Semisynthesis, Structure Elucidation and Anti-Mycobacterium marinum Activity of a Series of Marine-Derived 14-Membered Resorcylic Acid Lactones with Interesting Ketal Groups.

The incidence of Mycobacterium marinum infection is on the rise; however, the existing drug treatment cycle is lengthy and often requires multi-drug combination. Therefore, there is a need to develop new and effective anti-M. marinum drugs. Cochliomycin A, a 14-membered resorcylic acid lactone with an acetonide group at C-5' and C-6', exhibits a wide range of antimicrobial, antimalarial, and antifouling activities. To further explore the effect of this structural change at C-5' and C-6' on this compound's activity, we synthesized a series of compounds with a structure similar to that of cochliomycin A, bearing ketal groups at C-5' and C-6'. The R/S configuration of the diastereoisomer at C-13' was further determined through an NOE correlation analysis of CH3 or CH2 at the derivative C-13' position and the H-5' and H-6' by means of a 1D NOE experiment. Further comparative 1H NMR analysis of diastereoisomers showed the difference in the chemical shift (δ) value of the diastereoisomers. The synthetic compounds were screened for their anti-microbial activities in vitro. Compounds 15-24 and 28-35 demonstrated promising activity against M. marinum, with MIC90 values ranging from 70 to 90 µM, closely approaching the MIC90 of isoniazid. The preliminary structure-activity relationships showed that the ketal groups with aromatic rings at C-5' and C-6' could enhance the inhibition of M. marinum. Further study demonstrated that compounds 23, 24, 29, and 30 had significant inhibitory effects on M. marinum and addictive effects with isoniazid and rifampicin. Its effective properties make it an important clue for future drug development toward combatting M. marinum resistance.

Atranorin inhibits NLRP3 inflammasome activation by targeting ASC and protects NLRP3 inflammasome-driven diseases.

Aberrant NLRP3 activation has been implicated in the pathogenesis of numerous inflammation-associated diseases. However, no small molecular inhibitor that directly targets NLRP3 inflammasome has been approved so far. In this study, we show that Atranorin (C19H18O8), the secondary metabolites of lichen family, effectively prevents NLRP3 inflammasome activation in macrophages and dendritic cells. Mechanistically, Atranorin inhibits NLRP3 activation induced cytokine secretion and cell pyroptosis through binding to ASC protein directly and therefore restraining ASC oligomerization. The pharmacological effect of Atranorin is evaluated in NLRP3 inflammasome-driven disease models. Atranorin lowers serum IL-1ß and IL-18 levels in LPS induced mice acute inflammation model. Also, Atranorin protects against MSU crystal induced mice gouty arthritis model and lowers ankle IL-1ß level. Moreover, Atranorin ameliorates intestinal inflammation and epithelial barrier dysfunction in DSS induced mice ulcerative colitis and inhibits NLRP3 inflammasome activation in colon. Altogether, our study identifies Atranorin as a novel NLRP3 inhibitor that targets ASC protein and highlights the potential therapeutic effects of Atranorin in NLRP3 inflammasome-driven diseases including acute inflammation, gouty arthritis and ulcerative colitis.

STING inhibitor ameliorates LPS-induced ALI by preventing vascular endothelial cells-mediated immune cells chemotaxis and adhesion.

Acute lung injury (ALI) is a common and devastating clinical disorder featured by excessive inflammatory responses. Stimulator of interferon genes (STING) is an indispensable molecule for regulating inflammation and immune response in multiple diseases, but the role of STING in the ALI pathogenesis is not well elucidated. In this study, we explored the molecular mechanisms of STING in regulating lipopolysaccharide (LPS)-induced lung injury. Mice were pretreated with a STING inhibitor C-176 (15, 30 mg/kg, i.p.) before LPS inhalation to induce ALI. We showed that LPS inhalation significantly increased STING expression in the lung tissues, whereas C-176 pretreatment dose-dependently suppressed the expression of STING, decreased the production of inflammatory cytokines including TNF-α, IL-6, IL-12, and IL-1ß, and restrained the expression of chemokines and adhesion molecule vascular cell adhesion protein-1 (VCAM-1) in the lung tissues. Consistently, in vitro experiments conducted in TNF-α-stimulated HMEC-1cells (common and classic vascular endothelial cells) revealed that human STING inhibitor H-151 or STING siRNA downregulated the expression levels of adhesion molecule and chemokines in HMEC-1cells, accompanied by decreased adhesive ability and chemotaxis of immunocytes upon TNF-α stimulation. We further revealed that STING inhibitor H-151 or STING knockdown significantly decreased the phosphorylation of transcription factor STAT1, which subsequently influenced its binding to chemokine CCL2 and adhesive molecule VCAM-1 gene promoter. Collectively, STING inhibitor can alleviate LPS-induced ALI in mice by preventing vascular endothelial cells-mediated immune cell chemotaxis and adhesion, suggesting that STING may be a promising therapeutic target for the treatment of ALI.

