Antiproliferative ent-abietane diterpenoids from Euphorbia fischeriana.

Euphorfinoids M and N (1 and 2), two previously undescribed ent-abietane diterpenoids, together with seven known analogues (3-9), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, ECD, and comparison with structurally related known analogues. Bioassays against proliferative effects of HeLa cell line showed that compound 1 was the most active with IC50 3.62 ± 0.31 µM.

Euphorfinoids A and B, a pair of ent-atisane diterpenoid epimers from the roots of Euphorbia fischeriana, and their bioactivities.

Euphorfinoids A and B (1 and 2), a pair of ent-atisane diterpenoid epimers with a vicinal 2,3-diol moiety, together with four known analogues (3-6), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, NMR calculations, X-ray diffraction, and comparison with structurally related known analogues. Our bioassays have established that compound 1 displayed moderate anti-proliferative effects on Hcc1806 cell line with IC50 15.53 ± 0.21 µM, and compound 5 showed remarkable inhibitory effects against AChE with IC50 32.56 ± 2.74 µM by an in vitro screened experiment.

Regulation effect of koumine on T-helper cell polarization in rheumatoid arthritis.

Koumine, an alkaloid, exerts therapeutic effects against rheumatoid arthritis (RA), and thus may have a potential application in novel treatment strategies against this disease. Herein, we investigated the regulatory effect of koumine on Th cell polarization using a "pyramid" structure model to elucidate the mechanism underlying its therapeutic effect on RA. The third layer of the model comprises the cytokine secretion layer, in which the effects of koumine on the balance of Th-related cytokines were investigated in mice with collagen-induced arthritis (CIA). Koumine showed significant therapeutic effects and reversed the imbalance of Th1/Th2 and Th17/Treg cytokines. In the Th cell polarization layer, the effects of koumine on the relative numbers of Th cell subsets in splenocytes of rats with CIA were examined. Koumine attenuated both of the increased Th1/Th2 and Th17/Treg subset ratios accompanied with its therapeutic effects. Finally, the primary cultured splenocytes from BALB/c mice were used to further investigate the effect of koumine on Th cell activation by evaluating cell proliferation induced by concanavalin A (Con A), lipopolysaccharides (LPS) and phytohemagglutinin (PHA). Koumine inhibited the cell proliferation responses and its effects on proliferation induced by Con A and PHA were greater than those by LPS, showing the relatively selective inhibition on the proliferation of Th cells. Our results suggest that koumine might restore the homeostasis of the network system with Th subsets and cytokines by inhibiting the activation of T cells, subsequently regulating the polarization of Th subsets and the downstream imbalance of pro/anti-inflammatory cytokines in RA.

[Molecular Epidemiological Characteristics and Differential Diagnosis of Common 뫧-Thalassemia/HPFH].

OBJECTIVE: To investigate the molecular epidemiological characteristics of common δß-thalassemia/hereditary persistence of fetal hemoglobin(HPFH) in the prepregnant population in Huadu, and to provide a laboratory basis for prevention and control of thalassemia. METHODS: Blood samples of childbearing age people in Huadu District of Guangzhou who participated in free thalassemia testing from January 2016 to July 2021 were collected for hematological parameters analysis and hemoglobin electrophoresis. Chinese Gγ+(Aγδß)0-thalassemia, SEA-HPFH and Taiwanese deletion ß-thalassemia were detected by Gap-PCR in the samples with higher HbF(≥5%). Primers were designed for the proximal HBG1 and HBG2 promoter, and the point mutations in the proximal promoter region were detected by Sanger sequencing. Hematology parameters data were statistically analyzed. RESULTS: Among 27 088 samples, Thirteen cases of Chinese Gγ+(Aγδß)0-thalassemia and thirty-three cases of SEA-HPFH were detected, which including 3 cases of Chinese Gγ+(Aγδß)0/ßN compounded with --SEA/αα and three cases of SEA-HPFH/ßN compounded with --SEA/αα. 6 carriers with Aγ-196 C>T were also detected; No Taiwanese thalassemia genetype was detected. The total detection rate of common δß-thalassemia/HPFH was 0.19% (52/27 088). There were significant differences in the levels of MCV, MCH, HbA2, and HbF among Chinese Gγ+(Aγδß)0-thalassemia, SEA-HPFH, Aγ-196 C>T (P<0.001). The hematological parameters of Aγ-196C>T combined with α0-thalassemia were similar to those of Chinese Gγ+(Aγδß)0-thalassemia carriers, and only HbA2 was significantly lower than that of the latter, which was helpful for clinical identification. CONCLUSION: δß-thalassemia/HPFH should be included in the scope of thalassemia prevention program in the prepregnant population in Huadu District, and hematological parameters can provide some basis for identifying different types of δß-thalassemia/HPFH.

