Genome-Wide Identification of R2R3-MYB Transcription Factors: Discovery of a “Dual-Function” Regulator of Gypenoside and Flavonol Biosynthesis in Gynostemma pentaphyllum

The R2R3-MYB gene family participates in several plant physiological processes, especially the regulation of the biosynthesis of secondary metabolites. However, little is known about the functions of R2R3-MYB genes in Gynostemma pentaphyllum (G. pentaphyllum), a traditional Chinese medicinal herb that is an excellent source of gypenosides (a class of triterpenoid saponins) and flavonoids. In this study, a systematic genome-wide analysis of the R2R3-MYB gene family was performed using the recently sequenced G. pentaphyllum genome. In total, 87 R2R3-GpMYB genes were identified and subsequently divided into 32 subgroups based on phylogenetic analysis. The analysis was based on conserved exon–intron structures and motif compositions within the same subgroup. Collinearity analysis demonstrated that segmental duplication events were majorly responsible for the expansion of the R2R3-GpMYB gene family, and Ka/Ks analysis indicated that the majority of the duplicated R2R3-GpMYB genes underwent purifying selection. A combination of transcriptome analysis and quantitative reverse transcriptase-PCR (qRT-PCR) confirmed that Gynostemma pentaphyllum myeloblastosis 81 (GpMYB81) along with genes encoding gypenoside and flavonol biosynthetic enzymes exhibited similar expression patterns in different tissues and responses to methyl jasmonate (MeJA). Moreover, GpMYB81 could bind to the promoters of Gynostemma pentaphyllum farnesyl pyrophosphate synthase 1 (GpFPS1) and Gynostemma pentaphyllum chalcone synthase (GpCHS), the key structural genes of gypenoside and flavonol biosynthesis, respectively, and activate their expression. Altogether, this study highlights a novel transcriptional regulatory mechanism that suggests that GpMYB81 acts as a “dual-function” regulator of gypenoside and flavonol biosynthesis in G. pentaphyllum.

18β-Glycyrrhetinic Acid Inhibits TGF-β-Induced Epithelial-to-Mesenchymal Transition and Metastasis of Hepatocellular Carcinoma by Targeting STAT3

18[Formula: see text]-glycyrrhetinic acid (GA) is the active ingredient of the traditional Chinese medicinal herb Glycyrrhizae radix et rhizoma. We previously demonstrated that GA inhibited tumor growth in hepatocellular carcinoma (HCC). However, the effect of GA on transforming growth factor-[Formula: see text] (TGF-[Formula: see text]-induced epithelial-mesenchymal transition (EMT) and metastasis were still unclear. In this study, in vitro transwell assays and immunofluorescence (IF) demonstrated that GA inhibited TGF-[Formula: see text]-induced migration, invasion and EMT of HCC cells. However, it had little effect on the inhibition of proliferation by TGF-[Formula: see text]. Moreover, we confirmed that GA suppressed the metastasis of HCC cells in vivousing an ectopic lung metastasis model. Furthermore, we found that GA inhibited TGF-[Formula: see text]-induced EMT mainly by reducing the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which played an essential role in TGF-[Formula: see text]-induced EMT and cell mobility. Mechanistically, GA inhibited the phosphorylation of STAT3 by increasing the expression of Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 (SHP1 and SHP2). Therefore, we concluded that GA inhibited TGF-[Formula: see text]-induced EMT and metastasis via the SHP1&SHP2/STAT3/Snail pathway. Our data provide an attractive therapeutic target for future multimodal management of HCC.

Astragalus mongholicus Bunge Water Extract Exhibits Anti-inflammatory Effects in Human Neutrophils and Alleviates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice

Neutrophils are the primary immune cells in innate immunity, which are related to various inflammatory diseases. Astragalus mongholicus Bunge is a Chinese medicinal herb used to treat various oxidative stress-related inflammatory diseases. However, there are limited studies that elucidate the effects of Astragalus mongholicus Bunge in human neutrophils. In this study, we used isolated human neutrophils activated by various stimulants to investigate the anti-inflammatory effects of Astragalus mongholicus Bunge water extract (AWE). Cell-free assays were used to examine free radicals scavenging capabilities on superoxide anion, reactive oxygen species (ROS), and nitrogen-centered radicals. Imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model was used for investigating anti-psoriatic effects. We found that AWE inhibited superoxide anion production, ROS generation, and elastase release in human neutrophils, which exhibiting a direct anti-neutrophil effect. Moreover, AWE exerted a ROS scavenging ability in the 2,2’-Azobis (2-amidinopropane) dihydrochloride assay, but not superoxide anion in the xanthine/xanthine oxidase assay, suggesting that AWE exhibited anti-oxidation and anti-inflammatory capabilities by both scavenging ROS and by directly inhibiting neutrophil activation. AWE also reduced CD11b expression and adhesion to endothelial cells in activated human neutrophils. Meanwhile, in mice with psoriasis-like skin inflammation, administration of topical AWE reduced both the affected area and the severity index score. It inhibited neutrophil infiltration, myeloperoxidase release, ROS-induced damage, and skin proliferation. In summary, AWE exhibited direct anti-inflammatory effects by inhibiting neutrophil activation and anti-psoriatic effects in mice with IMQ-induced psoriasis-like skin inflammation. Therefore, AWE could potentially be a pharmaceutical Chinese herbal medicine to inhibit neutrophilic inflammation for anti-psoriasis.

