Norepinephrine (NA), a stress hormone, can accelerate hair graying by binding to β2 adrenergic receptors (β2AR) on melanocyte stem cells (McSCs). From this, NA-β2AR axis could be a potential target for preventing the stress effect. However, identifying selective blockers for β2AR has been a key challenge. Therefore, in this study, advanced computer-aided drug design (CADD) techniques were harnessed to screen natural molecules, leading to the discovery of rhynchophylline as a promising compound. Rhynchophylline exhibited strong and stable binding within the active site of β2AR, as verified by molecular docking and dynamic simulation assays. When administered to cells, rhynchophylline effectively inhibited NA-β2AR signaling. This intervention resulted in a significant reduction of hair graying in a stress-induced mouse model, from 28.5% to 8.2%. To gain a deeper understanding of the underlying mechanisms, transcriptome sequencing was employed, which revealed that NA might disrupt melanogenesis by affecting intracellular calcium balance and promoting cell apoptosis. Importantly, rhynchophylline acted as a potent inhibitor of these downstream pathways. In conclusion, the study demonstrated that rhynchophylline has the potential to mitigate the negative impact of NA on melanogenesis by targeting β2AR, thus offering a promising solution for preventing stress-induced hair graying.

Paxlovid is a nirmatrelvir (NMV) and ritonavir (RTV) co-packaged medication used for the treatment of coronavirus disease 2019 (COVID-19). The active component of Paxlovid is NMV and RTV is a pharmacokinetic booster. Our work aimed to investigate the drug/herb-drug interactions associated with Paxlovid and provide mechanism-based guidance for the clinical use of Paxlovid. By using recombinant human cytochrome P450s (CYPs), we confirmed that CYP3A4 and 3A5 are the major enzymes responsible for NMV metabolism. The role of CYP3A in Paxlovid metabolism were further verified in Cyp3a-null mice, which showed that the deficiency of CYP3A significantly suppressed the metabolism of NMV and RTV. Pregnane X receptor (PXR) is a ligand-dependent transcription factor that upregulates CYP3A4/5 expression. We next explored the impact of drug- and herb-mediated PXR activation on Paxlovid metabolism in a transgenic mouse model expressing human PXR and CYP3A4/5. We found that PXR activation increased CYP3A4/5 expression, accelerated NMV metabolism, and reduced the systemic exposure of NMV. In summary, our work demonstrated that PXR activation can cause drug interactions with Paxlovid, suggesting that PXR-activating drugs and herbs should be used cautiously in COVID-19 patients receiving Paxlovid.

BACKGROUND: Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary.
OBJECTIVE: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence.
METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence.
RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro.
CONCLUSIONS: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.

UNASSIGNED: Pulmonary fibrosis (PF) is a rapidly progressing and irreversible disease, and the currently available types of clinical drugs are limited and inefficient. In our previous study, we observed that Rhynchophylline (Rhy) hindered tendon adhesion and stimulated the healing of injured tendon structures. Considering the similar mechanisms between adhesion formation and PF, we explored the roles of Rhy in PF.
UNASSIGNED: The cytotoxicity of Rhy was tested by a Cell Counting Kit-8 (CCK-8) assay. The degree of PF was evaluated by Western blot (WB), Masson and hematoxylin-eosin (HE) staining, and hydroxyproline quantification. The Rhy-loaded nanoparticles were prepared through an emulsification sonication technique and characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The release of the Rhy-loaded nanoparticles was tested using the absorbance value of the supernatant. Transcriptome sequencing was performed to determine the downstream target and pathway of Rhy, which was then verified by WB.
UNASSIGNED: In vitro, Rhy decreased Transforming Growth Factor Beta 1 (TGF-β1)-induced abnormal overexpression of fibronectin (FN), collagen I (Col I), α-smooth muscle actin (α-SMA) in a dose-dependent manner in human lung fibroblast (HFL1) cells. In vivo, we confirmed (through Masson staining) that the intraperitoneal injection of Rhy reduced collagen deposition and the fibrotic area in a dose-dependent manner. Our results indicated that the Rhy-loaded nanoparticles intratracheal spray intuitively narrowed collagen deposition, shrank collagen deposition and the fibrotic area (Masson and HE staining), and reduced the expression of fibrosis-related markers (WB). Meanwhile, the lung index value and hydroxyproline content were markedly lower than the bleomycin (BLM)-treated group. By transcriptional sequencing analysis, we identified Receptor Tyrosine Kinase (TEK)-Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/AKT) as the downstream target and pathway of Rhy. It was also observed that Rhy could reverse the TGF-β1-induced TEK and phosphorylated AKT (p-AKT) elevated expression.
UNASSIGNED: Our findings indicate that Rhy constrained PF progression by inhibiting TEK-PI3K/AKT signaling pathway. Hence, this sustainable release system of Rhy is a highly effective therapy to limit PF and should be developed.

