Integrating network pharmacology, molecular docking, and experimental validation to reveal the mechanism of Radix Rehmanniae in psoriasis.

BACKGROUND: Radix Rehmanniae (RR) plays an important role in treating psoriasis. However, the active compounds of RR and potential mechanisms are unclear. The current study was designed to investigate the potential active ingredients, targets, and mechanisms of RR in treating psoriasis through network pharmacology, molecular docking, and vitro experiments. METHODS: Initially, the TCMSP database and literature retrieval were used to access the active ingredients of RR. The psoriasis target proteins were obtained from Therapeutic Target Database, OMIM, GeneCards, and DrugBank databases. The target proteins were then converted into target genes using Uniprot. Secondly, overlapping genes were obtained through Venn online tool. Then, protein-protein interactions network diagram is finished by STRING database. Next, Cytoscape software was used to acquire the top 10 hub proteins; gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis were then used to predict possible mechanisms. Afterwards, molecular docking validation of the active ingredients with the main targets was performed by AutoDock software. Finally, lipopolysaccharides induced RAW264.7, to assess the effects and molecular mechanisms by MTT, RT-qPCR, and Western blot assays. RESULTS: Overall, there are 20 effective compounds and 33 targets involved in biological processes including apoptosis, intracellular signaling, vasodilation, and mitogen-activated protein kinase (MAPK) signaling cascade. The docking results showed strong binding capacity between the active ingredients and targets. We verified aucubin as the key active ingredient, tumor necrosis factor α, and IL6 as the core targets, and focused on the p38MAPK protein pathway. Cellular experiments showed that aucubin down-regulated the phosphorylated p38MAP protein and reduced the expression of tumor necrosis factor α mRNA, IL6 mRNA, and IL1ßmRNA. CONCLUSION: In summary, RR is featured with multicomponent, multi-target, and multi-pathway in treating psoriasis; the preliminary mechanism may be associated with the down-regulation of p38MAPK phosphorylation and curbing the expression of inflammatory factor by aucubin. This paper provides the scientific basis for Traditional Chinese medicine treating psoriasis.

Enhancing protective immunity against bacterial infection via coating nano-Rehmannia glutinosa polysaccharide with outer membrane vesicles.

With the coming of the post-antibiotic era, there is an increasingly urgent need for safe and efficient antibacterial vaccines. Bacterial outer membrane vesicles (OMVs) have received increased attention recently as a potential subunit vaccine. OMVs are non-replicative and contain the principle immunogenic bacterial antigen, which circumvents the safety concerns of live-attenuated vaccines. Here, we developed a novel nano-vaccine by coating OMVs onto PEGylated nano-Rehmannia glutinosa polysaccharide (pRL) in a structure consisting of concentric circles, resulting in a more stable vaccine with improved immunogenicity. The immunological function of the pRL-OMV formulation was evaluated in vivo and in vitro, and the underlying mechanism was studied though transcriptomic analysis. The pRL-OMV formulation significantly increased dendritic cell (DC) proliferation and cytokine secretion. Efficient phagocytosis of the formulation by DCs was accompanied by DC maturation. Further, the formulation demonstrated superior lymph node targeting, contributing to a potent mixed cellular response and bacterial-specific antibody response against Bordetella bronchiseptica infection. Specifically, transcriptomic analysis revealed that the immune protection function correlated with T-cell receptor signalling and Th1/Th2/Th17 differentiation, among other markers of enhanced immunological activity. These findings have implications for the future application of OMV-coated nano-carriers in antimicrobial immunotherapy.

Rehmanniae Radix Praeparata aqueous extract improves hepatic ischemia/reperfusion injury by restoring intracellular iron homeostasis.

