Exploring the potential mechanisms of Rehmannia glutinosa in treating sepsis based on network pharmacology.

The present study utilized network pharmacology to identify therapeutic targets and mechanisms of Rehmannia glutinosa in sepsis treatment. RNA-sequencing was conducted on peripheral blood samples collected from 23 sepsis patients and 10 healthy individuals. Subsequently, the RNA sequence data were analyzed for differential expression. Identification of active components and their putative targets was achieved through the HERB and SwissTarget Prediction databases, respectively. Functional enrichment analysis was performed using GO and KEGG pathways. Additionally, protein-protein interaction networks were constructed and survival analysis of key targets was conducted. Single-cell RNA sequencing provided cellular localization data, while molecular docking explored interactions with central targets. Results indicated significant involvement of identified targets in inflammation and Th17 cell differentiation. Survival analysis linked several targets with mortality rates, while molecular docking highlighted potential interactions between active components and specific targets, such as rehmaionoside a with ADAM17 and rehmapicrogenin with CD81. Molecular dynamics simulations confirmed the stability of these interactions, suggesting Rehmannia glutinosa's role in modulating immune functions in sepsis.

First Report of Aspergillus westerdijkiae Causing Leaf Blight on Rehmannia glutinosa in China.

Rehmannia glutinosa (also known as Chinese foxglove) is a perennial dicotyledonous herb, which plays an important role in traditional Chinese medicine. Its active ingredients have a wide range of pharmacological effects on the blood system, endocrine system, immune system, cardiovascular system, and nervous system (Zhang et al. 2008). In May 2022, leaf blight was observed on 45-day-old R. glutinosa in a seedling nursery in Jiaozuo City (35°01'44.20″N, 113°05'30.63″E), Henan Province, China with an approximate disease incidence up to 54% (~1,300 plants). Irregular brown lesion initially appeared on the tips of basal leaves, then progressed to the entire leaf causing leaf drying out (Supple. Fig. 1-A, B, C). The same symptoms appeared successively in the leaves from the base to the top of the plant, which eventually caused the whole plant to die. To identify the pathogen, eight symptomatic leaves were randomly collected from eight individual plants, and cut into small pieces (5 × 5 mm) at the border of lesions. The pieces were surface disinfected in 75% ethanol for 15 s, followed by 1% NaClO for 1 min, rinsed in sterile water three times, and placed on potato dextrose agar (PDA) medium in the dark for 3 days at 25℃. Finally, 12 purified isolates (DHY1-DHY12) were obtained by using single spore method. Leaves of R. glutinosa seedlings were inoculated with conidial suspension (106 conidia/ml), three plants were inoculated per isolate. Controls were treated with sterilized water. All inoculated and control plants were incubated in a greenhouse at 25℃ under 80 ± 10% humidity and a 8-h/16-h dark/light cycle. This experiment was repeated three times. After 5 days, similar symptoms to those of diseased leaves in the seedling nursery appeared on leaves inoculated with DHY4-DHY10, while plants inoculated with DHY1-DHY3, DHY11-DHY12, and the controls remained asymptomatic (Supple. Fig.1-D, E). The same fungi were re-isolated from diseased leaves, fulfilling Koch's postulates. The causal agents DHY4 to DHY10, showed similar morphology, which were morphologically identified as Aspergillus sp. (Visagie et al. 2014). Isolate DHY5 was selected for further study. On PDA plates, the colonies were covered with white velutinous mycelia (Supple. Fig.1-F). Conidia were ochre yellow and outwards concentric circles. Vesicles were globose, and about 20.1-26.6 µm in diameter (Supple. Fig.1-G). Conidiophore stipes were smooth walled and hyaline, with conidial heads radiating. The conidia were light yellow to orange, exudate clear to orange droplets. The conidia were (2.53-3.25) µm × (2.58-3.47) µm in diameter (n=50) (Supple. Fig.1-H). For further molecular identification, the ITS and TUB gene sequences were amplified with primer pairs ITS1/ITS4 and BT2a/BT2b (Glass and Donaldson. 1995), respectively. BLASTn searches of the ITS (PP355445) and TUB (PP382788) sequences showed 100% and 98.42% similarity to those of A. westerdijkiae (OP237108 and OP700424), respectively. Phylogenetic analysis based on the concatenated sequences of ITS and TUB confirmed that the fungus was A. westerdijkiae, (Supple. Fig.2). A. westerdijkiae was mainly reported on its secondary metabolite ochratoxin A contamination of agricultural products, fruits, and various food products, such as coffee beans (Alvindia et al 2016), grapes (Díaz et al. 2009), oranges and fruit juice (Marino et al. 2009), etc. To our knowledge, this is the first report of A. westerdijkiae causing leaf blight on R. glutinosa in China.