Semi-Synthesis, Cytotoxic Evaluation, and Structure-Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity.

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2-16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure-activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC50 value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1-GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.

Structure-Activity Relationships and Potent Cytotoxic Activities of Terphenyllin Derivatives from a Small Compound Library.

A small library of 120 compounds was established with seventy new alkylated derivatives of the natural product terphenyllin, together with 45 previous reported derivatives and four natural p-terphenyl analogs. The 70 new derivatives were semi-synthesized and evaluated for cytotoxic activities against four cancer cell lines. Interestingly, 2',4''-diethoxyterphenyllin, 2',4,4''-triisopropoxyterphenyllin, and 2',4''-bis(cyclopentyloxy)terphenyllin showed potent activities with IC50 values in a range from 0.13 to 5.51 µM, which were similar to those of the positive control, adriamycin. The preliminary structure-activity relationships indicated that the introduction of alkyl substituents including ethyl, allyl, propargyl, isopropyl, bromopropyl, isopentenyl, cyclopropylmethyl, and cyclopentylmethyl are important for improving the cytotoxicity.

Artemisinin analogue SM934 ameliorates DSS-induced mouse ulcerative colitis via suppressing neutrophils and macrophages.

Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease (IBD) characterized by complicated and relapsing inflammation in the gastrointestinal tract. SM934 is a water-soluble artemisinin analogue that shows anti-inflammatory and immuno-regulatory effects. In this study, we investigated the effects of SM934 on UC both in vivo and in vitro. A mouse model of colitis was established in mice by oral administration of 5% dextran sulfate sodium (DSS). SM934 (3, 10 mg/kg per day, ig) was administered to the mice for 10 days. After the mice were sacrificed, colons, spleens and mesenteric lymph nodes (MLNs) were collected for analyses. We showed that SM934 administration restored DSS-induced body weight loss, colon shortening, injury and inflammation scores. Furthermore, SM934 administration significantly decreased the disease activity index (DAI), histopathological scores, and myeloperoxidase (MPO) activities in colonic tissues. Moreover, SM934 administration dose-dependently decreased the mRNA and protein levels of DSS-induced pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α), and the percentage of macrophages and neutrophils in colon tissues. The effects of SM934 on LPS-stimulated RAW 264.7 cells and THP-1-derived macrophages were examined in vitro. Treatment with SM934 (0.8, 8, 80 µmol/L) dose-dependently decreased the production of pro-inflammatory mediators in LPS-stimulated RAW264.7 cells and THP-1-derived macrophages via inhibiting activation of the NF-κB signaling. Our results reveal the protective effects of SM934 on DSS-induced colitis can be attributed to its suppressing effects on neutrophils and macrophages and its inhibitory role in the NF-κB signaling, suggests that SM934 might be a potential effective drug for ulcerative colitis.

(5R)-5-hydroxytriptolide ameliorates lupus nephritis in MRL/lpr mice by preventing infiltration of immune cells.