Euphorfiatnoids A-I: Diterpenoids from the roots of Euphorbia fischeriana with cytotoxic effects.

Nine previously undescribed diterpenoids, euphorfiatnoids A-I, together with seven known diterpenoids, were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by the interpretation of HRESIMS, UV, and NMR data. Their configurations were determined by electronic circular dichroism (ECD) spectroscopy analysis and the structure of euphorfiatnoid A was confirmed by X-ray crystallography. To further understand the antitumor effects of E. fischeriana, we tested the cytotoxicity of these compounds against H460, HepG2, and MCF-7 cell lines in vitro using MTT assays. Euphorfiatnoid B exhibited the most promising inhibitory effect against H460 cells with an IC50 value of 9.97 µM. Euphorfiatnoid A and C also exhibited moderate cytotoxicity against HepG2 cells with IC50 values of 11.64 and 13.10 µM, respectively.

APT1-Mediated Depalmitoylation Regulates Hippocampal Synaptic Plasticity.

Palmitoylation may be relevant to the processes of learning and memory, and even disorders, such as post-traumatic stress disorder and aging-related cognitive decline. However, underlying mechanisms of palmitoylation in these processes remain unclear. Herein, we used acyl-biotin exchange, coimmunoprecipitation and biotinylation assays, and behavioral and electrophysiological methods, to explore whether palmitoylation is required for hippocampal synaptic transmission and fear memory formation, and involved in functional modification of synaptic proteins, such as postsynapse density-95 (PSD-95) and glutamate receptors, and detected if depalmitoylation by specific enzymes has influence on glutamatergic synaptic plasticity. Our results showed that global palmitoylation level, palmitoylation of PSD-95 and glutamate receptors, postsynapse density localization of PSD-95, surface expression of AMPARs, and synaptic strength of cultured hippocampal neurons were all enhanced by TTX pretreatment, and these can be reversed by inhibition of palmitoylation with palmitoyl acyl transferases inhibitors, 2-bromopalmitate and N-(tert-butyl) hydroxylamine hydrochloride. Importantly, we also found that acyl-protein thioesterase 1 (APT1)-mediated depalmitoylation is involved in palmitoylation of PSD-95 and glutamatergic synaptic transmission. Knockdown of APT1, not protein palmitoyl thioesterase 1, with shRNA, or selective inhibition, significantly increased AMPAR-mediated synaptic strength, palmitoylation levels, and synaptic or surface expression of PSD-95 and AMPARs. Results from hippocampal tissues and fear-conditioned rats showed that palmitoylation is required for synaptic strengthening and fear memory formation. These results suggest that palmitoylation and APT1-mediated depalmitoylation have critical effects on the regulation of glutamatergic synaptic plasticity, and it may serve as a potential target for learning and memory-associated disorders.SIGNIFICANCE STATEMENT Fear-related anxiety disorders, including post-traumatic stress disorder, are prevalent psychiatric conditions, and fear memory is associated with hyperexcitability in the hippocampal CA1 region. Palmitoylation is involved in learning and memory, but mechanisms coupling palmitoylation with fear memory acquisition remain poorly understood. This study demonstrated that palmitoylation is essential for postsynapse density-95 clustering and hippocampal glutamatergic synaptic transmission, and APT1-mediated depalmitoylation plays critical roles in the regulation of synaptic plasticity. Our study revealed that molecular mechanism about downregulation of APT1 leads to enhancement of AMPAR-mediated synaptic transmission, and that palmitoylation cycling is implicated in fear conditioning-induced synaptic strengthening and fear memory formation.

Euphorfistrines A-G, cytotoxic and AChE inhibiting triterpenoids from the roots of Euphorbia fischeriana.