Hepatic ischemia/reperfusion injury (HIRI) is a common pathophysiological condition occurring during or after liver resection and transplantation, leading to hepatic viability impairment and functional deterioration. Recently, ferroptosis, a newly recognized form of programmed cell death, has been implicated in IRI. Rehmanniae Radix Praeparata (RRP), extensively used in Chinese herbal medicine for its hepatoprotective, anti-inflammatory, and antioxidant properties, presents a potential therapeutic approach. However, the mechanisms by which RRP mitigates HIRI, particularly through the regulation of ferroptosis, remain unclear. In this study, we developed a HIRI mouse model and monocrotaline (MCT)- and erastin-induced in vitro hepatocyte injury models. We conducted whole-genome transcriptome analysis to elucidate the protective effects and mechanisms of RRP on HIRI. The RRP aqueous extract was characterized by the presence of acteoside, rehmannioside D, and 5-hydroxymethylfurfural. Our results demonstrate that the RRP aqueous extract ameliorated oxidative stress, reduced intracellular iron accumulation, and attenuated HIRI-induced liver damage. Additionally, RRP significantly inhibited hepatocyte death by restoring intracellular iron homeostasis both in vivo and in vitro. Mechanistically, the RRP aqueous extract reduced intrahepatocellular iron accumulation by inhibiting ZIP14-mediated iron uptake, promoting hepcidin- and ferroportin-mediated iron efflux, and ameliorating mitochondrial iron aggregation through upregulation of Cisd1 expression. Moreover, siRNA-mediated inhibition of hamp synergistically enhanced the RRP aqueous extract's inhibitory effect on ferroptosis. In conclusion, our study elucidates the mechanisms by which RRP aqueous extracts alleviate HIRI, highlighting the restoration of iron metabolic balance. These findings position RRP as a promising candidate for clinical intervention in HIRI treatment.

Radix rehmanniae praeparata extracts ameliorate hepatic ischemia-reperfusion injury by reversing LRP1-NOTCH1-C/EBPß axis-mediated senescence fate of LSECs.

BACKGROUND: Hepatic ischemia-reperfusion injury (HIRI) is commonly observed in cases of extensive hepatic resection and involves complex mechanisms. Cell senescence has been recognized as a factor in liver injury including HIRI, where it presents as a pro-inflammatory phenotype called senescence-associated secretory phenotype (SASP). Radix Rehmanniae Praeparata (RRP) is a commonly utilized traditional Chinese medicine known for its hepatoprotective, anti-aging and antioxidant qualities. Despite its recognized benefits, the specific mechanisms by which RRP may impede the progression of HIRI through the regulation of cell senescence and the identification of the most potent anti-aging extracts from RRP remain unclear. MATERIALS AND METHODS: Here, we first applied different chemical analysis methods to identify the RRP aqueous extract (RRPAE) and active fractions of RRP. Next, we constructed a surgically established mouse model and a hypoxia-reoxygenation (HR)-stimulated liver sinusoidal endothelial cells (LSECs) model to explore the underlying mechanism of RRP against HIRI through transcriptomics and multiple molecular biology experiments. RESULTS: After identifying active ingredients in RRP, we observed that RRP and its factions effectively restored LSECs fenestration and improved inflammation, cellular swelling and vascular continuity in the hepatic sinusoidal region during HIRI. Transcriptomic results revealed that RRP might reverse HIRI-induced senescence through the NOTCH signaling pathway and cell categorization further showed that the senescent cell population in HIRI liver was primarily LSECs rather than other cell types. Different RRPAE, especially RRP glucoside (RRPGLY), improved LSECs senescence and suppressed the expression of pro-inflammatory SASP genes either induced by HR insult or NOTCH1 activator, which was accompanied with the inhibition of LRP1-NOTCH1-C/EBPß pathways. Additionally, the specific inhibition of NOTCH1 by siRNA synergistically enhanced the hepatoprotective effect of RRPGLY. The ChIP-qPCR results further showed that C/EBPß was enriched at the promoter of a representative SASP, Il-1ß, in hypoxic LSECs but was significantly inhibited by RRPGLY. CONCLUSION: Our study not only clarified the potential mechanism of RRP active extractions in alleviating HIRI, but also highlighted RRPGLY was the main component of RRP that exerted anti-aging and anti-HIRI effects, providing a fresh perspective on the use of RRP to improve HIRI.

[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.

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.

Evaluating stability and bioactivity of Rehmannia-derived nanovesicles during storage.