(5R)-5-hydroxytriptolide (LLDT-8), a triptolide derivative with low toxicity, was previously reported to have strong immunosuppressive effects both in vitro and in vivo, but it remains unknown whether LLDT-8 has a therapy effect on systemic lupus erythematosus. In this study, we aimed to investigate the therapeutic effects of LLDT-8 on lupus nephritis in MRL/lpr mice, a model of systemic lupus erythematosus. Compared with the vehicle group, different clinical parameters were improved upon LLDT-8 treatment as follows: prolonged life span of mice, decreased proteinuria, downregulated blood urea nitrogen and serum creatinine, reduced glomerular IgG deposits, and ameliorated histopathology. A decreased expression of the inflammatory cytokines IFN-γ, IL-17, IL-6, and TNF-α was also observed in the kidney of LLDT-8 treated MRL/lpr mice. Moreover, infiltration of T cells in the kidney was mitigated after LLDT-8 treatment, corresponding with decreased expression of related chemokines IP-10, Mig, and RANTES in the kidney. The proportion of macrophage and neutrophil cells and related chemokines expression was also reduced in kidneys of LLDT-8-treated mice. In the human proximal tubule epithelial cell line and mouse mesangial cell line, consistent with our in vivo experimental results, LLDT-8 suppressed the expression of related chemokines and IL-6. In summary, LLDT-8 has a therapeutic benefit for lupus nephritis via suppressing chemokine expression and inhibiting immune cell infiltration in kidneys of MRL/lpr mice.

Toll-like receptors: potential targets for lupus treatment.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the loss of tolerance to self-nuclear antigens. Accumulating evidence shows that Toll-like receptors (TLRs), previously proven to be critical for host defense, are implicated in the pathogenesis of autoimmune diseases by recognition of self-molecules. Genome-wide association studies, experimental mouse models and clinical sample studies have provided evidence for the involvement of TLRs, including TLR2/4, TLR5, TLR3 and TLR7/8/9, in SLE pathogenesis. A number of downstream proteins in the TLR signaling cascade (such as MyD88, IRAKs and IFN-α) are identified as potential therapeutic targets for SLE treatment. Numerous antagonists targeting TLR signaling, including oligonucleotides, small molecular inhibitors and antibodies, are currently under preclinical studies or clinical trials for SLE treatment. Moreover, the emerging new manipulation of TLR signaling by microRNA (miRNA) regulation shows promise for the future treatment of SLE.

Therapeutic effects of DZ2002, a reversible SAHH inhibitor, on lupus-prone NZB×NZW F1 mice via interference with TLR-mediated APC response.

AIM: To examine the therapeutic effects and underlying mechanisms of DZ2002, a reversible S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitor, on lupus-prone female NZB×NZW F1 (NZB/W F1) mice. METHODS: Female NZB/W F1 mice were treated orally with DZ2002 (0.5 mg·kg(-1)·d(-1)) for 11 weeks, and the proteinuria level and body weight were monitored. After the mice ware euthanized, serum biochemical parameters and renal damage were determined. Splenocytes of NZB/W F1 mice were isolated for ex vivo study. Toll-like receptor (TLR)-stimulated human peripheral blood mononuclear cells (PBMCs) or murine bone marrow-derived dendritic cells (BMDCs) were used for in vitro study. RESULTS: Treatment of the mice with DZ2002 significantly attenuated the progression of glomerulonephritis and improved the overall health. The improvement was accompanied by decreased levels of nephritogenic anti-dsDNA IgG2a and IgG3 antibodies, serum IL-17, IL-23p19 and TGF-ß. In ex vivo studies, treatment of the mice with DZ2002 suppressed the development of pathogenic Th17 cells, significantly decreased IL-17, TGF-ß, IL-6, and IL-23p19 production and impeded activation of the STAT3 protein and JNK/NF-κB signaling in splenocytes. DZ2002 (500 µmol/L) significantly suppressed TLR agonists-stimulated up-regulation in IL-6, IL-12p40, TNF-α, and IgG and IgM secretion as well as in HLA-DR and CD40 expression of dendritic cells among human PBMCs in vitro. DZ2002 (100 µmol/L) also significantly suppressed TLR agonists-stimulated up-regulation in IL-6 and IL-23p19 production in murine BMDCs, and prevented Th17 differentiation and suppressed IL-17 secretion by the T cells in a BMDC-T cell co-culture system. CONCLUSION: DZ2002 effectively ameliorates lupus syndrome in NZB/W F1 mice by regulating TLR signaling-mediated antigen presenting cell (APC) responses.