Seven new pentasaccharides from the roots of Rehmannia glutinosa.

Seven new pentasaccharides (1-7), rehmaglupentasaccharides A-G, were isolated from the air-dried roots of Rehmannia glutinosa. Their structures were established from the spectroscopic data obtained and by chemical evidence. The known verbascose (8) and stachyose (9) were also obtained in the current investigation, and the structure of stachyose was unequivocally defined using X-ray diffraction data. Compounds 1-9 were tested for their cytotoxicity against five human tumor cell lines, influence on dopamine receptor activation, and proliferation effects against Lactobacillus reuteri.

Four new iridoid glycosides from the roots of Rehmannia glutinosa.

Four new iridoid glycosides (1-4), rehmaglutosides L-O, were isolated from the air-dried roots of Rehmannia glutinosa. Their structures were established from the spectroscopic data obtained and by chemical evidence. The known mellittoside (5) and ajugol (6) were also obtained in the current investigation, and the structure of mellittoside was unequivocally defined using X-ray diffraction data. Compounds 1-6 were tested for their cytotoxicity against five human tumor cell lines and proliferation effects on Lactobacillus Reuteri.

[Role of mitochondrial autophagy and the curative effect of rehmannia glutinosa leaves total glycoside capsules on nucleos(t)ide drug-induced renal injury].

Objective: To study the curative effect of rehmannia glutinosa leaves total glycoside capsules and the role of mitochondrial autophagy on nucleos(t)ide drug-induced renal injury. Methods: Adefovir dipivoxil (ADV) was used to construct a hepatitis B virus (HBV) transgenic mouse model for renal injury. Renal function was measured in each group at one and two weeks of modeling. Mitochondrial autophagy indicators were measured at two weeks of modeling in renal tissue. Transmission electron microscopy was used to detect mitochondrial autophagy phenomena in renal tissue. The model was established for two weeks. Mouse with renal injury were treated with rehmannia glutinosa leaves total glycoside capsules or isotonic saline for eight weeks by intragastric administration. Renal function was measured. Renal tissue morphology was observed. Mitochondrial autophagy indicators were detected in renal tissue. The protective effect of different concentrations of verbascoside (the main active ingredient of rehmannia glutinosa capsule) was observed on HK-2 cell damage induced by ADV. HK-2 cells were divided into control, ADV, and ADV plus verbascoside groups. The effects of verbascoside at different times and concentrations were observed on the HK-2 mitochondrial autophagy indicators. Fifty patients with chronic hepatitis B were collected who presented with renal injury after treatment with nucleos(t)ide analogs. The random number method was used to divide 29 cases into a control group that received conventional treatment. The treatment group of 21 cases was treated with rehmannia glutinosa leaves total glycoside capsules on the basis of the control group. Serum creatinine (Scr) and urinary protein were detected at eight weeks.The χ(2) test or t-test was used for statistical analysis. Results: Compared with the control group, two weeks of modeling in the ADV group induced renal function injury in HBV mice. The expression of autophagy indicators was higher in the renal tissue of the ADV group than that of the control group. Transmission electron microscopy had revealed mitochondrial autophagy in the renal tissue of the ADV group. Compared with the control group, the renal function of HBV mice treated with rehmannia glutinosa leaves total glycoside capsules improved for two months, and the expressions of autophagy indicators were down-regulated.Verbascoside promoted proliferation in ADV-damaged HK-2 cells, and the expression of autophagy indicators was down-regulated compared with the ADV alone group. In 50 patients with renal function injury, the urinary protein improvement was significantly superior in the treatment group than that in the control group, with eighteen and three cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with a statistically significant difference (χ(2) = 9.975 0, P = 0.001 6). Serum creatinine was decreased in the treatment group compared with the control group, with 11 and 10 cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with no statistically significant difference (χ(2) = 0.593 5, P = 0.441 1). Conclusion: Rehmannia glutinosa leaves total glycoside capsule can improve the nucleos(t)ide drug-induced renal function injury in chronic hepatitis B, possibly playing a role via inhibiting PINK1/Parkin-mediated mitochondrial autophagy.