Seven new triterpenoids including two cycloartanes (1-2), a lanostane (3), a tirucallane (4), a dammarane (5), an ursane (6), and an oleanane (7), along with nineteen known triterpenoids (8-26), have been obtained from the roots of Euphorbia fischeriana. Their structures were established by NMR, HRESIMS, single-crystal X-ray diffraction analysis, Mosher's method, NMR calculations, ECD analysis, and comparison with structurally related known analogues. Among them, compounds 1 and 8 were a pair of cycloartane-type triterpenoids epimers. Our bioassays have established that compounds 1-5 and 10 displayed moderate cytotoxic effects, and the structure-activity relationships of cycloartane-type triterpenoids (CTTs) were further examined. Notably, some triterpenoids displayed moderate inhibitory effects against AChE by an in vitro screened experiment. Triterpenoid 7 (Euphorfistrine G, ETG) displayed the potent inhibitory effect with IC50 = 2.45 and Ki = 2.30 µM (inhibition kinetic). And, in silico docking analyses have been performed to investigate the inhibitory mechanism of compound 7.

Koumine modulates spinal microglial M1 polarization and the inflammatory response through the Notch-RBP-Jκ signaling pathway, ameliorating diabetic neuropathic pain in rats.

BACKGROUND: Diabetic neuropathic pain (DNP), a complication of diabetes, has serious impacts on human health. As the pathogenesis of DNP is very complex, clinical treatments for DNP is limited. Koumine (KM) is an active ingredient extracted from Gelsemium elegans Benth. that exerts an inhibitory effect on neuropathic pain (NP) in several animal models. PURPOSE: To clarify the anti-NP effect of KM on rats with DNP and the molecular mechanisms involving the Notch- Jκ recombination signal binding protein (RBP-Jκ) signaling pathway. METHODS: Male Sprague-Dawley rats were administered streptozocin (STZ) by intraperitoneal injection to induce DNP. The effect of KM on mechanical hyperalgesia in rats with DNP was evaluated using the Von Frey test. Microglial polarization in the spinal cord was examined using western blotting and quantitative real-time PCR. The Notch-RBP-Jκ signaling pathway was analysed using western blotting. RESULTS: KM attenuated DNP during the observation period. In addition, KM alleviated M1 microglial polarization in STZ-induced rats. Subsequent experiments revealed that Notch-RBP-Jκ signaling pathway was activated in the spinal cord of rats with DNP, and the activation of this pathways was decreased by KM. Additionally, KM-mediated analgesia and deactivation of the Notch-RBP-Jκ signaling pathway were inhibited by the Notch signaling agonist jagged 1, indicating that the anti-DNP effect of KM may be regulated by the Notch-RBP-Jκ signaling pathway. CONCLUSIONS: KM is a potentially desirable candidate treatment for DNP that may inhibit microglial M1 polarization through the Notch-RBP-Jκ signaling pathway.

Streptozotocin-Induced Hyperglycemia Affects the Pharmacokinetics of Koumine and its Anti-Allodynic Action in a Rat Model of Diabetic Neuropathic Pain.

Koumine (KM), the most abundant alkaloid in Gelsemium elegans, has anti-neuropathic, anti-inflammatory, and analgesic activities; thus, it has the potential to be developed as a broad-spectrum analgesic drug. However, factors determining the relationship between analgesic efficacy and the corresponding plasma KM concentration are largely unclear. The pharmacokinetics and pharmacodynamics of KM and their optimization in the context of neuropathic pain have not been reported. We investigated the pharmacokinetics and pharmacodynamics of KM after oral administration in a streptozotocin-induced rat model of diabetic neuropathic pain (DNP) using a population approach. A first-order absorption and elimination pharmacokinetics model best described the plasma KM concentration. This pharmacokinetic model was then linked to a linear pharmacodynamic model with an effect compartment based on the measurement of the mechanical withdrawal threshold. KM was rapidly absorbed (time to maximum plasma concentration: 0.14-0.36 h) with similar values in both DNP and naïve rats, suggesting that DNP did not influence the KM absorption rate. However, the area under the curve (AUC0-∞) of KM in DNP rats was over 3-fold higher than that in naïve rats. The systemic clearance rate and volume of KM distribution were significantly lower in DNP rats than in naïve rats. Blood glucose value prior to KM treatment was a significant covariate for the systemic clearance rate of KM and baseline value of the threshold. Our results suggest that streptozotocin-induced hyperglycemia is an independent factor for decreased KM elimination and its anti-allodynic effects in a DNP rat model. To the best of our knowledge, this is the first study to investigate the role of DNP in the pharmacokinetics and pharmacokinetics-pharmacodynamics of KM in streptozotocin-induced diabetic rats.