Hemin protects UVB-induced skin damage through inhibiting keratinocytes apoptosis and reducing neutrophil infiltration.

Ultraviolet-B (UVB) exposure on the skin triggers apoptosis, oxidative stress and acute inflammatory responses, which eventually increases the risk of various skin disorders. Hemin, an iron-binding porphyrin, has been clinically used for porphyria treatment. However, whether hemin contributes to the skin protection against UVB injury remains to be elucidated. Here, we found that hemin treatment (10 and 20 mg/kg) by intraperitoneal administration could dramatically relieve UVB irradiation-induced skin damage featured by erythema, edema, epidermal hyperplasia and collagen loss in C57BL/6 J mice. Importantly, hemin treatment attenuated UVB irradiation-triggered cell apoptosis in skin epidermis. Consistently, hemin (10, 20 µM) treatment decreased Caspase-3 activation and protected against UVB-induced apoptosis in HaCaT cells. Besides, hemin treatment reduced the infiltration of neutrophils in skin under UVB irradiation, thus restrained neutrophil extracellular traps (NET) formation and myeloperoxidase (MPO) release. We further revealed that hemin inhibited the expression of inflammation associated cytokines and chemokines in UVB-induced HaCaT cells and blocked the chemotaxis of dHL-60 cells to preconditioned media from HaCaT culture upon UVB irradiation. Furthermore, hemin inhibited the excessive maturation and mobilization of bone marrow neutrophils and rectified the proportion of abnormally elevated neutrophils in the blood under UVB irradiation. In conclusion, our study showed that hemin treatment protects against UVB-induced skin damage through inhibiting keratinocytes apoptosis, and suppressing neutrophils infiltration in the skin via externally restraining the keratinocyte attraction and internally regulating bone marrow neutrophil maturation and mobilization, suggesting that hemin is an effective drug candidate for the therapy of UVB damage.

A receptor-binding domain-based nanoparticle vaccine elicits durable neutralizing antibody responses against SARS-CoV-2 and variants of concern.

Numerous vaccines have been developed to address the current COVID-19 pandemic, but safety, cross-neutralizing efficacy, and long-term protectivity of currently approved vaccines are still important issues. In this study, we developed a subunit vaccine, ASD254, by using a nanoparticle vaccine platform to encapsulate the SARS-CoV-2 spike receptor-binding domain (RBD) protein. As compared with the aluminum-adjuvant RBD vaccine, ASD254 induced higher titers of RBD-specific antibodies and generated 10- to 30-fold more neutralizing antibodies. Mice vaccinated with ASD254 showed protective immune responses against SARS-CoV-2 challenge, with undetectable infectious viral loads and reduced typical lesions in lung. Besides, neutralizing antibodies in vaccinated mice lasted for at least one year and were effective against various SARS-CoV-2 variants of concern, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, particle size, polydispersity index, and zeta-potential of ASD254 remained stable after 8-month storage at 4°C. Thus, ASD254 is a promising nanoparticle vaccine with good immunogenicity and stability to be developed as an effective vaccine option in controlling upcoming waves of COVID-19.

Positively charged nanocomplex modulates dendritic cell differentiation to enhance Th1 immune response.

Most existing vaccines use activators that polarize the immune response to T-helper (Th) 2 response for antibody production. Our positively charged chitosan (Cs)-based nanocomplex (CNC) drives the Th1 response through unknown mechanisms. As receptors for the positively charged CNC are not determined, the physico-chemical properties are hypothesized to correlate with its immunomodulatory effects. To clarify the effects of surface charge and size on the immune response, smaller CNC and negatively charged CNC encapsulating ovalbumin are tested on dendritic cell (DC) 2.4 â€‹cells. The negatively charged CNC loses activity, but the smaller CNC does not. To further evaluate the material effects, we replace Cs by poly-amino acids. Compared with the negatively charged nanocomplex, the positively charged one preserves its activity. Using immature bone marrow-derived DCs (BMDC) enriched from BALB/c mice as a model to analyze DC differentiation, treatments with positively charged nanocomplexes evidently increase the proportions of Langerin+ dermal DC, CD11blo interstitial DC, and CD8a+ conventional DC. Additionally, vaccination with two doses containing positively charged nanocomplexes are safe and increase ovalbumin-specific IgG and recall T-cell responses in mice. Overall, a positive charge seems to contribute to the immunological effect of nanocomplexes on elevating the Th1 response by modulating DC differentiation.