[Gene cloning and functional analysis of an iridoid oxidase gene in Rehmannia glutinosa].

Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, which has activities of heat-clearing,blood-cooling, Yin-nourishing, and body fluid-promoting. Iridoid glycosides are the main bioactive in R. glutinosa. Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides. In this study, an iridoid oxidase gene Rg IO was screened based on the transcriptome data, followed by bioinformatics analysis, expression characteristic detection, and subcellular localization analysis. The results show that the coding region of Rg IO is 1 536 bp, with 511 amino acids encoded, and the molecular weight is about 58 258. 01. The protein sequence of Rg IO contains the conserved domains and motifs of cytochrome P450 oxidases. Rg IO has the highest sequence identities with its ortholog proteins in Striga asiatica, Striga hermonthica, and Centranthera grandiflora and has good sequence identities(77. 28%) with Catharanthus roseus Cr IO. Rg IO shows specific expression in the leaf of R. glutinosa. In response to MeJA induction, the expression of MeJA in leaves and roots after treatment increases by 3. 15 and 1. 3 times at 3 h and 6 h,respectively. The result of subcellular localization shows that Rg IO is distributed in the endoplasmic reticulum. Agrobacterium-mediated transient expression of Rg IO gene in leaves of R. glutinosa makes the content of catalpol increase by 0. 82 times compared with the transient expression of the empty vector. This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R. glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.

[Effect of 2-phenylethyl-beta-glucopyranoside isolated from Huaizhong No. 1 Rehmannia glutinosa on hypoxic pulmonary hypertension by regulating PI3K/Akt/m TOR/HIF-1α pathway].

The study investigated the protective effect and mechanism of 2-phenylethyl-beta-glucopyranoside(Phe) from Huaizhong No.1 Rehmannia glutinosa on hypoxic pulmonary hypertension(PH), aiming to provide a theoretical basis for clinical treatment of PAH. Male C57BL/6N mice were randomly divided into normal group, model group, positive drug(bosentan, 100 mg·kg~(-1)) group, and low-and high-dose Phe groups(20 and 40 mg·kg~(-1)). Except for the normal group, all other groups were continuously subjected to model induction in a 10% hypoxic environment for 5 weeks, with oral administration for 14 days starting from the 3rd week. The cardiopulmonary function, right ventricular pressure, cough and asthma index, lung injury, cell apoptosis, oxidative stress-related indicators, immune cells, and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/hypoxic inducible factor 1α(HIF-1α) pathway-related proteins or mRNA levels were examined. Furthermore, hypoxia-induced pulmonary arterial smooth muscle cell(PASMC) were used to further explore the mechanism of Phe intervention in PH combined with PI3K ago-nist(740Y-P). The results showed that Phe significantly improved the cardiopulmonary function of mice with PH, decreased right ventricular pressure, cough and asthma index, and lung injury, reduced cell apoptosis, oxidative stress-related indicators, and nuclear levels of phosphorylated Akt(p-Akt) and phosphorylated mTOR(p-mTOR), inhibited the expression levels of HIF-1α and PI3K mRNA and proteins, and maintained the immune cell homeostasis in mice. Further mechanistic studies revealed that Phe significantly reduced the viability and migration ability of hypoxia-induced PASMC, decreased the expression of HIF-1α and PI3K proteins and nuc-lear levels of p-Akt and p-mTOR, and this effect was blocked by 740Y-P. Therefore, it is inferred that Phe may exert anti-PH effects by alleviating the imbalance of oxidative stress and apoptosis in lung tissues and regulating immune levels, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR/HIF-1α pathway. This study is expected to provide drug references and research ideas for the treatment of PH.

