A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42-mediated GSK-3ß/ß-catenin Signaling.

Kidney fibrosis is a common fate of chronic kidney diseases (CKDs), eventually leading to renal dysfunction. Yet, no effective treatment for this pathological process has been achieved. During the bioassay-guided chemical investigation of the medicinal plant Wikstroemia chamaedaphne, a daphne diterpenoid, daphnepedunin A (DA), is characterized as a promising anti-renal fibrotic lead. DA shows significant anti-kidney fibrosis effects in cultured renal fibroblasts and unilateral ureteral obstructed mice, being more potent than the clinical trial drug pirfenidone. Leveraging the thermal proteome profiling strategy, cell division cycle 42 (Cdc42) is identified as the direct target of DA. Mechanistically, DA targets to reduce Cdc42 activity and down-regulates its downstream phospho-protein kinase Cζ(p-PKCζ)/phospho-glycogen synthase kinase-3ß (p-GSK-3ß), thereby promoting ß-catenin Ser33/37/Thr41 phosphorylation and ubiquitin-dependent proteolysis to block classical pro-fibrotic ß-catenin signaling. These findings suggest that Cdc42 is a promising therapeutic target for kidney fibrosis, and highlight DA as a potent Cdc42 inhibitor for combating CKDs.

Study of the mechanism underlying the anti-inflammatory effect of Miao medicine comprising raw and processed Radix Wikstroemia indica using the "sweat soaking method".

ETHNOPHARMACOLOGICAL RELEVANCE: To explore the differences in the anti-inflammatory efficacy and mechanisms of the Miao medicine, both raw and after processing, using the "sweat soaking method" of Radix Wikstroemia indica (RWI). AIM OF THE STUDY: The purpose of this study was to explore the differences in the anti-inflammatory efficacy and mechanism of action before and after the processing of the Miao medicine (RWI) using the "sweat soaking method." MATERIALS AND METHODS: Network pharmacology technology was used to construct the "drug-component target-pathway-disease" network, and the main anti-inflammatory pathways of RWI were identified. Rat models of collagen-induced arthritis were established. The changes in body weight, swelling rate of the foot pad and ankle joint, arthritis index, thymus index, spleen index, pathological changes of the ankle joint, and the content of inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-10, TNF-α, and NO) were used as indices to evaluate the effect of RWI on rats with collagen-induced arthritis before and after its processing. Plasma and urine samples were collected from the rats, and the potential biomarkers of, and metabolic pathways underlying the anti-inflammatory effects of RWI before and after processing were identified using 1H-Nuclear magnetic resonance metabolomics combined with a multivariate statistical analysis. RESULTS: Eleven key anti-inflammatory targets of IL6, IL-1ß, TNF, ALB, AKT1, IFNG, INS, STAT3, EGFR, TP53, and SRC were identified by network pharmacology. The PI3K-Akt signaling pathway, steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, tryptophan metabolism, and other pathways were mainly involved in these effects. Pharmacodynamic studies found that both raw and processed RWI products downregulated inflammatory factors in rats with collagen-induced arthritis and alleviated the pathological changes. A total of 41 potential pathways for the anti-inflammatory effects of raw RWI products and 36 potential pathways for the anti-inflammatory effects of processed RWI products were identified by plasma and urine metabolomics. The common pathways of network pharmacology and metabolomics were steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. CONCLUSIONS: The anti-inflammatory effect of RWI was mainly related to the regulation of steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. Finally, the "sweat soaking method" enhanced the anti-inflammatory effect of RWI.

Chemical constituents of Wikstroemia alternifolia and their neuroprotective activities.

Phytochemical investigation on the plant of Wikstroemia alternifolia led to the isolation of 26 compounds including two new ones, wikstralternifols A and B (1 and 7). Their structures including the absolute configuration were elucidated by spectroscopic data together with analysis of experimental and calculated ECD data. All compounds were isolated from this plant for the first time, and their main structural types were lignans, sesquiterpenoids, and flavonoids. In the sodium nitroprusside-induced rat pheochromocytoma PC-12 cell model, the neuroprotective activities of the selected sesquiterpenoids (1 and 4) and lignans (7 - 14) were screened at the concentration of 10 µM, and 7 - 14 displayed better activities than the positive control edaravone.

A High-Performance Liquid Chromatography with Photodiode Array Detection Method for Simultaneous Determination of Three Compounds Isolated from Wikstroemia ganpi: Assessment of the Effects on Cytochrome P450-Mediated Metabolism In Vitro and In Vivo.