BACKGROUND: Uncaria rhynchophylla ([Mi] Jack) (gouteng) exerts antidepressive effects. Rhynchophylline (RH), a major component of U. rhynchophylla, exerts similar pharmacological effects to those of gouteng. Thus, RH may have antidepressive effects.
OBJECTIVE: To investigate the anti-depressive effects of RH in chronic unpredictable mild stress (CUMS)-induced depressive mice. The anti-depressive mechanism of RH determined by measuring the 5-HT levels, the expressions of cAMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in cortex and hippocampus.
METHODS: The behaviors of CUMS-induced depressive mice were measured using an open field test (OFT), forced swimming test (FST), and tail suspension test (TST). 5-HT levels were measured using an ELISA kits. The expressions of BDNF and CREB were determined using western blot test.
RESULTS: RH increased the frequency of rearing and grooming in the OFT and decreased the immobility time in the FST and TST. RH effectively increased the 5-HT level and BDNF and CREB expressions in the cortex and hippocampus.
CONCLUSIONS: Our findings indicate that the antidepressive mechanism of RH is related to increased levels of 5-HT from regulating CREB and BDNF expressions in cortex and hippocampus.

The aim of this study is to overcome the obstacle of the blood-brain barrier (BBB) in therapeutic drugs of Parkinson\'s disease (PD), like rhynchophylline (RIN) entry by intranasal administration and to solve the problem of short residence time of drugs in the nasal cavity by the dosage form design of thermosensitive gel. We first conducted a study of the screening of absorption enhancers and 3% hydroxypropyl-β-cyclodextrin (HP-β-CD) was effective to improve the nasal mucosal permeability of RIN. By adjusting the ratio of different components in order to make the gel with adhesion and rapid gelation which were determined to be Poloxamer 407 (P407) 20%, Poloxamer 188 (P188) 1%, polyethylene glycol 6000 (PEG-6000) 1% and HP-β-CD 3%. In addition, the characterization showed that the thermosensitive gel was network cross-linked, rapidly gelation upon entry into the nasal cavity and was stable as semi-solid state with adhesion as well as sustained release properties. Moreover, pharmacokinetic study was performed to evaluate the bioavailability and brain targeting of RIN thermosensitive gel and which were 1.6 times and 2.1 times higher than those of oral administration. We also evaluated the anti-PD effects of RIN thermosensitive gel in-vitro as well as in-vivo. The results showed that RIN thermosensitive gel was effective in repairing the motor function impairment, dysregulated expression levels of oxidative stress factors, and positive neuronal damage within the substantia nigra and dopamine caused by PD. The constructed intranasal drug administration strategy through thermosensitive gel provided a new choice for targeted treatment of PD together with other central nervous system diseases.

BACKGROUND: Previous studies have documented that rhynchophylline exerts antioxidative and anti-inflammatory effects on ischemic neuronal damage in vitro or in vivo. There is a considerable lack of direct evidence for its role in neural function and neuroplasticity after ischemic stroke.
OBJECTIVE: This study aims to explore the role of rhynchophylline in middle cerebral artery occlusion (MCAO) induced ischemic stroke model and the potential mechanisms.
METHODS: Mice were randomly divided into the following three groups: Sham, MCAO + ddH2O, and MCAO + Rhy(40 mg/kg by oral gavage) groups. Cerebral ischemia was induced by MCAO. Cerebral blood flow was monitored to indicate the success of the ischemic model. The neurological severity score and a series of related behavior tests were performed(after MCAO 3d,7d,14d,21d,28d). Golgi staining and Sholl analysis were used to evaluate the complexity of dendrites and the density of dendritic spines. Immunohistochemistry was used to detect the expression of synapsin I and NeuN.
RESULTS: Administration of rhynchophylline for 7 consecutive days after the onset of cerebral ischemia alleviated the sensory-motor functional defects and ameliorated hippocampus-dependent spatial memory injury as well as reduced the infarct volume induced by MCAO. However, golgi staining and sholl analysis showed that rhynchophylline improved dendritic complexity and spine density as well as the synaptic plasticity. Furthermore,the expression of synapsin I and Neun was significantly reduced after cerebral ischemia and rhynchophylline administration ameliorated the loss of synapsin I.
CONCLUSIONS: Rhynchophylline is a promising treatment for ischemic stroke via improving synaptic plasticity and ameliorating the sensory-motor function.