Angiotensin I-Converting Enzyme-Inhibitory Activity and Phytochemical Profile of Constituents of the Leaves of Rehmannia glutinosa f. hueichingensis.

The root of Rehmannia glutinosa Liboschitz forma hueichingensis HSIAO has been used as a tonic and treatment for urinary and skin disorders in Japanese Kampo medicine. Phytochemical investigation of the root has been well reported, but that of the leaves is limited. To explore the potential value of R. glutinosa leaves, we focused on the angiotensin I-converting enzyme (ACE)-inhibitory activity. The leaf extract exhibited ACE-inhibitory activity, and the inhibitory potency of leaves was stronger than that of roots. Using this activity as an indicator, we isolated linaride (1), 6-O-hydroxybenzoyl ajugol (2), acteoside (3), leucosceptoside A (4), martynoside (5), luteolin (6), apigenin (7), and chrysoeriol (8) by separating and purifying the extract. We then examined the ACE-inhibitory activities of 1-8, catalpol (9), aucubin (10), ajugol (11), and echinacoside (12). Among them, 3, 6, and 12 displayed the most potent inhibitory activity. A simultaneous analytical method was also developed using compounds contained in R. glutinosa leaves and roots, and their contents were compared. The method consisted of extraction with 50% aqueous methanol under sonication for 60 min and LC/MS measurement. R. glutinosa leaves tended to have higher levels of majority of the analytes than the roots, including 3 and 6, which had higher ACE-inhibitory activity. These results suggest that 3 and 6 contribute to the ACE-inhibitory activity of R. glutinosa leaves, which may represent a useful medicinal resource for hypertension.

The Effects of NAA on the Tuberous Root Yield and Quality of Rehmannia glutinosa and Its Regulatory Mechanism by Transcriptome and Metabolome Profiling.

Naphthylacetic acid (NAA) was used to increase the tuberous root yield of Rehmannia glutinosa, but the differences between its NAA-treated and control tuberous roots (NT and CG) and the regulatory mechanism of NAA effect remain unclear. In order to investigate them, NTs and CGs were used as materials, and both yield-related indices were measured; the metabolomics and transcriptomics were used to capture differentially accumulated metabolites (DAM) and to validate them via mining differentially expressed genes (DEGs), respectively. The effects of NAA treatment: increased NT mass per plant by 21.14%, through increasing the number of roots and increasing the mean root diameter; increased catalpol content by 1.2234% (p < 0.05); up-regulated 11DAMs and 596DEGs; and down-regulated 18 DAMs and 517DEGs. In particular, we discovered that NAA regulated its DAMs and biomass via 10 common metabolic pathways, and that the number of NAA-down-regulated DAMs was more than that of NAA-up-regulated DAMs in its tuberous root. Furthermore, HPLC validated the changes of several DAMs and 15 DEGs (4CL, ARF, CCoAOMT, ARGOS, etc.) associated with the yield increase and DAMs were verified by RT-qPCR. This study provided some valuable resources, such as tuberous root indices, key genes, and DAMs of Rehmannia glutinosa in response to NAA for distinguishing the CGs from NTs, and novel insights into the regulatory mechanism of NAA effects on both at the transcriptomic and metabolomic levels, so it will lay a theoretical foundation for NAA-regulated plant yield and quality, and provide references for prohibiting the uses of NAA as a swelling agent in medicinal tuber plants in China.

Functional characterization of tyrosine decarboxylase genes that contribute to acteoside biosynthesis in Rehmannia glutinosa.