In natural products, the content and quality of the marker components differ depending on the part, production area, collection period, and extraction method; therefore, a standardized analysis method is required to obtain consistent results. This study developed a simultaneous analysis method for three marker components (7-methoxylutolin-5-O-glucoseide, pilloin 5-O-ß-d-glucopyranoside, rutarensin) isolated and purified from Wikstroemia ganpi (W. ganpi). Simultaneous analysis was performed using high-performance liquid chromatography with photodiode array detection (HPLC-PDA) method that was validated according to the International Council for Harmonisation (ICH) guidelines. The developed analytical method exhibited linearity (r2 > 0.999), detection limits (0.72-3.34 µg/mL), and quantification limits (2.19-10.22 µg/mL). The relative standard deviation (RSD) value of intra- and inter-day precisions was less than 1.68%, and analyte recoveries (93.42-117.55%; RSD < 1.86%) were validated according to the analytical procedures, and all parameters were within the allowable range. Quantitative analysis of the three marker components from W. ganpi MeOH extract (WGM) showed 7-methoxylutolin-5-O-glucoseide with the highest content (51.81 mg/g). The inhibitory effects of WGM on cytochrome P450 (CYP) substrate drugs were further investigated. The in vitro study revealed that WGM inhibited the CYP3A-mediated metabolism of buspirone and that 7-methoxylutolin-5-O-glucoseide and pilloin 5-O-ß-d-glucopyranoside inhibited the metabolism of buspirone with IC50 values of 2.73 and 18.7 µM, respectively. However, a single oral dose of WGM did not have significant effects on the pharmacokinetics of buspirone in rats, suggesting that WGM cannot function as an inhibitor of CYP3A-mediated metabolism in vivo.

Potential inhibitors of microglial activation from the roots of Wikstroemia lichiangensis W. W. Sm.

Research on natural inhibitors of microglial overactivation derived from members of the Wikstroemia genus revealed that the extract of W. lichiangensis W. W. Sm. Has a remarkable inhibitory effect on nitric oxide production in overactivated microglia. In the present study, thirty-four compounds, including five undescribed sesquiterpenoids [wiksdauctins A-B (1-2) and wikscarotins A-C (3-5)] and one undescribed lignan [wikstroeminasin A (8)], were isolated from a 95% EtOH extract of W. lichiangensis roots using bio-guided phytochemical research. The structures of the isolated compounds were elucidated using comprehensive spectroscopic analyses. Furthermore, their anti-neuroinflammatory effects were evaluated in lipopolysaccharide-stimulated BV-2 microglia. Seventeen isolated compounds exhibited stronger inhibitory effects than positive control minocycline (IC50 values of 67.08 ± 1.95 µM), with IC50 values ranging from 7.35 ± 2.51 to 64.49 ± 3.38 µM. The findings of this study imply that the isolated compounds might serve as potential therapeutic agents for neurodegenerative diseases.

Potential HIV latency-reversing agents with STAT1-activating activity from the leaves of Wikstroemia chamaedaphne.

Developing highly effective HIV latency-reversing agent is an inportmant approach for the treatment of AIDS via the "shock and kill" of latent HIV. In this study, two unreported modified daphnane-type diterpenes (chamaedaphnelide A and epi-chamaedaphnelide A) and one unreported tigliane-type diterpene (chamaedaphnelide B), along with four known daphnane-type diterpenes and one known tigliane-type diterpene were obtained from the leaves of Wikstroemia chamaedaphne. Chamaedaphnelide A and epi-chamaedaphnelide A represents the first A ring cleavage daphnane-type backbone. Chamaedaphnelide A, epi-chamaedaphnelide A, chamaedaphnelide B, and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate showed HIV latency-reversing activity, especially chamaedaphnelide B and 6α,7α-epoxy-5ß-hydroxy-12-deoxyphorbol-13-decanoate displayed equally potential to positive drugs prostratin with reversing latent HIV on more than 100-fold compared to unstimulated cells. Furthermore, the activation of STAT1 was involved in the HIV latency-reversing activity of these diterpenes, firstly demonstrating that daphnane- and tigliane-type diterpenes can rapidly activate STAT1 activity. Indeed, these results also supported that activating STAT1 activity is a pathway for reversing latent HIV.

Anti-HIV Tigliane-Type Diterpenoids from the Aerial Parts of Wikstroemia lichiangensis.