Highly stable gold nanoparticle-antigen conjugates with self-adjuvanting property for induction of robust antigen-specific immune responses.

Poor long-term stability and formation of irreversible aggregates when subjected to a freeze-drying process greatly limits the clinical application of gold nanoparticles (GNPs) as a vaccine carrier. In this study, we synthesized a GNP-antigen conjugate with high colloidal stability by using a thiolated polyethylene glycol (PEG) linker to conjugate a model antigen (ovalbumin; OVA) onto the GNP surface (i.e. GNP-OVA) and demonstrated this conjugate had self-adjuvanting properties to augment antigen-specific immune responses. The synthesized GNP had an average hydrodynamic size of 13.8 ± 2.1 nm (n = 3); after conjugation of OVA, the diameter increased to 28.6 ± 7.3 nm (n = 3). The obtained GNP-OVA can maintain a stable dispersion state in aqueous solutions for at least 12 months and withstand stresses during lyophilization without creating irreversible aggregates. Compared with OVA alone or a mixture of PEG-functionalized GNP (GNP-PEG) and OVA (i.e. GNP-PEG/OVA), the chemical conjugation of OVA onto GNP-PEG substantially increased antigen uptake and upregulated major histocompatibility complex class II expression in macrophages. This indicated that the GNP can function as not only an adjuvant to promote the phagocytic activity of macrophages but also a carrier to deliver the conjugated antigen into the immune cells for the enhancement of its antigen presentation capability. Importantly, OVA-specific immunoglobulin G levels in the mice immunized with GNP-OVA were 4.1 and 2.9 times higher than those in the mice injected with OVA and GNP-PEG/OVA, respectively. These results demonstrated that the GNP-antigen conjugate exhibited remarkable stability either in liquid or freeze-dried form, which makes it attractive for further pharmaceutical applications. Moreover, covalently linking antigens onto the GNP surface was enabled to enhance the immunogenicity of antigens and boost immune responses, showing the potential of the GNP conjugation strategy for vaccine development.

Hemin protects against Zika virus infection by disrupting virus-endosome fusion.

Zika virus (ZIKV) is a flavivirus that causes severe neuropathology in newborns and adults. There is no ZIKV-specific treatment or preventative. Therefore, it is urgent to develop safe and effective anti-ZIKV agents. Hemin, an iron-binding porphyrin, has been authorized by FDA to treat acute porphyria since the 1970s. Here, we aim to evaluate the anti-ZIKV effect of hemin in SNB-19 cells (a human glioma cell line) and explore the underlying mechanism based on the virus life cycle and functions of the host cell. Our study found that hemin has a strong activity to protect SNB-19 cells from ZIKV infection presented by decreased expression of viral proteins and virus yield. Meanwhile, ZIKV infection caused STAT1/IRF1 signaling activation and induced inflammatory responses in SNB-19 cells, which was relieved by hemin treatment. HO-1 has been reported to be potently induced by hemin and play a broad-spectrum antiviral effect. Intriguingly, hemin could still exert anti-ZIKV activity upon HO-1 siRNA treatment. Then, we conducted a time-of-addition assay, the result indicated hemin works mainly by interfering with the virus entry process. Further experiments excluded the effects of hemin on AXL-dependent viral adsorption and clathrin-mediated endocytosis processes. Subsequently, by fluorescence spectroscopy studies, intracellular fusion assay and syncytia formation assay, we revealed that hemin acts on the process of virus-endosome fusion. This study elaborated that hemin could play anti-ZIKV activity by disrupting the virus-endosome fusion process and shed new light on developing novel agents against ZIKV infection.