Plant-derived nanovesicles (PDNVs) have garnered growing attention in the biomedical field owing to their abundance in plant-derived ribonucleic acids (RNA), proteins, lipids and metabolites. The question about the preservation of PDNVs is a crucial and unavoidable concern in both experiments' settings and their potential clinical application. The objective of this research was to examine the impact of varying storage temperatures on the stability and bioactivity of Rehmannia-derived nanovesicles (RDNVs). The results showed that RDNVs aggregated after 2 weeks of storage period at 4 °C, and the particle size of some RDNVs gradually increased with time, along with the increase of solution potential. After 2 months of storage, all RDNVs exhibited varying levels of aggregation irrespective of storage temperature. The bioactivities of nanovesicles under different temperature storage conditions revealed a gradual decline in cell proliferation inhibition bioactivity over time, significantly lower than that of freshly prepared RDNVs. In contrast, the preservation of anti-migratory activity in RDNVs was found to be more effective when subjected to rapid freezing in liquid nitrogen followed by storage at - 80 °C, as opposed to direct storage at - 80 °C. These findings suggest that temperature alone may not be sufficient in safeguarding the activity and stability of RDNVs, highlighting the necessity for the development of novel protective agents for PDNVs.

Radix Rehmanniae Praeparata extracts ameliorate hepatic ischemia-reperfusion injury by restoring lipid metabolism in hepatocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatic ischemia/reperfusion injury (HIRI) is a common occurrence during or after liver surgery, representing a major cause for postoperative complications or increased morbidity and mortality in liver diseases. Rehmanniae Radix Praeparata (RRP) is a traditional Chinese medicine frequently used and has garnered extensive attention for its therapeutic potential treating cardiovascular and hepatic ailments. Recent studies have indicated the possibility of RRP in regulating lipid accumulation and apoptosis in hepatocytes. AIM OF THE STUDY: This study aimed to investigate the specific mechanisms by which RRP may impede the progression of HIRI through the regulation of lipid metabolism. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC) was used to identify the major components of RRP water extract. C57BL/6J mice were orally given RRP at doses of 2.5 g/kg, 5 g/kg, and 10 g/kg for a duration of 7 days before undergoing HIRI surgery. Furthermore, we established a lipid-loaded in vitro model by exposing hepatocytes to oleic acid and palmitic acid (OAPA). The anti-HIRI effect of RRP was determined through transcriptomics and various molecular biology experiments. RESULTS: After identifying active ingredients in RRP, we observed that RRP exerted lipid-lowering and hepatoprotective effects on HIRI mice and OAPA-treated hepatocytes. RRP activated AMP-activated protein kinase (AMPK) and inhibited mammalian target of rapamycin (mTOR), which further on the one hand, inhibited the cleavage and activation of sterol regulatory element binding protein 2 (SREBP2) by limiting the movement of SREBPs cleavage-activating protein (SCAP)-SREBP2 complex with the help of endoplasmic reticulum lipid raft-associated protein 1 (ERLIN1) and insulin-induced gene 1 (INSIG1), and on the other hand, promoted liver X receptor α (LXRα) nuclear transportation and subsequent cholesterol efflux. Meanwhile, the anti-lipotoxic effect of RRP can be partly reversed by an LXRα inhibitor but largely blocked by the application of compound C, an AMPK inhibitor. CONCLUSION: Our study elucidated that RRP served as a potential AMPK activator to alleviate HIRI by blocking SREBP2 activation and cholesterol synthesis, while also activating LXRα to facilitate cholesterol efflux. These findings shed new light on the potential therapeutic use of RRP for improving HIRI.

[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.

Alleviated diabetic osteoporosis and peripheral neuropathic pain by Rehmannia glutinosa Libosch polysaccharide via increasing regulatory T cells.