Tigliane-type diterpenoids have attracted much attention in drug discovery since they have been reported to exhibit remarkable biological effects, such as tumor-promoting, antineoplastic, and anti-HIV activities. In continuing our efforts to discover novel biologically important diterpenoids from Wikstroemia species, Wikstroemia lichiangensis was investigated phytochemically for the first time. As a result, four new (1-4) and one known (5) tigliane-type diterpenoid were isolated, and their structures were elucidated by spectroscopic data analysis. Tiglianes (1-5) showed potent anti-HIV activity against HIV-1 infection of MT4 lymphocytes with IC50 values of 1.1-65.4 nM.

Retusone A, a Guaiane-Type Sesquiterpene Dimer from Wikstroemia retusa and Its Inhibitory Effects on Histone Acetyltransferase HBO1 Expression.

Retusone A (1), a new sesquiterpene dimer consisting of two guaiane-type sesquiterpenoids, and oleodaphnal (2) were isolated from heartwood of Wikstroemia retusa (Thymelaeaceae). The planar structure of 1 was elucidated on the basis of HRESIMS and NMR spectroscopic data, and the relative stereochemistry was established by X-ray diffraction analysis. The absolute configuration of 1 was determined by electronic circular dichroism. Compound 1 suppressed luciferase reporter gene expression driven by the HBO1 (histone acetyltransferase binding to ORC1) gene promoter in human breast cancer MCF7 cells. Compound 1 also decreased the expression of endogenous HBO1 mRNA and protein, and inhibited proliferation of the cells. These results suggest that retusone A (1), which has a unique dimeric sesquiterpenoid structure with inhibitory activity against HBO1 expression, may contribute to the development of a novel therapeutic candidate for the treatment of breast cancer.

A feruloylated acylglycerol isolated from Wikstroemia pilosa and its distribution in ten plants of Wikstroemia species.

Feruloylated acylglycerols (FAGs) have recently garnered a lot of interest as water-oil-miscible ferulic acid derivatives. A novel FAG derivate, 1-feruloyl-3-hexadecanoylglycerol (1), was isolated from Wikstroemia pilosa and its structure was elucidated from extensive physiochemical and spectroscopic analysis. Since the limited distribution of FAGs in plant species has been reported, a high-resolution accurate mass (HRAM) LC-MS quantitative analysis was carried out to determine the contents of 1 in ten plants of Wikstroemia species. As a result, compound 1 was detected in all species at contents of 1.29-50.96 mg/kg dry weight and W. dolichantha contained 1 at the highest content.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Three new compounds from the dried root bark of Wikstroemia indica and their cytotoxicity against HeLa cells.

Three new compounds (1-3) and twelve known compounds (4-15) were isolated from the n-butanol extraction of the 70% ethanol extract of the dried root barks of Wikstroemia indica. The structures of new compounds were identified by chemical evidence and extensive analysis of spectroscopic data (HR-ESI-MS, 1 D and 2 D NMR) and ECD calculations. All isolated compounds were evaluated for their cytotoxicity against HeLa cells using MTT assay and the results showed that daphnoretin (compound 5) owned the highest cytotoxicity against HeLa cells (IC50=28.89 µmol/L).

Inhibitory Effects of Luteolin 7-Methyl Ether Isolated from Wikstroemia ganpi on Tnf-A/Ifn-Γ Mixture-Induced Inflammation in Human Keratinocyte.

Plants of the genus Wikstroemia are traditionally used in China to treat various inflammatory diseases. The purpose of this study was to isolate the components of Wikstroemia ganpi (Siebold & Zucc.) Maxim., to evaluate their anti-atopic activities and to identify candidates with anti-atopic therapeutics. A total of 24 compounds were isolated by bioassay-guided separation, including one novel compound, which was tilianin 5-methyl ether. The anti-atopic activities of the isolated compounds were determined using TNF-α-treated RBL-2H3 cells and HaCaT cells. The mRNA expressions of IL-4, IL-6, GM-CSF, G-CSF and TRPV1 were reduced by luteolin 7-methyl ether. The study shows that the luteolin 7-methyl ether isolated from W. ganpi is a potential therapeutic agent for the treatment of atopic dermatitis.

Diarylpentanones from the root of Wikstroemia indica and their cytotoxic activity against human lung A549 cells.