Cloning, prokaryotic expression, and purification of acetyl-CoA C-acetyltransferase from Atractylodes lancea

Background: Cangzhu (Atractylodes lancea), a valuable and common traditional Chinese medicinal herb, is primarily used as an effective medicine with various health-promoting effects. The main pharmacological bioactive ingredients in the rhizome of A. lancea are terpenoids. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpenoid synthesis pathway and catalyzes two units of acetyl-CoA into acetoacetyl-CoA. Objective: The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea. Method: A full-length cDNA clone of AlAACT was isolated using PCR and expressed in Escherichia coli. The expressed protein was purified using Ni-NTA agarose column using standard protocols. AlAACT was transiently expressed in N. benthamiana leaves to determine their subcellular location. The difference in growth between recombinant bacteria and control bacteria under different stresses was observed using the droplet plate experiment. Result: In this study, a full-length cDNA of AACT (AlAACT) was cloned from A. lancea, which contains a 1,227 bp open reading frame and encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that AlAACT shared high similarity with AACTs from other plants. The recombinant protein pET32a(+)/AlAACT was successfully expressed in Escherichia coli BL21(DE3) cells induced with 0.4 mM IPTG at 30°C as the optimized condition. The recombinant enzyme pET-32a-AlAACT was purified using the Ni-NTA column based on the His-tag, and the molecular weight was determined to be 62 kDa through SDS-PAGE and Western Blot analysis. The recombinant protein was eluted with 100, 300, and 500 mM imidazole; most of the protein was eluted with 300 mM imidazole. Under mannitol stress, the recombinant pET-32a-AlAACT protein showed a substantial advantage in terms of growth rates compared to the control. However, this phenomenon was directly opposite under NaCl abiotic stress. Subcellular localization showed that AlAACT localizes to the nucleus and cytoplasm. Conclusion: The expression and purification of recombinant enzyme pET-32a-AlAACT were successful, and the recombinant strain pET-32a-AlAACT in showed better growth in a drought stress. The expression of AlAACT-EGFP fusion protein revealed its localization in both nuclear and cytoplasm compartments. This study provides an important foundation for further research into the effects of terpenoid biosynthesis in A. lancea.

Tissue and Growth Specific Chemical Profiles and Antimicrobial Activity of Libanotis buchtormensis (Fisch.) DC Extracts

Libanotis buchtormensis (Fisch.) DC. is a traditional Chinese medicinal herb used for the treatment of rheumatism, articular pain, and symptoms of the common cold. To evaluate the possible medicinal value of the aerial parts of L. buchtormensis, the chemical composition and antibacterial activity were assessed on a tissue and growth specific basis. Results based on high performance liquid chromatography (HPLC) showed that the samples were divided into two main clusters, one associated with roots and the other associated with the aerial parts. Analysis of the bioactive chemical contents revealed that coumarin was the most abundant compound in leaves (0.53 to 0.82%), while osthole and isoimperatorin exhibited relatively high concentrations in roots. Two-year growth samples, which correspond with the harvest period of L. buchtormensis, contained the highest amounts of total coumarin, osthole, and isoimperatorin. Broth microdilution tests showed that Gram-positive bacterial strains ( Staphylococcus aureus, S. epidermidis and Streptococcus agalactiae) were more sensitive to L. buchtormensis extracts than the Gram-negative bacterial strain ( Escherichia coli). Staphylococcus aureus and S. epidermidis were more sensitive to the extracts, exhibiting significant correlation with osthole and isoimperatorin contents. Comparative analysis of the chemical profiles and antibacterial activity revealed significant differences between the root and the aerial parts, which implied that the clinical application of the aerial parts needed further study. In addition, the aerial parts could serve as a material source for osthole, having comparable content to that of the root.

In Vitro Gingival Wound Healing Activity of Extracts from Reynoutria japonica Houtt Rhizomes

Rhizomes of Reynoutria japonica Houtt. are a traditional Chinese medicinal herb (Polygoni cuspidati rhizoma, hu zhang) used for treatment of numerous diseases including wound healing support. The aim of this study was to provide evidence for the value of this herbal drug’s traditional use as a gingival healing treatment as well as to obtain the most active extract. In vitro studies were performed using primary human gingival fibroblasts (HGFs) with determination of viability (MTT assay), cell proliferation (the confocal laser scanning microscope (CLSM) was used to visualize histone 3 expression), cell migration (wound healing assay), and evaluation of the expression of collagen type III (immunocytochemical staining) after incubation with extracts from R. japonica rhizomes (25% or 40% ethanol or 60% acetone). In addition to these extracts, commercial dental rinse (containing chlorhexidine digluconate 0.2%) was tested as the gold standard of choice for gum healing in dental practice. The studied extracts were qualitatively and quantitatively characterized using the validated HPLC/DAD/ESI-HR-QTOF-MS method. Total phenols and tannins content were determined using the Folin–Ciocalteu assay. Low concentration of all extracts after 24 h incubation caused significant increase in HGF viability. This effect was most pronounced at a concentration of 50 µg/mL, which was selected for further experiments. All extracts (at 50 µg/mL) stimulated HGF to proliferate, migrate, and increase collagen III synthesis, but with different strength. The highest stimulated proliferation and migration activity was observed after incubation with 25% EtOH, which according to phytochemical analysis may be related to the highest content of resveratrol and an appropriate composition of procyanidins. The 25% EtOH extract from R. japonica rhizomes appears to be a promising gingival wound healing agent worthy of animal and clinical trials.