MAIN CONCLUSION: The RgTyDCs possess typical decarboxylase functional activity in vitro and in vivo and participate in acteoside biosynthesis in R. glutinosa, positively controlling its production via activated acteoside/tyrosine-derived pathways. Acteoside is an important ingredient in Rehmannia glutinosa and an active natural component that contributes to human health. Tyrosine decarboxylase (TyDC) is thought to play an important role in acteoside biosynthesis. Several plant TyDC family genes have been functionally characterized and shown to play roles in some bioactive metabolites' biosynthesis by mediating the decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-DOPA); however, one TyDC (named RgTyDC1) in R. glutinosa has been identified to date, but the family genes that contribute to acteoside biosynthesis remain largely characterized. Here, by in silico and experimental analyses, we isolated and identified three RgTyDCs (RgTyDC2 to RgTyDC4) in this species; these genes' sequences showed 50.92-82.55% identity, included highly conserved domains with homologues in other plants, classified into two subsets, and encoded proteins that localized to the cytosol. Enzyme kinetic analyses of RgTyDC2 and RgTyDC4 indicated that they both efficiently catalysed L-tyrosine and L-dopa. The overexpression of RgTyDC2 and RgTyDC4 in R. glutinosa, which was associated with enhanced TyDC activity, significantly increased tyramine and dopamine contents, which was positively correlated with improved acteoside production; moreover, the overexpression of RgTyDCs led to upregulated expression of some other genes-related to acteoside biosynthesis. This result suggested that the overexpression of RgTyDCs can positively activate the molecular networks of acteoside pathways, enhancing the accumulation of tyramine and dopamine, and promoting end-product acteoside biosynthesis. Our findings provide an evidence that RgTyDCs play vital molecular roles in acteoside biosynthesis pathways, contributing to the increase in acteoside yield in R. glutinosa.

Study on Catalpol Content in Rehmannia glutinosa Root, an Important Ingredient in Kampo Prescriptions.

Rehmannia glutinosa is an important medicinal plant in Asia, and its roots are used as an ingredient in herbal medicine. However, the roots exhibit different medicinal effects depending on the processing conditions. Since the catalpol content differs greatly during the process, the catalpol content is an essential index for quality evaluation. R. glutinosa roots have various weights, diameters, and lengths, and there are differences between individuals and within an individual immediately after harvest. We found that, catalpol content in the roots tended to increase as root diameter increased. Furthermore, it has been reported that catalpol content decreased with drying, and our results also supported this phenomenon. To clarify the reason for the decrease in catalpol content, we investigated the effect of ß-glucosidase in R. glutinosa cells. An in situ assay for ß-glucosidase activity revealed that the activity in the tissue inside the cambium disappeared one month after drying under natural conditions, and the activity in the tissue outside the cambium completely disappeared after two months. Because catalpol content remained almost unchanged even after drying for two months, it was clarified that ß-glucosidase activity had minimal involvement in the decrease in catalpol content in R. glutinosa roots. Based on the above results, we proposed that slicing the roots and rapidly removing water by natural drying is best to obtain dry root with little loss of catalpol content.

First Report of Tobacco Mild Green Mosaic Virus Infecting Rehmannia glutinosa in China.