One new diarylpentanone, 4(S)-hydroxy-1, 5-diphenyl -2(E)-en-1-pentanone (1), two diarylpentanones isolated from Wikstroemia indica for the first time (3 and 5) and two other known diarylpentanones were isolated from petroleum ether fraction of root of Wikstroemia indica. The structure of the new compound including absolute configuration was elucidated by extensive spectroscopic techniques, including 1D, 2D NMR, HR-ESI-MS and ECD spectroscopy. All isolated compounds were tested for their cytotoxic activity against cancer-derived cell lines A549. Compound 5 exhibited remarkable cytotoxic activity comparable to that of positive control cisplatin.

LC-MS Identification, Isolation, and Structural Elucidation of Anti-HIV Tigliane Diterpenoids from Wikstroemia lamatsoensis.

Structurally diverse tigliane diterpenoids have drawn significant research interest for drug discovery over many decades. Using LC-MS-guided fractionation and separation, the first phytochemical investigation on Wikstroemia lamatsoensis led to the isolation of eight tiglianes (1-8), including two new compounds, wikstrocin D (1) and wikstrocin E (2). The new structures were elucidated based on extensive physicochemical and spectroscopic analyses. The characteristic ESIMS/MS fragmentations of tiglianes 1-8 were also summarized. Among the isolated tiglianes, three compounds (8, 5, and 7) showed the most potent anti-HIV activity, with IC50 values of 0.18, 3.8, and 12.8 nM, respectively.

Identification of anti-HIV macrocyclic daphnane orthoesters from Wikstroemia ligustrina by LC-MS analysis and phytochemical investigation.

Macrocyclic daphnane orthoesters (MDOs) have attracted significant research interest for the drug discovery to cure HIV infection based on the "Shock and Kill" strategy. In the present study, the first chemical study on Wikstroemia ligustrina (Thymelaeaceae) was carried out by LC-MS analysis and phytochemical investigation. Nine daphnane diterpenoids (1-9) including seven MDOs were detected by LC-MS analysis. Further phytochemical investigation resulted in the isolation and structural elucidation of five daphnanes (1, 2, 5, 8, and 9) with potent anti-HIV activity. Taking the isolated MDO (1) as a model compound, the MS/MS fragmentation pathway was also elucidated.

Complete plastomes of six species of Wikstroemia (Thymelaeaceae) reveal paraphyly with the monotypic genus Stellera.

Wikstroemia (Thymelaeaceae) is a diverse genus that extends from Asia to Australia and has been recorded on the Hawaiian Islands. Despite its medicinal properties and resource utilization in pulp production, genetic studies of the species in this important genus have been neglected. In this study, the plastome sequences of six species of Wikstroemia were sequenced and analysed. The plastomes ranged in size between 172,610 bp (W. micrantha) and 173,697 bp (W. alternifolia) and exhibited a typical genome structure consisting of a pair of inverted repeat (IR) regions separated by a large single-copy (LSC) region and a small single-copy (SSC) region. The six plastomes were similar in the 138 or 139 genes predicted, which consisted of 92 or 93 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The overall GC contents were identical (36.7%). Comparative genomic analyses were conducted with the inclusion of two additional published species of Wikstroemia in which the sequence divergence and expansion of IRs in the plastomes were determined. When compared to the coding sequences (CDSs) of Aquilaria sinensis, five genes, namely, rpl2, rps7, rps18, ycf1 and ycf2, indicated positive selection in W. capitata. The plastome-based phylogenetic analysis inferred that Wikstroemia in its current state is paraphyletic to Stellera chamaejasme, while the ITS-based tree analyses could not properly resolve the phylogenetic relationship between Stellera and Wikstroemia. This finding rekindled interest in the proposal to synonymize Stellera with Wikstroemia, which was previously proposed but rejected due to taxonomic conflicts. Nevertheless, this study provides valuable genomic information to aid in the taxonomic implications and phylogenomic reconstruction of Thymelaeaceae.

Comprehensive investigation of pharmacodyamic material basis of Wikstroemia indica (L.) C. A. Mey. by serum pharmacochemistry and bivariate correlation analysis.