Orally Administered Koumine Persists Longer in the Plasma of Aged Rats Than That of Adult Rats as Assessed by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Aging leads to changes in nearly all pharmacokinetic phases. Koumine (KM), an alkaloid derived from Gelsemium elegans Benth., is effective against age-associated chronic diseases, but its dose proportionality following oral administration in aged individuals remains unknown. Herein, we established and validated a simple method that requires low sample volumes to determine KM concentration in rats using ultra-performance liquid chromatography-tandem mass spectrometry. The maximum plasma concentration (Cmax) of 7 mg·kg-1 KM was ~12-fold and ~24-fold higher than that of 0.28 mg·kg-1 KM in adult and aged rats, respectively (P < 0.01). Time to reach Cmax (Tmax) for 7 mg·kg-1 KM was 4-fold longer in aged rats (P < 0.05). The area under the curve (AUC) of 7 mg·kg-1 KM was >17-fold and >43-fold higher than those of 0.28 mg·kg-1 KM in adult and aged rats, respectively (P < 0.01). The half-life (t1/2) of 7 mg·kg-1 KM was over 4-fold longer than that of 0.28 mg·kg-1 KM in adult rats (P < 0.01). The t1/2 of 1.4 and 7 mg·kg-1 KM were 1.5~2-fold longer, than that of 0.28 mg·kg-1 KM in aged rats (P < 0.05). The clearance rate of 7 mg·kg-1 KM was significantly lower in aged than in adult rats (P < 0.05). For 7.0 mg·kg-1 KM, the Cmax in aged rats was higher than in adult rats during the Tmax period (P < 0.05). In aged rats, the AUC for KM was >2.5-fold higher (P < 0.05) and the t1/2 was >60% longer than in adult rats (P < 0.05). These results help interpret the pharmacokinetics of KM in aging-associated diseases.

Pathological cardiac remodeling occurs early in CKD mice from unilateral urinary obstruction, and is attenuated by Enalapril.

Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD) and end-stage renal disease. Despite increasing recognition of a close interplay between kidney dysfunction and cardiovascular disease, termed cardiorenal syndrome (CRS), the underlying mechanisms of CRS remain poorly understood. Here we report the development of pathological cardiac hypertrophy and fibrosis in early stage non-uremic CKD. Moderate kidney failure was induced three weeks after unilateral urinary obstruction (UUO) in mice. We observed pathological cardiac hypertrophy and increased fibrosis in UUO-induced CKD (UUO/CKD) animals. Further analysis indicated that this cardiac fibrosis was associated with increased expression of transforming growth factor ß (TGF-ß) along with significant upregulation of Smad 2/3 signaling in the heart. Moreover early treatment of UUO/CKD animals with an angiotensin-converting-enzyme inhibitor (ACE I), Enalapril, significantly attenuated cardiac fibrosis. Enalapril antagonized activation of the TGF-ß signaling pathway in the UUO/CKD heart. In summary our study demonstrates the presence of pathological cardiac hypertrophy and fibrosis in mice early in UUO-induced CKD, in association with early activation of the TGF-ß/Smad signaling pathway. We also demonstrate the beneficial effect of ACE I in alleviating this early fibrogenic process in the heart in UUO/CKD animals.

Effects of Koumine on Adjuvant- and Collagen-Induced Arthritis in Rats.

To examine the effect of koumine, a Gelsemium alkaloid, on two experimental models of rheumatoid arthritis (RA), rats with adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) were administered koumine (0.6, 3, or 15 mg/kg/day) or vehicle through gastric gavage (i.g.). Clinical evaluation was performed via measurements of hind paw volume, arthritis index (AI) score, mechanical withdrawal threshold, organ weight, and by radiographic and histological examinations. Levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and antitype II collagen (CII) antibody were also examined. In rats with AIA, koumine reduced the AI score and mechanical allodynia of the injected hind paw in a dose-dependent manner and significantly inhibited increase in thymus and liver weights. In rats with CIA, koumine inhibited increase in hind paw volume, AI score, and mechanical allodynia in a dose-dependent manner and reduced joint space narrowing. Furthermore, koumine also attenuated the increase in the expression of IL-1ß and TNF-α, as well as the robust increase of serum anti-CII antibodies in response to immunization. These results suggested that koumine effectively attenuated arthritis progression in two rat models of RA and that this therapeutic effect may be associated with its immunoregulatory action.

