POPDC1 Variants Cause Atrioventricular Node Dysfunction and Arrhythmogenic Changes in Cardiac Electrophysiology and Intracellular Calcium Handling in Zebrafish.

Popeye domain-containing (POPDC) proteins selectively bind cAMP and mediate cellular responses to sympathetic nervous system (SNS) stimulation. The first discovered human genetic variant (POPDC1S201F) is associated with atrioventricular (AV) block, which is exacerbated by increased SNS activity. Zebrafish carrying the homologous mutation (popdc1S191F) display a similar phenotype to humans. To investigate the impact of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling, homozygous popdc1S191F and popdc1 knock-out (popdc1KO) zebrafish larvae and adult isolated popdc1S191F hearts were studied by functional fluorescent analysis. It was found that in popdc1S191F and popdc1KO larvae, heart rate (HR), AV delay, action potential (AP) and calcium transient (CaT) upstroke speed, and AP duration were less than in wild-type larvae, whereas CaT duration was greater. SNS stress by ß-adrenergic receptor stimulation with isoproterenol increased HR, lengthened AV delay, slowed AP and CaT upstroke speed, and shortened AP and CaT duration, yet did not result in arrhythmias. In adult popdc1S191F zebrafish hearts, there was a higher incidence of AV block, slower AP upstroke speed, and longer AP duration compared to wild-type hearts, with no differences in CaT. SNS stress increased AV delay and led to further AV block in popdc1S191F hearts while decreasing AP and CaT duration. Overall, we have revealed that arrhythmogenic effects of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling in zebrafish are varied, but already present in early development, and that AV node dysfunction may underlie SNS-induced arrhythmogenesis associated with popdc1 mutation in adults.

Comparison of animal welfare assessment tools and methodologies: need for an effective approach for captive elephants in Asia.

Welfare is a fundamental aspect of animal management and conservation. In light of growing public awareness and welfare concerns about captive elephants, there is an urgent need for comprehensive, globally coordinated efforts for Asian elephants (Elephas maximus) that participate in religious, logging, or tourist activities in range countries where the majority reside, and where welfare issues have been identified but not addressed. This review provides a comparative analysis of available animal assessment tools. Each offers distinct features for assessment that allow institutions to select criteria for specific needs and available resources. Most are applied to general animal welfare assessments, although some are tailored to particular species, including elephants. The tools span diverse formats, from digital to primarily paper-based assessments. Assessments operate at individual and institutional levels and across multiple welfare domains. Methodologies rely on keeper ratings or expert evaluations, incorporate numerical scoring and Likert scales for welfare grading, and encompass inputs including behaviors, health, and physiological indicators. For tourist camp elephants, one challenge is that the tools were developed in zoos, which may or may not have application to non-zoological settings. Digital tools and assessment methodologies such as keeper ratings face logistical challenges when applied across tourist venues. As with any tool, reliability, validity, and repeatability are essential and must address the unique welfare challenges of diverse captive settings. We propose that a holistic, context-specific, evidence-based, and practical tool be developed to ensure high elephant welfare standards in non-zoological facilities throughout Asia.

Improving the meiotic competence of small antral follicle-derived porcine oocytes by using dibutyryl-cAMP and melatonin.

OBJECTIVE: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. METHODS: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. RESULTS: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. CONCLUSION: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

Optimized New Shengmai Powder modulation of cAMP/Rap1A signaling pathway attenuates myocardial fibrosis in heart failure.

BACKGROUND: Optimized New Shengmai Powder (ONSMP) is a traditional Chinese medicine formula with significant anti-heart failure and myocardial fibrosis effects, but the specific molecular biological mechanisms are not fully understood. METHODS: In this study, we first used network pharmacology to analyze the ONSMP's active ingredients, core signaling pathways, and core targets. Second, calculate the affinity and binding modes of the ONSMP components to the core targets using molecular docking. Finally, the heart failure rat model was established by ligating the left anterior descending branch of the coronary artery and assessing the effect of ONSMP on myocardial fibrosis in heart failure using echocardiography, cardiac organ coefficients, heart failure markers, and pathological sections after 4 weeks of drug intervention. The cAMP level in rat myocardium was determined using Elisa, the α-SMA and FSP-1 positive expression determined by immunohistochemistry, and the protein and mRNA levels of the cAMP/Rap1A signaling pathway were detected by Western Blotting and quantitative real-time PCR, respectively. RESULTS: The result shows that the possible mechanism of ONSMP in reducing myocardial fibrosis also includes the use of 12 active ingredients such as baicalin, vitamin D, resveratrol, tanshinone IIA, emodin, 15,16-dihydrotanshinone-i to regulate ß1-AR, AC6, EPAC1, Rap1 A, STAT3, and CCND1 on the cAMP/Rap1A signaling pathway, thereby inhibiting the proliferation of cardiac fibroblasts and reduce the excessive secretion of collagen, effectively improve cardiac function and ventricular remodeling in heart failure rats. CONCLUSION: This research shows that ONSMP can inhibit myocardial fibrosis and delay heart failure through the cAMP/Rap1A signaling pathway.