Rehmannia glutinosa (family Scrophulariaceae) is an important traditional medicinal plant, whose root is used to treat anemia, hemoptysis, and gynecological diseases in China (Matsumoto et al. 1989). This plant is native to China and cultivated in China, Korea, Japan, and northern Vietnam (Kwak et al. 2020). Viral diseases caused remarkable loss in the yield and quality of R. glutinosa (Ling et al. 2009). To date, ten viruses have been identified globally to infect R. glutinosa and seven of these viruses reported in China (Liu et al. 2018; Zhang et al. 2021). Most plants of R. glutinosa are infected with one or more of these viruses (Kwak et al. 2018; Zhang et al. 2004). In July 2020, a survey of the viral disease infecting R. glutinosa was conducted in commercial plantations of Wenxian, Wuzhi, Mengzhou, and Yuzhou counties in Henan Province, China. The disease symptoms included mosaic, chlorosis, leaf distortion, and the percentage of symptomatic plants was over 70% in the surveyed fields (n=9). Sixty leaf samples of symptomatic R. glutinosa plants were collected from nine cultivation fields in Wenxian, Wuzhi, Mengzhou, and Yuzhou counties (five to seven plants for each field). Total RNA was extracted from one pooled sample containing a portion of all above-mentioned leaf samples using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) and analyzed by high-throughput sequencing (HTS) to identify viral pathogens. A transcriptome library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), and sequenced on an Illumina NovaSeq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 27,664,949 high-quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs (n=109,180) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. BLASTn analysis showed that the R. glutinosa plants were infected with known viruses, including broad bean wilt virus, rehmannia mosaic virus, youcai mosaic virus, and cucurbit chlorotic yellows virus. In addition, one contig (6,418 nt in length) had a nucleotide sequence identity of 99.64% with the TN29 isolate of tobacco mild green mosaic virus (TMGMV, GenBank accession no. MF139550). To confirm the presence of this virus, sixty above-mentioned samples were screened by reverse transcription-polymerase chain reaction (RT-PCR) using the specific primer pairs (Supplementary Table1) TMGMG-CPF/TMGMG-CPR targeting a 545-nt fragment within the CP gene. Amplicons with expected sizes were detected from 47 of 60 samples but not from the negative control (virus-free healthy plant through the tip meristem culture). Seventeen amplicons (11#, 13#, 14#, 21#, 22#, 23#, 25#, 26#, 27#, 31#, 32#, 33#, 37#, 52#, 57#, 59#, and 60#) of TMGMV-CP were selected, and purified. The PCR products were cloned into the pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The sequences were deposited into the GenBank (accession nos. MZ395944 to MZ395960). The near-full-length genomic sequence of TMGMV-Rg14 isolate was obtained from one positive sample (sample no. 14) by RT-PCR amplification of two overlapping fragments using the following primer pairs: TMGMV-40F/TMGMV-3570R and TMGMV-3220F/TMGMV-6400R. The near-full-length genomic sequence of the TMGMV-Rg14 isolate was 6 304 nucleotides (nt) in length and deposited into GenBank (accession no. MZ395975). BLASTn analysis demonstrated that the TMGMV-Rg14 isolate shared a sequence identity ranging from 96.89% (AB078435) to 99.60% (MF139550) with the other TMGMV isolates. Furthermore, the virus-free healthy R. glutinosa plants were inoculated with sap from the positive sample (14#) to confirm the infection of TMGMV. Mosaic symptoms were induced on the systemically infected leaves of the inoculated plants 14 days post inoculation. The systemically infected leaves of inoculated plants were assayed by RT-PCR using the primer pairs TMGMV-CPF/CPR. Amplicons of expected size were detected from the inoculated plants but not from non-inoculated plants. To our knowledge, this is the first report of TMGMV infection on R. glutinosa. Further studies are necessary to select a suitable indicator plant for this TMGMV, its host range, and the symptoms it induces in single infection. Since R. glutinosa is cultivated by vegetative propagation, production of virus-free healthy plants is necessary. This study will help to generate virus-free healthy plants and prevent viral disease on R. glutinosa. Further study is needed to determine its pathological implications and economic impact on R. glutinosa in China.

Three new pyridine alkaloids and one new iridoid analogue from the leaves of Rehmannia glutinosa.

As part of an ongoing project on Rehmannia species, three new pyridine alkaloides (glutinosines A - C), and one new iridoid analogue (rehmaglutin E), were isolated from the leaves of Rehmannia glutinosa. The structures of the new compounds were established by extensive spectroscopic analysis and electronic circular dichroism calculations.

Renoprotective activity of a new amide and a new hydroxycinnamic acid derivative from the fresh roots of Rehmannia glutinosa.

A new amide, named rehmagluamide (1), and a new hydroxycinnamic acid derivative, named nepetoidin F (2), together with six known compounds, 2'-O-methyluridine (3), puroglutamic acid (4), biliverdic acid (5), peterolactam (6), nicotinic acid (7), nicotinamide (8), were isolated from the fresh roots of Rehmannia glutinosa. All the structures of compounds were identified by the interpretation of their spectroscopic data and comparison with those reported in the literatures. The protective effects of compounds 1-7 on normal rat kidney tubule epithelioid (NRK-52e) cells injury induced by LPS were investigated. The results indicated that compounds 1, 2, and 7 exhibited protective effects against LPS-induced NRK 52e cells injury.

Identification and Functional Characterization of Tyrosine Decarboxylase from Rehmannia glutinosa.