Diabetic peripheral neuropathy (DPN) and diabetic osteoporosis (DOP) are conditions that significantly impact the quality of life of patients worldwide. Rehmanniae Radix Preparata, a component of traditional Chinese medicine with a history spanning thousands of years, has been utilized in the treatment of osteoporosis and diabetes. Specifically, Rehmannia glutinosa Libosch polysaccharide (RGP), a key bioactive compound of Rehmanniae Radix Preparata, has demonstrated immune-modulating properties and beneficial effects on hyperglycemia, hyperlipidemia, and vascular inflammation in diabetic mice. Despite these known actions, the precise mechanisms of RGP in addressing DOP and DPN remain unclear. Our study aimed to explore the impact of RGP on osteoporosis and peripheral neuropathic pain in diabetic mice induced by streptozotocin (STZ). The findings revealed that RGP not only improved hyperglycemia and osteoporosis in STZ-induced diabetic mice but also enhanced osteogenesis, insulin production, and nerve health. Specifically, RGP alleviated distal pain, improved nerve conduction velocity, nerve fiber integrity, and immune cell balance in the spleen. Mechanistically, RGP was found to upregulate HDAC6 mRNA expression in regulatory T cells, potentially shedding light on novel pathways for preventing DOP and DPN. These results offer promising insights for the development of new therapeutic approaches for diabetic complications.

Study on the mechanism of macrophages activated by phosphoesterified rehmanniae polysaccharide on human gastric cancer cells.

Gastric cancer(GC)is one of the most common gastrointestinal malignant tumors in the world, requiring the development of novel therapeutic agents with reduced toxicity. Rehmannia polysaccharide (RPS) possesses immunomodulatory and anti-tumor properties, yet its efficacy is suboptimal. To enhance its biological activity, we subjected RPS to molecular modifications, resulting in phosphorylated Rehmannia polysaccharides (P-RPS). Using the mixed phosphate method, we synthesized P-RPS and optimized the synthesis conditions through a combination of single-factor and response surface methodologies. In vitro studies on P-RPS's anti-tumor activity showed no direct influence on the viability of GC cells. However, P-RPS induced the transformation of PMA-activated THP-1 cells into the M1 phenotype. We collected conditioned medium (CM) of THP-1 cells to stimulate gastric cancer cells and CM-P-RPS significantly promoted apoptosis of gastric cancer cells and inhibited cell proliferation, and reduced cell migration. Mechanistically, CM-P-RPS inhibits the Wnt/ß-catenin signaling pathway through LGR6, leading to the suppression of tumor growth. Furthermore, P-RPS demonstrated a significant inhibitory effect on tumor growth in vivo, suggesting its potential as a promising therapeutic agent for GC treatment.

Catalpol from Rehmannia glutinosa Targets Nrf2/NF-κB Signaling Pathway to Improve Renal Anemia and Fibrosis.

Rehmannia glutinosa is widely recognized as a prominent medicinal herb employed by practitioners across various generations for the purpose of fortifying kidney yin. Within Rehmannia glutinosa, the compound known as catalpol (CAT) holds significant importance as a bioactive constituent. However, the protective effects of CAT on kidneys, including ameliorative effects on chronic kidney disease - most prominently renal anemia and renal fibrosis - have not been clearly defined. In this study, the kidney injury model of NRK-52E cells and C57BL/6N male mice was prepared by exposure to aristolochic acid I (AA-I), and it was discovered that CAT could ameliorate oxidative stress injury, inflammatory injury, apoptosis, renal anemia, renal fibrosis, and other renal injuries both in vivo and in vitro. Further treatment of NRK-52E cells with Nrf2 inhibitors (ML385) and activators (ML334), as well as NF-κB inhibitors (PDTC), validated CAT's ability to target Nrf2 activation. Furthermore, the expression of phosphorylated NF-κB p65, IL-6, and Cleaved-Caspase3 protein was inhibited. CAT also inhibited NF-κB, and then inhibited the expression of IL-6, p-STAS3, TGF-ß1 protein. Therefore, CAT can regulate Nrf2/NF-κB signaling pathway, significantly correct renal anemia and renal fibrosis, and is conducive to the preservation of renal structure and function, thus achieving a protective effect on the kidneys.

Silk fibroin microspheres loaded Rehmannia Liuwei extract for the protection of endothelial cells from the inhibitory effects.