Muscular atrophy (MA) is a disease of various origins, i.e., genetic or the most common, caused by mechanical injury. So far, there is no universal therapeutic model because this disease is often progressive with numerous manifested symptoms. Moreover, there is no safe and low-risk therapy dedicated to muscle atrophy. For this reason, our research focuses on finding an alternative method using natural compounds to treat MA. This study proposes implementing natural substances such as celastrol and Rhynchophylline on the cellular level, using a simulated and controlled atrophy process. Methods: Celastrol and Rhynchophylline were used as natural compounds against simulated atrophy in C2C12 cells. Skeletal muscle C2C12 cells were stimulated for the differentiation process. Atrophic conditions were obtained by the exposure to the low concertation of doxorubicin and validated by FoxO3 and MAFbx. The protective and regenerative effect of drugs on cell proliferation was determined by the MTT assay and MT-CO1, VDAC1, and prohibitin expression. Results: The obtained results revealed that both natural substances reduced atrophic symptoms. Rhynchophylline and celastrol attenuated atrophic cells in the viability studies, morphology analysis by diameter measurements, modulated prohibitin VDAC, and MT-CO1 expression. Conclusions: The obtained results revealed that celastrol and Rhynchophylline could be effectively used as a supportive treatment in atrophy-related disorders. Thus, natural drugs seem promising for muscle regeneration.

A prominent characteristic of Alzheimer\'s disease (AD) is the deposition of both amyloid-β (Aβ) peptide and tau protein in the brain. Aβ and tau not only induce toxicity through self-aggregation but also induce more potent toxicity through the synergistic action of Aβ and tau. In particular, neurotoxic aggregates of Aβ and tau directly affect several AD pathologies including neuroinflammation and cognitive decline. Therefore, there is increasing interest in strategies to modulate the aggregation and dissociation of Aβ and tau for treatment of AD. Our recent study found that Uncaria rhynchophylla (UR) has a therapeutic effect on AD via the inhibition of Aβ aggregation and attenuating Aβ-mediated pathogenesis of AD. However, no studies have investigated whether UR has anti- and disaggregation effects on both Aβ and tau. In this study, we showed the significant effects of UR on aggregation and dissociation of Aβ42 and tau K18 using a thioflavin T (ThT) assay. In addition, histological study revealed an inhibitory effect of UR on the accumulation of Aβ and tau and AD-related pathologies in 3xTg mice with both Aβ and tau pathology. Furthermore, we found that rhynchophylline and corynoxeine, bioactive components of UR, could modulate the aggregation and dissociation of both Aβ and tau using molecular docking simulation, isothermal titration calorimetry, and ThT assays. In conclusion, our results demonstrate that UR can inhibit the aggregation of Aβ and tau and promote the degradation of their aggregates in AD.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes that can lead to heart failure and death, for which there is no effective treatment. Rhynchophylline (Rhy) is the main effective component of the Chinese herbal medicine Uncaria rhynchophylla, which mainly acts on the cardiovascular and nervous systems. However, its role in protecting against DCM remains unexplored. The present study sought to reveal the mechanism of Rhy in improving type 2 diabetes mellitus (T2DM) myocardial lesions from the perspective of regulating calcium homeostasis in cardiomyocytes. We prepared a mouse model of T2DM using a high-fat diet combined with low doses of streptozotocin. The T2DM mice were given 40 mg/kg of Rhy for 8 weeks. The results showed that Rhy can attenuate cardiac pathological changes, slow down the heart rate, decrease serum cardiac enzyme levels, reduce cardiomyocyte apoptosis, enhance cardiomyocyte contractility, and raise the calcium transient amplitude in T2DM mice. Further, Rhy downregulated the phosphorylation level of ryanodine receptor 2, upregulated the phosphorylation level of phospholamban, protected mitochondrial structure and function, and increased adenosine triphosphate levels in the cardiac tissue of T2DM mice. Our results demonstrated that Rhy may protect against myocardial damage in T2DM mice and promote cardiomyocyte contraction, and its mechanism of action seems to be related to the regulation of intracellular calcium homeostasis.