New brefeldin A-cinnamic acid ester derivatives as potential antitumor agents: Design, synthesis and biological evaluation.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and ranks third in mortality rate worldwide. Brefeldin A (BFA, 1), a natural Arf1 inhibitor, qualifies extremely superior antitumor activity against HCC while its low aqueous solubility, poor bioavailability, and high toxicity have greatly hindered its translation to the clinic. Herein, a series of BFA-cinnamic acid ester derivatives was rationally designed and synthesized via introducing active cinnamic acid and its analogues into the structure of 1. Their in vitro cytotoxic activities on five cancer cell lines, including HepG2, BEL-7402, HeLa, Eca-109 and PANC-1, were evaluated using MTT assay. As expected, favorable cytotoxic activity was observed on majority of the mono-substituted derivatives. Especially, the most potent brefeldin A 4-O-(4)-dimethylaminocinnamate (CHNQD-01269, 33) with improved aqueous solubility, demonstrated the strong cytotoxic activity against HepG2 and BEL-7402 cell lines with IC50 values of 0.29 and 0.84 µM, respectively. More importantly, 33 performed low toxicity on normal liver cell line L-02 with the selectivity index (SI) of 9.69, which was more than 17-fold higher than that of 1. Results from mechanistic studies represented that 33 blocked the cell cycle in the G1 phase, and induced apoptosis via elevating reactive oxygen species (ROS) production and increasing expression of apoptosis-related proteins of HepG2 cells. Docking experiment also suggested 33 a promising Arf1 inhibitor, which was confirmed by the cellular thermal shift assay that 33 displayed a significant effect on the stability of Arf1 protein. Furthermore, 33 possessed high safety profile (MTD >100 mg/kg, ip) and favorable pharmacokinetic properties. Notably, the superior antiproliferative activity was verified in HepG2 tumor-bearing xenograft model in which 33 markedly suppressed the tumor growth (TGI = 46.17%) in nude mice at a dose of 10 mg/kg once a day for 16 d. The present study provided evidence of exploiting this series of highly efficacious derivatives, especially 33, for the treatment of HCC.

Structure modification, antialgal, antiplasmodial, and toxic evaluations of a series of new marine-derived 14-membered resorcylic acid lactone derivatives.

Marine natural products play critical roles in the chemical defense of many marine organisms and are essential, reputable sources of successful drug leads. Sixty-seven 14-membered resorcylic acid lactone derivatives 3-27 and 30-71 of the natural product zeaenol (1) isolated from the marine-derived fungus Cochliobolus lunatus were semisynthesized by chlorination, acylation, esterification, and acetalization in one to three steps. The structures of these new derivatives were established by HRESIMS and NMR techniques. All the compounds (1-71) were evaluated for their antialgal and antiplasmodial activities. Among them, 14 compounds displayed antifouling activities against adhesion of the fouling diatoms. In particular, 9 and 34 exhibited strong and selective inhibitory effects against the diatoms Navicula laevissima and Navicula exigua (EC50 = 6.67 and 8.55 µmol/L), respectively, which were similar in efficacy to those of the positive control SeaNine 211 (EC50 = 2.90 and 9.74 µmol/L). More importantly, 38, 39, and 69-71 showed potent antiplasmodial activities against Plasmodium falciparum with IC50 values ranging from 3.54 to 9.72 µmol/L. Very interestingly, the five antiplasmodial derivatives displayed non-toxicity in the cytotoxicity assays and the zebrafish embryos model, thus, representing potential promising antiplasmodial drug agents. The preliminary structure-activity relationships indicated that biphenyl substituent at C-2, acetonide at positions C-5' and C-6', and tri- or tetra-substituted of acyl groups increased the antiplasmodial activity. Therefore, combining evaluation of chemical ecology with pharmacological models will be implemented as a systematic strategy, not only for environmentally friendly antifoulants but also for structurally novel drugs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-021-00103-0.

Epigallocatechin gallate/L-ascorbic acid-loaded poly-γ-glutamate microneedles with antioxidant, anti-inflammatory, and immunomodulatory effects for the treatment of atopic dermatitis.