Liuwei Dihuang (LWDH) is a multi-component and multi-target Chinese herbal compound widely used for treating chronic conditions such as diabetes, diabetic nephropathy, hypertension, osteoporosis, and chronic kidney disease. However, traditional Chinese medicine (TCM) preparations like decoction and pill face limitations, including low active component concentration, limited bioavailability, short half-life, and the need for high dosage, which may increase the burden on liver and kidney functions and reduce clinical efficacy. In this study, LWDH was further purified using D101 macroporous adsorption resin, resulting in a soluble extract with an active component content 53.6 times higher than that of LWDH itself. The freeze-dried LWDH extract was then encapsulated within silk fibroin (SF) microspheres to significantly enhance the sustained release performance of the drug. In a human umbilical vein endothelial cell (HUVEC) model cultured under high glucose conditions, methanol vapor-treated SF/LWDH microspheres demonstrated a decrease in the 24-hour drug release rate from 61.88 % to 34.81 %, augmenting their protective effect on endothelial cells.

The role of TrkB signaling-mediated synaptic plasticity in the antidepressant properties of catalpol, the main active compound of Rehmannia glutinosa Libosch.

ETHNOPHARMACOLOGICAL RELEVANCE: Rehmannia glutinosa Libosch. (RGL) is a famous ethnic medicine contained in antidepressant Chinese medicine formulas and is traditionally clinically used for depression. We have recently confirmed that RGL enhanced synaptic plasticity in a mouse model of Chinese medical syndrome and that catalpol may be the representatively pharmacological component responsible for its improvement in synaptic plasticity and treatment of depression. Impaired synaptic plasticity is closely linked to major depression. Tyrosine kinase receptor B (TrkB) signaling has recently been discovered as a key pathway for synaptic plasticity improvement and antidepressant discovery. However, to date, it is unknown whether the target of catalpol to improve synaptic plasticity involves TrkB and whether its antidepressant mechanism involves synaptic plasticity mediated by TrkB signaling. AIM OF STUDY: This study aims to elucidate the potential antidepressant target and mechanisms of catalpol, the main active compound of RGL, through TrkB signaling-mediated synaptic plasticity. MATERIALS AND METHODS: We have recently predicted through molecular networking strategy (including network pharmacology, molecular docking, and molecular dynamics simulation) that catalpol may exert its antidepressant effects by regulating TrkB signaling and thus modulating essential synaptic plasticity proteins. Then, this study used classic behavioral tests, targeted diagnostic reagents, Nissl and Golgi staining, immunohistochemical analysis, immunofluorescence analysis, Western blot, enzyme-linked immunosorbent assay, and Real-time PCR to confirm the potential target and signaling of catalpol to improve synaptic plasticity for the treatment of depression. RESULTS: The data showed that catalpol could improve synaptic plasticity and depressive behaviors, and its action pathway was predicted to involve TrkB signaling. Subsequently, the blockade of TrkB abolished the improvement of synaptic plasticity by catalpol and its antidepressant properties, which validated that TrkB signaling was the key pathway for catalpol to improve synaptic plasticity and exert antidepressant properties. Inhibition of COX-2 was likely to be a necessary facilitator for the antidepressant efficacy of catalpol via the TrkB target and TrkB-mediated synaptic plasticity. CONCLUSION: TrkB signaling-mediated synaptic plasticity plays a key role in the antidepressant properties of catalpol. This study provides critical information for the development of new and targeted antidepressant therapies or treatment strategies by catalpol. However, considering the existence of sex differences in depression (female depression is 2-3 times than that of males) and not exploring the antidepressant sex specificity of catalpol is a limitation, we will investigate the sex specificity of the antidepressant effects and molecular mechanisms of catalpol on sex-specific animals in the future to provide a preclinical basis for more accurate and targeted medication of catalpol.

Co-decoction of Lilii bulbus and Radix Rehmannia Recens and its key bioactive ingredient verbascoside inhibit neuroinflammation and intestinal permeability associated with chronic stress-induced depression via the gut microbiota-brain axis.