Rehmannia glutinosa is an important medicinal plant that has long been used in Chinese traditional medicine. Acteoside, one of the bioactive components from R. glutinosa, possessed various pharmacological activities for human health; however, the molecular mechanism of acteoside formation is not fully understood. In the current study, a novel tyrosine decarboxylase (designated as RgTyDC2) was identified from the R. glutinosa transcriptome. Biochemical analysis of RgTyDC2 showed RgTyDC2 uses tyrosine and dopa as the substrate to produce tyramine and dopamine, respectively, and it displays higher catalytic efficiency toward tyrosine than dopa. Moreover, the transcript level of RgTyDC2 was consistent with the accumulation pattern of acteoside in R. glutinosa, supporting its possible role in the biosynthesis of acteoside in vivo.

Acteoside and ursolic acid synergistically protects H2O2-induced neurotrosis by regulation of AKT/mTOR signalling: from network pharmacology to experimental validation.

CONTEXT: Ursolic acid (UA) and acteoside (ATS) are important active components that have been used to treat Alzheimer's disease (AD) because of their neuroprotective effects, but the exact mechanism is still unclear. OBJECTIVE: Network pharmacology was used to explore the mechanism of UA + ATS in treating AD, and cell experiments were used to verify the mechanism. MATERIALS AND METHODS: UA + ATS targets and AD-related genes were retrieved from TCMSP, STITCH, SwissTargetPrediction, GeneCards, DisGeNET and GEO. Key targets were obtained by constructing protein interaction network through STRING. The neuroprotective effects of UA + ATS were verified in H2O2-treated PC12 cells. The subsequent experiments were divided into Normal, Model (H2O2 pre-treatment for 4 h), Control (H2O2+ solvent pre-treatment), UA (5 µM), ATS (40 µM), UA (5 µM) + ATS (40 µM). Then apoptosis, mitochondrial membrane potential, caspase-3 activity, ATG5, Beclin-1 protein expression and Akt, mTOR phosphorylation levels were detected. RESULTS: The key targets of UA + ATS-AD network were mainly enriched in Akt/mTOR pathway. Cell experiments showed that UA (ED50: 5 µM) + ATS (ED50: 40 µM) could protect H2O2-induced (IC50: 250 µM) nerve damage by enhancing cells viability, combating apoptosis, restoring MMP, reducing the activation of caspase-3, lessening the phosphorylation of Akt and mTOR, and increasing the expression of ATG5 and Beclin-1. CONCLUSIONS: ATS and UA regulates multiple targets, bioprocesses and signal pathways against AD pathogenesis. ATS and UA synergistically protects H2O2-induced neurotrosis by regulation of AKT/mTOR signalling.

[Synthesis, characterization, and immunological activity of Rehmannia glutinosa seleno-polysaccharides].

The present study explored the optimum synthesis process of Rehmannia glutinosa seleno-polysaccharides with acetic acid as a catalyst, characterized the structure of R. glutinosa seleno-polysaccharides by Fourier transform infrared spectroscopy(FT-IR), scanning electron microscopy(SEM), thermogravimetry(TG), and atomic force microscopy(AFM), and preliminarily investigated the immunological activity of R. glutinosa seleno-polysaccharides. The results showed that the optimal conditions for the synthesis of R. glutinosa seleno-polysaccharides included m(acetic acid)∶m(R. glutinosa polysaccharides)=0.80, m(Na_2SeO_3)∶m(R. glutinosa polysaccharides)=1.25, reaction temperature of 80.0 ℃, and reaction time of 7.0 h. Under these conditions, the selenium content of R. glutinosa seleno-polysaccharides was 2.239 mg·g~(-1). The acetic acid catalysis method was milder than the nitric acid method, without affecting the structure of polysaccharides. The results of IR, SEM, TG, and AFM showed that R. glutinosa seleno-polysaccharides were properly prepared. The results of immunological activity showed that compared with the control group, R. glutinosa seleno-polysaccharides could significantly promote the phagocytic capacity of mouse monocyte macrophages and improve the spleen index and thymus index of mice. In the concentration range of 15-240 µg·mL~(-1), the proliferation of spleen lymphocytes of mice was strengthened, and the IL-2 and IFN-γ secretion by Th1 cytokines was promoted. This study can provide references for the further development and application of R. glutinosa polysaccharides.