Epigallocatechin gallate (EGCG) is a potential therapeutic agent for treatment of atopic dermatitis (AD) due to its antioxidant and anti-inflammatory activities. However, inherent instability of EGCG greatly limits its bioavailability and clinical efficacy. In this study, we developed a poly-γ-glutamate (γ-PGA)-based microneedle (MN) formulation capable of maintaining EGCG's stability and efficiently delivering EGCG into the skin to ameliorate AD symptoms. The γ-PGA MN can not only protect EGCG from oxidation, but also serve as an immunomodulator to downregulate T helper type 2 (Th2)-type immune responses. Encapsulation of EGCG into the γ-PGA MN and utilization of L-ascorbic acid (AA) as a stabilizer preserved 95% of its structural stability and retained 93% of its initial antioxidant activity after 4 weeks of storage. Once-weekly administration of EGCG/AA-loaded MNs to an Nc/Nga mouse model of AD for 4 weeks significantly ameliorated skin lesions and epidermal hyperplasia by reducing serum IgE (from 12156 ± 1344 to 5555 ± 1362 ng/mL) and histamine levels (from 81 ± 18 to 40 ± 5 pg/mL) and inhibiting IFN-γ (from 0.10 ± 0.01 to 0.01 pg/mg total protein) and Th2-type cytokine production, when compared to the AD (no treatment) group (p < 0.05). Notably, once-weekly MN therapy was at least as effective as the daily topical application of an EGCG + AA solution but markedly reduced the administration frequency and required dose. These results show that EGCG/AA-loaded γ-PGA MNs may be a convenient and promising therapeutic option for AD treatment. STATEMENT OF SIGNIFICANCE: This study describes epigallocatechin gallate (EGCG)/L-ascorbic acid (AA)-loaded poly-γ-glutamate (γ-PGA) microneedles (MN) capable of providing antioxidant, anti-inflammatory, and immunomodulatory effects on inflamed skin for ameliorating atopic dermatitis (AD) symptoms in Nc/Nga mice. After skin insertion, the γ-PGA MN can be quickly dissolved in the skin and remain in the dermis for sustained release of encapsulated active ingredients for 6 days. We demonstrated that once-weekly MN therapy effectively alleviated skin lesions and modulated immune response to relieve Th2-polarized allergic response in mice. Once-weekly MN dosing regimen may provide patients with a more convenient, therapeutically equivalent option to daily topical dosing, and may increase compliance and long-term persistence with AD therapy.

CHNQD-00603 Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by the miR-452-3p-Mediated Autophagy Pathway.

Background: Periodontitis is a chronic and progressive disease accompanied by bone loss. It is still a challenge to restore the bone structure. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a decisive role in bone restoration and regeneration. Marine natural products (MNPs) have multiple biological activities, including anti-tumor and anti-inflammatory properties. However, the exploration of MNPs in osteogenesis is far from sufficient. Methods: We obtained a series of derivatives through structural optimization from 4-phenyl-3,4-dihydroquinolin-2(1H)-one alkaloid isolated from Scopulariopsis sp. Some preliminary cytological experiments showed that CHNQD-00603, obtained by adding a methoxy group to the position C3 and a hydroxyl group to the position C4 of 4-phenyl-3,4-dihydroquinolin-2(1H)-one, might promote the osteogenic differentiation of BMSCs. To further investigate the effects of CHNQD-00603 on BMSCs, we performed a CCK-8 assay and qRT-PCR, alkaline phosphatase staining (ALP), and alizarin red S staining to assess the cytotoxicity and the ability of osteogenic differentiation of CHNQD-00603. The autophagy level was assessed and validated by WB, qRT-PCR, and transmission electron microscopy. Then, 3-methyladenine (3-MA) was added to further examine the role of autophagy. Based on the expression of autophagy-related genes, we predicted and examined the potential miRNAs by bioinformatics. Results: CCK-8 assay showed that CHNQD-00603 at 1 µg/ml did not influence BMSCs activity. However, the proliferation rate decreased from the seventh day. qRT-PCR, ALP staining, ALP activity assay, and Alizarin red S staining showed that the best concentration of CHNQD-00603 to promote osteogenic differentiation was 1 µg/ml. Further investigations indicated that CHNQD-00603 activated autophagy, and the inhibition of autophagy by 3-MA attenuated CHNQD-00603-enhanced osteogenic differentiation. Subsequently, the findings from bioinformatics and qRT-PCR indicated that miR-452-3p might be a regulator of autophagy and osteogenesis. Furthermore, we transfected BMSCs with miR-452-3p NC and mimics separately to further determine the function of miR-452-3p. The data showed that the overexpression of miR-452-3p moderated the level of autophagy and osteogenic differentiation of CHNQD-00603-treated BMSCs. Conclusion: Our data suggested that CHNQD-00603 promoted the osteogenic differentiation of BMSCs by enhancing autophagy. Meanwhile, miR-452-3p played a regulatory role in this process.