BACKGROUND: Gut microbiota plays a critical role in the pathogenesis of depression and are a therapeutic target via maintaining the homeostasis of the host through the gut microbiota-brain axis (GMBA). A co-decoction of Lilii bulbus and Radix Rehmannia Recens (LBRD), in which verbascoside is the key active ingredient, improves brain and gastrointestinal function in patients with depression. However, in depression treatment using verbascoside or LBRD, mechanisms underlying the bidirectional communication between the intestine and brain via the GMBA are still unclear. PURPOSE: This study aimed to examine the role of verbascoside in alleviating depression via gut-brain bidirectional communication and to study the possible pathways involved in the GMBA. METHODS: Key molecules and compounds involved in antidepressant action were identified using HPLC and transcriptomic analyses. The antidepressant effects of LBRD and verbascoside were observed in chronic stress induced depression model by behavioural test, neuronal morphology, and synaptic dendrite ultrastructure, and their neuroprotective function was measured in corticosterone (CORT)-stimulated nerve cell injury model. The causal link between the gut microbiota and the LBRD and verbascoside antidepressant efficacy was evaluate via gut microbiota composition analysis and faecal microbiota transplantation (FMT). RESULTS: LBRD and Verbascoside administration ameliorated depression-like behaviours and synaptic damage by reversing gut microbiota disturbance and inhibiting inflammatory responses as the result of impaired intestinal permeability or blood-brain barrier leakiness. Furthermore, verbascoside exerted neuroprotective effects against CORT-induced cytotoxicity in an in vitro depression model. FMT therapy indicated that verbascoside treatment attenuated gut inflammation and central nervous system inflammatory responses, as well as eliminated neurotransmitter and brain-gut peptide deficiencies in the prefrontal cortex by modulating the composition of gut microbiota. Lactobacillus, Parabacteroides, Bifidobacterium, and Ruminococcus might play key roles in the antidepressant effects of LBRD via the GMBA. CONCLUSION: The current study elucidates the multi-component, multi-target, and multi-pathway therapeutic effects of LBRD on depression by remodeling GMBA homeostasis and further verifies the causality between gut microbiota and the antidepressant effects of verbascoside and LBRD.

Radix Rehmanniae Praeparata promoted zebrafish fin regeneration through aryl hydrocarbon receptor-dependent autophagy.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Rehmanniae Radix Praeparata (RRP), a staple in traditional Chinese medicine, is derived from Rehmannia glutinosa Libosch and is renowned for its wound-healing properties. Despite its clinical prevalence, the molecular mechanisms underlying RRP's wound-healing effects have not been fully elucidated. AIM OF THE STUDY: This research endeavored to delineate the molecular and cellular mechanisms underlying the beneficial effects of RRP on wound healing, utilizing a zebrafish model. MATERIALS AND METHODS: Zebrafish larvae at 3 days post-fertilization were amputated at the fin and subsequently treated with RRP. The pro-wound healing and regenerative effects of RRP were evaluated through morphological analysis, assessment of cell proliferation and apoptosis, Additionally, mechanistic insights were gained through a comprehensive approach encompassing network pharmacology analysis, cell tracing, RNA-sequencing, CRISPR/Cas9 gene editing, and pharmacological inhibition. RESULTS: Our findings demonstrate that RRP significantly accelerates caudal fin regeneration in zebrafish following injury by suppressing cell apoptosis, promoting cell proliferation, and upregulating the expression of regenerative-related genes. Furthermore, RRP triggers autophagy signals during the regenerative process, which is attenuated by the autophagy inhibitor chloroquine (CQ). Notably, the administration of RRP enhances the expression of ahr1 and ahr2 in the regenerating fin. Genetic knockout of ahr1a, ahr1b, or ahr2 using CRISPR/Cas9, or pharmacological blockade of AHR signals with the antagonist CH-223191, diminishes the regenerative potential of RRP. Remarkably, zebrafish lacking ahr2 completely lose their fin regeneration ability. Additionally, inhibition of AHR signaling suppresses autophagy signaling during fin regeneration. CONCLUSIONS: This study uncovers that RRP stimulates fin regeneration in zebrafish by inducing AHR signals and, at least partially, activating the autophagy process. These findings provide novel insights into the molecular mechanisms underlying the wound-healing effects of RRP and may pave the way for the development of novel therapeutic strategies.

Preparation of Rehmanniae Radix Praeparata Polysaccharide Iron(III) Complex and Evaluation of Its Biological Activity.