Saikosaponin D Attenuates Pancreatic Injury Through Suppressing the Apoptosis of Acinar Cell via Modulation of the MAPK Signaling Pathway

Chronic pancreatitis (CP) is a progressive fibro-inflammatory syndrome. The damage of acinar cells is the main cause of inflammation and the activation of pancreatic stellate cells (PSCs), which can thereby possibly further aggravate the apoptosis of more acinar cells. Saikosaponind (SSd), a major active ingredient derived from Chinese medicinal herb bupleurum falcatum, which exerted multiple pharmacological effects. However, it is not clear whether SSd protects pancreatic injury of CP via regulating the apoptosis of pancreatic acinar cells. This study systematically investigated the effect of SSd on pancreatic injury of CP in vivo and in vitro. The results revealed that SSd attenuate pancreatic damage, decrease the apoptosis and suppress the phosphorylation level of MAPK family proteins (JNK1/2, ERK1/2, and p38 MAPK) significantly in the pancreas of CP rats. In addition, SSd markedly reduced the apoptosis and inflammation of pancreatic acinar AR42J cells induced by cerulein, a drug induced CP, or Conditioned Medium from PSCs (PSCs-CM) or the combination of PSCs-CM and cerulein. Moreover, SSd significantly inhibited the activated phosphorylation of JNK1/2, ERK1/2, and p38 MAPK induced by cerulein or the combination of PSCs-CM and cerulein in AR42J cells. Furthermore, SSd treatment markedly decreased the protein levels of p-JNK and p-p38 MAPK caused by PSCs-CM alone. In conclusion, SSd ameliorated pancreatic injury, suppressed AR42J inflammation and apoptosis induced by cerulein, interrupted the effect of PSCs-CM on AR42J cells inflammation and apoptosis, possibly through MAPK pathway.

Metabolomics Reveals the Mechanisms for the Pulmonary Toxicity of Siegesbeckia orientalis L. and the Toxicity-Reducing Effect of Processing

Siegesbeckia orientalis L. (SO) is a commonly used Chinese medicinal herb. It has long been used as a remedy in traditional Chinese medicine (TCM) for symptoms that resemble inflammatory joint disorders. However, it is slightly toxic. According to the TCM theory, processing can reduce the toxicity of the herbs. Here, we performed metabolomics to determine whether processing with rice wine reduces the toxicity of raw SO, and to explore the mechanisms underlying the raw SO–induced toxicity and the toxicity-reducing effect of processing. Our results showed that raw SO has long-term toxicity in rats. It significantly elevated the serum level of LDH and caused histopathological damages in the lung tissues. It is worth noting that the LDH level in the PSO group was lower than that in the raw SO group, and the damages in lung tissues were relatively mild in PSO-treated rats, suggesting that processing reduces the pulmonary toxicity of the raw. Moreover, a total of 32 significantly changed metabolites were identified. Based on the MetaboAnalyst pathway analysis, we found that two characteristic metabolic pathways including alanine, aspartate and glutamate metabolism and glycerophospholipid metabolism were only changed in the raw SO group, while histidine metabolism was only changed in the PSO group, which suggests that induction of oxidative stress contributes to raw SO–induced pulmonary toxicity, and free radical scavenging might be responsible for the toxicity-reducing effect of processing. Our data shed new light on how raw SO induces pulmonary toxicity and how the toxicity can be reduced by processing. This study not only provides scientific justifications for the traditional processing theory of SO, but also helps to optimize the processing protocol and the clinical drug combination of SO.

Effect of Huangqin Decoction on Improving the Mouse Model of Ulcerative Colitis by Inhibiting NF-κB p65 Signal Pathway

Natural medicine, such as traditional Chinese medicine, plays a role in treating ulcerative colitis (UC) because of its action on multiple targets. Here we have improved the effectiveness of a traditional Chinese medicinal herb, Huangqin decoction (HQD), on UC via the nuclear factor-κB (NF-κB) p65 signaling pathway in a mouse model. NF-κB is a crucial regulator of inflammation, cancer, and autoimmunity. It may be related to the initiation and development of UC. Histopathological changes in colon show that Huangqin Decoction can improve colon tissue environment In addition, the Western blots of the inflammatory cytokines related to the NF-κB p65 pathway suggest that HQD inhibits the development of UC by regulating the NF-κB p65 signaling pathway. These findings provide a basis for the clinical application of HQD and suggest a new strategy for treating UC in the future.