Poly-γ-Glutamate microneedles as transdermal immunomodulators for ameliorating atopic dermatitis-like skin lesions in Nc/Nga mice.

Atopic dermatitis (AD), a common, relapsing, inflammatory disorder of the skin, is associated with T helper type 2 (Th2)-biased immune responses. Despite the efficacy of existing drugs for AD treatment, their safety and side effects cause concern. The present study describes the use of dissolvable poly-γ-glutamate (γ-PGA) microneedles (MNs) with immunomodulatory effects for effectively relieving AD-like symptoms in Nc/Nga mice. γ-PGA MNs can easily penetrate the epidermis and release γ-PGA into the dendritic cell-rich dermis to interact with dendritic cells for modulating immune responses. Transdermal administration of high-molecular-weight (HMW, 1100 kDa) γ-PGA MNs significantly reduced clinical dermatitis scores, epidermal thickness, and mast cell infiltration in mice by downregulating immunoglobulin (Ig)E and IgG1 levels (Th2-associated antibodies) compared with the AD control group. However, low-molecular-weight (200-400 kDa) γ-PGA MNs ameliorated AD-like skin lesions less effectively than HMW γ-PGA MNs, thus indicating that the MW of γ-PGA may affect its immunomodulatory properties. Notably, the mouse skin quickly recovered its barrier function within 4 h after MN application. No weight loss or abnormality was observed in the MN-treated mice during the 8-week treatment period. These results suggest that the γ-PGA MNs represent an innovative, safe, and reliable therapeutic strategy for AD management. STATEMENT OF SIGNIFICANCE: This study is the first to explore the feasibility of using poly-γ-glutamate (γ-PGA) microneedles (MNs) as transdermal immunomodulators for improving atopic dermatitis (AD) symptoms and to evaluate their immunomodulatory effect in mice with spontaneously developed AD. Transdermal administration of γ-PGA MNs enables the γ-PGA to localize in the skin for activation of dermal dendritic cells, thus modulating immune responses. We demonstrate that high-molecular-weight γ-PGA MNs can be retained in the skin for at least 6 days and effectively suppress AD-like skin lesions in mice by reducing infiltration of mast cells and downregulating Th2-associated antibody production (IgE and IgG1). The developed MN device has the potential to replace conventional therapy and to become an innovative treatment strategy for AD.

[Determination of baicalin and total flavonoids in Radix scutellariae by near infrared diffuse reflectance spectroscopy].

The objective of the present study was to develop a method for rapid determination of baicalin and total flavonoids in radix scutellariae by near infrared diffuse reflectance spectroscopy. Sixty one samples of radix scutellariae from different areas containing baicalin of 12.24%-21.34% and total flavonoids of 16.08%-26.52% were used. The range of 8000-4000 cm(-1) of near infrared spectra (NIRS) was selected. Calibration models were established using the PLS(partial least squares). Different spectra pretreatment methods were compared and the optimal model was selected. The study showed that first derivative pretreatments and minimum-maximum normalization methods can be used to extracted spectra information thoroughly to analyze the contents of baicalin and total flavonoids, respectively. The correlation coefficient (r) of baicalin was 0.9024, SEC was 1.01 (standard deviation of the calibration sets) and SEP was 0.8764 (standarddeviation ofthe prediction sets). The correlation coefficient(r) of total flavonoids was 0.9527, SEC was 0.7850 and SEP was 0.5211. Results indicated that near infrared diffuse reflectance spectroscopy method can be used to analyze the main active components in radix scutellariae rapidly.