This study extracted and purified a polysaccharide from Rehmanniae radix praeparata (RGP) with an average molecular weight. The structural characteristics of RGP and its iron (III) complex, RGP-Fe(III), were examined for their antioxidant properties and potential in treating iron deficiency anemia (IDA). Analysis revealed that RGP comprised Man, Rha, Gal, and Xyl, with a sugar residue skeleton featuring 1→3; 1→2, 3; and 1→2, 3, 4 linkages, among others. RGP-Fe(III) had a molecular weight of 4.39×104 Da. Notably, RGP-Fe(III) exhibited superior antioxidant activity compared to RGP alone. In IDA rat models, treatment with RGP-Fe(III) led to increased weight gain, restoration of key blood parameters including hemoglobin, red blood cells, and mean hemoglobin content, elevated serum iron levels, and decreased total iron-binding capacity. Histological examination revealed no observable toxic effects of RGP-Fe(III) on the liver and spleen. These findings suggest the potential of RGP-Fe(III) as a therapeutic agent for managing IDA and highlight its promising antioxidant properties.

Integrated Network Pharmacology and Experimental Validation Approach to Investigate the Mechanisms of Radix Rehmanniae Praeparata - Angelica Sinensis - Radix Achyranthis Bidentatae in Treating Knee Osteoarthritis.

Background: Knee osteoarthritis (KOA) is a persistent degenerative condition characterized by the deterioration of cartilage. The Chinese herbal formula Radix Rehmanniae Praeparata- Angelica Sinensis-Radix Achyranthis Bidentatae (RAR) has often been used in effective prescriptions for KOA as the main functional drug, but its underlying mechanism remains unclear. Therefore, network pharmacology and verification experiments were employed to investigate the impact and mode of action of RAR in the treatment of KOA. Methods: The destabilization of the medial meniscus model (DMM) was utilized to assess the anti-KOA effect of RAR by using gait analysis, micro-computed tomography (Micro-CT), and histology. Primary chondrocytes were extracted from the rib cartilage of a newborn mouse. The protective effects of RAR on OA cells were evaluated using a CCK-8 assay. The antioxidative effect of RAR was determined by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) production. Furthermore, network pharmacology and molecular docking were utilized to propose possible RAR targets for KOA, which were further verified through experiments. Results: In vivo, RAR significantly ameliorated DMM-induced KOA characteristics, such as subchondral bone sclerosis, cartilage deterioration, gait abnormalities, and the degree of knee swelling. In vitro, RAR stimulated chondrocyte proliferation and the expression of Col2a1, Comp, and Acan. Moreover, RAR treatment significantly reduced ROS accumulation in an OA cell model induced by IL-1ß and increased the activity of antioxidant enzymes (SOD and GSH). Network pharmacology analysis combined with molecular docking showed that Mapk1 might be a key therapeutic target. Subsequent research showed that RAR could downregulate Mapk1 mRNA levels in IL-1ß-induced chondrocytes and DMM-induced rats. Conclusion: RAR inhibited extracellular matrix (ECM) degradation and oxidative stress response via the MAPK signaling pathway in KOA, and Mapk1 may be a core target.

Two new iridoid glycosides from the whole plant of Rehmania piasezkii.

Two new iridoid glycosides, piasezkiiosides A (1) and B (2), were isolated from aqueous extract of the whole plant of Rehmannia piasezkii. Their structures were established from the spectroscopic data, chemical transformation, and X-ray diffraction analysis. Compound 1 exhibited weak hepatoprotective activity against APAP-induced HepG2 cell damage.

Five new eremophilane-type sesquiterpenes from the fresh roots of Rehmannia glutinosa.

Five undescribed eremophilane-type sesquiterpenes, remophilanetriols E-I (1-5), along with seven known compounds (6-12) were isolated from the fresh roots of Rehmannia glutinosa. Their structures were characterized by extensive spectroscopic data analysis and their absolute configurations were determined by comparing their calculated electronic circular dichroism (ECD) spectra and experimental ECD spectra. The anti-pulmonary fibrosis activities of all compounds were evaluated in vitro by MTT methods, and compounds 2, 8, 10, and 12 exhibited excellent anti-pulmonary fibrosis activities. In addition, compound 2 can reduce the levels of ROS and apoptosis in TGF-ß1-induced BEAS-2B cells.