[Acetylation of Rehmannia glutinosa polysaccharides and antioxidant activity of acetylated derivatives].

This study aims to acetylate Rehmannia glutinosa polysaccharides by acetic anhydride method, optimize process parameters and evaluate their antioxidant activity. With the degree of substitution(D_s) as a criterion, the effects of reaction time, acetic anhydride-to-polysaccharides ratio and temperature were investigated. Process parameters were optimized by single-factor experiment and response surface methodology. The infrared spectroscopy(IR) and scanning electron microscopy(SEM) proved the successful acetylation and were employed to preliminarily analyze the structural characteristics of acetylated derivatives. The results showed that the D_s was 0.327 under the optimal technological conditions, including m(acetic anhydride):m(R. glutinosa polysaccharides)=2.70, reaction time 3.0 h and temperature 48 ℃. Further, the antioxidant properties of acetylated derivatives were investigated in vitro and acetylation was found effective to improve the antioxidant activity of R. glutinosa polysaccharides. This study provides a reference for the further development and application of R. glutinosa polysaccharides.

[Identification and biological characteristics of a pathogen causing leaf blight of Rehmannia glutinosa].

Leaf blight outbroke in Rehmannia glutinosa plantation in Wenxian county, Henan province in 2019. R. glutinosa plants with diseased leaves were collected from the plantation, and three strains were isolated from the diseased leaf samples. Pathogenicity test, morphological observation, and phylogenetic analysis of ITS, EF1-α, and Tub suggested that they were respectively Fusarium proliferatum, F. oxysporum, and F.acuminatum. Among them, F. acuminatum, as a pathogen of R. glutinosa leaf disease, had never been reported. To clarify the biological characteristics of F. acuminatum, this study tested the influence of light, pH, temperature, medium, carbon source, and nitrogen source on the mycelial growth rate of the pathogen during a 5-day culture period, and explored the lethal temperature. The results showed that the mycelia grew well under the photoperiod of 12 h light/12 h darkness, at 5-40 ℃(optimal temperature: 25 ℃), at pH 4-11(optimal pH: 7.0), on a variety of media(optimal medium: oatmeal agar), and in the presence of diverse carbon and nitrogen sources(optimal carbon source: soluble starch; optimal nitrogen source: sodium nitrate). The lethal temperature was verified to be 51 ℃(10 min). The conclusion is expected to lay a scientific basis for diagnosis and control of R. glutinosa leaf diseases caused by F. acuminatum.

[Identification of miR160 family and their target genes in Rehmannia glutinosa in response to endophytic fungus GG22 infection].

This study aims to reveal the possible role of miR160 family in Rehmannia glutinosa in response to the infection of endophytic fungus Fusarium oxysporum GG22. Specifically, miR160 precursors and mature miR160 were retrieved from the small RNA database yielded by high-throughput sequencing. RNAfold was used to analyze the precursor structure, and DNAMAN and MEGA to analyze conservation and evolution of miR160 precursors and mature miR160. The target genes of miR160 were predicted and annotated, and the interaction was analyzed. Based on degradome sequencing, the target genes were further identified. The results showed that miR160 precursors had intact stem-loop structures. The precursor and mature sequences were conserved, particularly the 3 rd-16 th bases of the 5'-terminal. According to the phylogenetic tree, R. glutinosa had close evolutionary relationship with Arabidopsis thaliana, Oryza sativa, Salvia miltiorrhiza, and Sesamum indicum. A total of 22 target genes of miR160 were predicted and most of them were auxin response factor(ARF) genes. The target genes were involved in the Gene Ontology(GO) terms of biological processes, cellular components, and molecular functions. According to the degradome sequencing results, four target genes of miR160 were ARF(ARF18, ARF22) genes. R. glutinosa regulated its growth in response to the infection of endophytic fungus by changing the expression of miR160 and the target genes. qRT-PCR result of the differentially expressed rgl-miR160a and rgl-miR160a-3p was consistent with the sequencing result. This study clarifies the molecular mechanism of R. glutinosa in response to GG22 stress, laying a theoretical basis for the improvement and future research of R. glutinosa.