In this study, the theory of serum pharmacochemistry of traditional Chinese medicine was used to analyze the constituents absorbed into serum after oral administration of Wikstroemia indica (L.) C. A. Mey. by ultra high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The micro-liquid dilution method was used to determine the minimum inhibitory concentration of the serum containing Wikstroemia indica. The bivariate correlation analysis method was used to study the spectral-efficiency relationship between the drug-containing serum and the antibacterial activity, and find the main antibacterial active components in serum containing Wikstroemia indica. A total of 26 serum migration components were identified or speculated in the samples, including 11 prototype components and 15 metabolites. Of which, syringic acid, caffeic acid, dihydrocaffeic acid, 4-hydroxybenzoic acid, hippuric acid, 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid, triumbelletin, (7R)-3-hydroxy-1-methyl-2-oxo-7-(prop-1-en-2-yl)-2,3,5,6,7,8- hexahydroazulene-4- carbaldehyde and (1S,3aS,8aS)-1,3,5-trihydroxy-1,4-dimethyl-7-(propan-2- ylidene) octahydroazulen-6(1H)-one were bacteriostatic active substances. It is the first time to study the constituents in serum containing Wikstroemia indica and reveal its antibacterial pharmacodyamic material basis. The above works provide scientific reference for the in-depth study of Wikstroemia indica.

Discovering the Mechanisms of Wikstroelide E as a Potential HIV-Latency-Reversing Agent by Transcriptome Profiling.

The discovery of efficient and specific HIV-latency-reversing agents is critical for HIV therapy. Here, we developed wikstroelide E, a daphnane diterpene from the buds of Wikstroemia chamaedaphne, as a potential HIV-latency-reversing agent that is 2500-fold more potent than the drug prostratin. Based on transcriptome analysis, the underlying mechanism was that wikstroelide E regulated the MAPK, PI3K-Akt, JAK-Stat, TNF, and NF-κB signaling pathways. We clearly demonstrated that wikstroelide E reversed latent HIV infection by activating PKC-NF-κB signals, serving as a proxy for verifying the transcriptome data. Strikingly, the Tat protein contributes to the robust activation of latent HIV in wikstroelide-E-treated cells, producing an unexpected latency-reversing effect against latent HIV. This study provides the basis for the potential development of wikstroelide E as an effective HIV-latency-reversing agent.

Wikstromol from Wikstroemia indica induces apoptosis and suppresses migration of MDA-MB-231 cells via inhibiting PI3K/Akt pathway.

Triple negative breast cancer (TNBC) is the most severe type of breast cancer due to the lack of specific targets and rapid metastasis, which result in the poor prognosis. Recently, phosphatidylinositol 3-kinase (PI3K)/Akt pathway has emerged as a potential target for the treatment of TNBC. In our research interest to discover phytochemicals targeting TNBC, we have investigated wikstromol from Wikstroemia indica using the human TNBC MDA-MB-231 cells. The results showed wikstromol at 10 µM inhibited cell growth of MDA-MB-231 cells which was confirmed by MTT assay. Further DAPI staining has revealed wikstromol at 10 µM induced apoptosis of cancer cells, which was associated with the activation of caspase-3 following down-regulation of Bcl-2 as well as up-regulation of Bax, cleaved PARP and phosphorylated p53. Meanwhile, it was observed at 0.1 µM wikstromol suppressed the migration of the cancer cells via decreasing transcription of NF-κB and reducing activity and secretion of downstream MMP-9. In addition, p-PI3K and p-Akt were down-regulated in MDA-MB-231 cells in the presence of wikstromol at 0.1 µM, which indicated inactivation of PI3K/Akt pathway was involved in these inhibitory effects.

Bioactive daphnane diterpenes from Wikstroemia chuii with their potential anti-inflammatory effects and anti-HIV activities.

A phytochemical investigation on the stems and leaves of Wikstroemia chuii resulted in the isolation of three new daphnane diterpenes, wikstroechuins A-C (1-3), together with eight known analogues (4-11). The structures of new daphnane diterpenes (1-3) were determined on the basis of extensive spectroscopic methods and the known daphnane diterpenes (4-11) were identified by comparing their observable spectroscopic data with those reported spectral data in the literature. The anti-inflammatory effects as well as anti-HIV activities in vitro of all isolated daphnane diterpenes 1-11 were assessed. As a consequence, daphnane diterpenes 1-11 displayed remarkable inhibitory activities on NO (nitric oxide) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells showing IC50 values in the range of 0.12 ± 0.03 to 10.58 ± 0.16 µM. Meanwhile, daphnane diterpenes 1-11 displayed significant anti-HIV-1 reverse transcriptase (RT) effects showing EC50 values ranging from 0.09509 to 8.62356 µM. These research results indicated that the discovery of these new daphnane diterpenes with remarkable anti-inflammatory and anti-HIV activities from W. chuii, especially these new ones, could be extremely meaningful to the discovery of new anti-inflammatory agents and anti-HIV drugs as well as their potential practical values in the health and pharmaceutical products.