Anti-allodynic and neuroprotective effects of koumine, a Benth alkaloid, in a rat model of diabetic neuropathy.

Diabetic neuropathy is characterized by progressive degeneration of nerve fibers associated with diabetes mellitus. Antidepressants and anticonvulsants are the mainstay of pharmacological treatment, but are often limited in effectiveness against the core clinical feature of pain. In the current study, we examined the potential effects of koumine, a Gelsemium elegans Benth alkaloid, using a rat model of diabetic neuropathy. Rats were administered intraperitoneally a single dose of streptozocin (60 mg/kg) to induce type 1 diabetes. Koumine was given at a dose range of 0.056-7 mg/kg subcutaneously for one week starting 3 weeks after streptozocin adminstration. Behavioral responses to mechanical stimuli were evaluated every day after streptozocin injection. At 4 weeks after streptozocin injection, sensory nerve conduction velocity (SNCV) and morphological alternation of sciatic nerves were assessed by electron microscopy. Diabetic rats developed mechanical hyperalgesia within 3 weeks after streptozocin injection and exhibited reduced SNCV and impaired myelin/axonal structure. Koumine treatment of diabetic rats decreased neuropathic pain behavior as early as after the first administration. At a dose of 7 mg/kg, koumine was more effective than gabapentin (100 mg/kg), and decreased mechanical sensitivity threshold to a level comparable to healthy control. Repeated treatment of koumine significantly reduced the damage to axon and myelin sheath of the sciatic nerve and increased SNCV, without affecting body weight and blood glucose. These findings encourage the use of koumine in the treatment of diabetic neuropathy.

The active alkaloids of Gelsemium elegans Benth. are potent anxiolytics.

RATIONALE: An increasing number of herbal products has been introduced to treat anxiety and depression. Gelsemium elegans Benth (G. elegans) is a well-known herbal plant in Asia. Four major alkaloids (gelsemine, koumine, gelsevirine, and gelsenicine) have been isolated from G. elegans. Recently, interest has arisen to investigate the pharmaceutical potential of G. elegans alkaloids in the context of neuropsychopharmacology. OBJECTIVES: We investigated whether G. elegans alkaloids are capable of producing anxiolytic and antidepressant effects in mouse models. In particular, we examined whether the anxiolytic action of G. elegans alkaloids is due to the agonist effects of glycine receptor in the brain. METHODS: Two mouse models (elevated plus-maze and light-dark transition model) were used to examine potential anxiolytic effects. Forced swim test and tail suspension test were used to test the antidepressive action of G. elegans alkaloids. Moreover, we also explored the anxiolytic mechanisms of G. elegans alkaloids by intracerebroventricular administration of strychnine, an antagonist of glycine receptor, in the elevated plus-maze. RESULTS: Gelsemine, koumine, and gelsevirine, but not gelsenicine, exhibited potent anxiolytic effects in the two anxiety models. None of the four G. elegans alkaloids exerted antidepressant effects in the two depression models. None of G. elegans alkaloids impaired spontaneous motor activities. The intracerebroventricular administration of strychnine significantly antagonized the anxiolytic effects of gelsemine, koumine, and gelsevirine administrated subcutaneously. CONCLUSIONS: Gelsemine, koumine, and gelsevirine could be developed as the treatment of anxiety-related disorders in human patients. Their anxiolytic mechanism may be involved in the agonist action of glycine receptor in the brain.

Anion binding in aqueous solutions by N-(Isonicotinamido)-N'-phenylthiourea-based simple synthetic neutral receptors. role of the hydrophobic microenvironment of the receptor molecule.

N-(Isonicotinamido)-N'-(substituted-phenyl)thioureas (1a-e, substituent X = p-OCH3, p-CH3, H, m-Br, and m-CF3) have been designed as neutral receptors, in order to prove the influence of conformational issues on the ability to bind anions in aqueous solutions. Compounds 1a-e were shown to create a hydrophobic microenvironment around the thiourea group, favoring hydrogen bonding interactions, by evidence from quantum mechanic calculations, thermodynamic analysis, NMR aromatic current shielding, and comparative anion binding. Referring to N-(substituted-benzamido)thioureas (2a-e, substituent Y = H, m-Cl, m-NO2, m,m-Cl,Cl, and p-NO2), we showed that, for the hydrophobic microenvironment to be operative in aqueous solutions, the amido -NH proton needs to be acidic enough.