FR10) was established using different methods to explore which methods can shorten the modeling time and test the dose-response relationship and median effective dose (ED₅₀) value. Animal welfare and experimental procedures are in accordance with the provision of the Animal Ethics Committee of Shanghai InnoStar Bio-tech Co., Ltd. Forty rats were initially shaped to press lever under a fixed-ratio schedule of food reinforcement. The animals that were successfully trained under a FR10 schedule of food reinforcement were divided into two groups, namely the single-lever + double-lever training group 1 and the double-lever training group 2. In each group, rats were trained to discriminate morphine at 5.6 mg·kg^-1 from saline by the intraperitoneal route. After training, different doses of morphine were used to substitute for training dose of morphine, the dose-response curve for morphine were identified in rats, and the ED₅₀ value was calculated. The results showed that, in food training phase: 34 rats successfully entered the discrimination training during food training; in discrimination training phase: 14 animals in group 1 met the discrimination training standard for the first time, which took about (40.71±2.93) days, and there were 13 animals in group 2 that met the discrimination training criteria for the first time, and it took about (51.15±2.55) days. It can be seen that the method of single-lever + double-lever training is better than single-lever training, and the difference is significant compared with group 1 (P < 0.05); in generalization test phase: there are 17 rats completed morphine generalization test, and the percentages of morphine-lever responses produced by the generalization test of different doses of morphine (0, 0.1, 0.5, 1, 3, 5.6, and 10 mg·kg^-1) were (9.56±3.13)%, (9.01±5.83)%, (13.82±7.95)%, (29.04±10.13)%, (41.70±10.65)%, (85.36±7.16)%, (94.56±2.76)%, respectively. The results showed that the discriminative stimulative effect induced by morphine dose between 0-10 mg·kg^-1 increased in a dose-dependent manner, producing a good dose-response curve, and the ED₅₀ value of morphine was 4.74 mg·kg^-1 by linear fitting. The above results showed that, the FR10 morphine drug discrimination model has been successfully established using different methods; the single-lever + double-lever training method is better than the single-lever training, and can relatively shorten the discrimination training cycle.

ABC transporters on the intestinal barrier, blood-brain barrier and on tumor cells will affect drug bioavailability, transport across the blood-brain barrier and multidrug resistance. The active ingredients of traditional Chinese medicines can affect the function and expression of ABC transporters. When combined with pharmaceuticals the potential interaction between the two can change the efficacy of the medicines. We review the ABC transporter superfamily and their distribution with regard to their relationship and interactions with traditional Chinese medicine on the intestinal barrier and the blood-brain barrier, as well as their role in tumor multidrug resistance mediated by ABC transporters. We summarize the research progress over the past five years.