Multi-omics reveals the testosterone promotion effect mechanism of Cordyceps Sobolifera on Leydig cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sobolifera (CS) has been traditionally utilized as an ethnic remedy for various health conditions, including chronic kidney diseases, anti-fatigue interventions, and management of chronic inflammation. Notably, CS is recognized for its substantial content of bioactive compounds, among which nucleosides prominently feature as constituents with diverse therapeutic advantages. AIM OF THE STUDY: This study aims to investigate the effects of CS on testosterone secretion in Leydig cells and explore the underlying mechanism. MATERIALS AND METHODS: Leydig cells were isolated from rat testes to establish a primary rat Leydig cells model. Cell proliferation and testosterone secretion were assessed via the methyl-piperidino-pyrazole (MTT) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Samples earmarked for RNA sequencing (RNA-Seq) analysis facilitated the identification of significantly differentially expressed genes (DEGs), and we conducted Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analyses. The veracity of our findings was validated through quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. RESULTS: The results showed that CS and guanosine could promote Leydig cell proliferation and bolster testosterone secretion. Our integrative analysis of metabolomics and transcriptomics has unveiled the potential mechanisms governing testosterone synthesis. Specifically, metabolomics has illuminated striking correlations within cholesterol metabolism, and bile secretion. Concurrently, transcriptomics has underscored the pivotal roles played by the cyclic adenosine monophosphate (cAMP) signaling pathway and steroid hormone biosynthesis. Furthermore, our investigation has demonstrated CS's aptitude in elevating the expression of proteins and genes. Notably, our findings have elucidated that these effects can be mitigated by protein kinase A (PKA) and adenylate cyclase (AC) specific inhibitors. CONCLUSION: This study delineates the cAMP-PKA pathways as plausible mechanisms underpinning the testosterone-enhancing properties of CS, with guanosine emerging as a fundamental bioactive constituent.

Hypersampsonone H attenuates ulcerative colitis via inhibition of PDE4 and regulation of cAMP/PKA/CREB signaling pathway.

BACKGROUND AND OBJECTIVES: Ulcerative colitis (UC) is a recurrent intestinal inflammatory disease which poses a serious threat to the life of patients. However, there are no specific drugs for UC yet. Hypericum sampsonii Hance (HS) is a Chinese herbal medicine traditionally used to treat enteritis and dysentery. Our previous studies have demonstrated that HS holds potential anti-UC effects, and a novel compound named Hypersampsonone H (HS-1) isolated from HS possesses significant anti-inflammatory activity. However, the beneficial effects of HS-1 on UC remain unclear. This study aimed to investigate the therapeutic effects of HS-1 on UC and its potential mechanisms, both in vitro and in vivo. METHODS: The in vitro model was employed using LPS-induced RAW264.7 cells to investigate the anti-inflammatory effects of HS-1 and its possible mechanisms. Furthermore, the therapeutic efficacy and potential mechanisms of HS-1 against dextran sulfate sodium (DSS)-induced acute colitis were assessed through histopathological examination, biochemical analysis, and molecular docking. RESULTS: In vitro, HS-1 significantly reduced LPS-induced inflammatory responses, as indicated by inhibiting NO production, down-regulating the overexpression of COX-2 and iNOS, as well as regulating the imbalanced levels of IL-6, TNF-α, and IL-10. Moreover, HS-1 also inhibited the expression of PDE4, elevated the intracellular cAMP level, and promoted the phosphorylation of CREB, thereby activating the PKA/CREB pathway in RAW264.7 cells. In vivo, HS-1 demonstrated therapeutic capacity against DSS-induced colitis by alleviating the symptoms of colitis mice, regulating the abnormal expression of inflammatory mediators, protecting the integrity of intestinal epithelial barrier, and reducing tissue fibrosis. Consistently, HS-1 was found to decrease the expression of PDE4 isoforms, subsequently activating the cAMP/PKA/CREB signaling pathway. Furthermore, the molecular docking results indicated that HS-1 exhibited a high affinity for PDE4, particularly PDE4D. Further mechanistic validation in vitro demonstrated that HS-1 possessed a synergistic effect on forskolin and an antagonistic effect on H-89 dihydrochloride, thereby exerting anti-inflammatory effects through the cAMP/PKA/CREB signaling pathway. CONCLUSION: We disclose that HS-1 serves as a promising candidate drug for the treatment of UC by virtue of its ability to reduce DSS-induced colitis via the inhibition of PDE4 and the activation of cAMP/PKA/CREB signaling pathway.

Jiao-tai-wan and its effective component-berberine improve diabetes and depressive disorder through the cAMP/PKA/CREB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiao-tai-wan (JTW), a classic herbal formula of traditional Chinese medicine recorded in Han Shi Yi Tong, has been used to alleviate sleep disorders since ancient times. In modern pharmacological research, JTW has been adopted for treating diabetes mellitus and even exerts antidepressant effects. However, the potential mechanisms deserve further elucidation. AIM OF THE STUDY: The prevalence of diabetes mellitus combined with depressive disorder (DD) is continuing to increase, yet it is currently under-recognized and its treatment remains inadequate. The present study aims to explore the underlying therapeutics and mechanisms of JTW on DD. MATERIALS AND METHODS: Chronic restraint stress was used on db/db mice to construct a mouse model of DD. The therapeutic effects of JTW were assessed by glucolipid metabolic indexes, behavioral tests, and depression-related neurotransmitter levels. The inflammatory status and cell apoptosis of different mice were investigated and the changes in the cAMP/PKA/CREB pathway were detected. Combining the results of fingerprinting with molecular docking, the active components of JTW were screened. A cellular model was constructed by intervention of glucose combined with corticosterone (CORT). The levels of apoptosis and depression-related neurotransmitters in HT-22 cells were examined, and the changes in the cAMP/PKA/CREB pathway were tested. Finally, the activator and inhibitor of the PKA protein were used for reverse validation experiments. RESULTS: JTW could improve the impaired glucose tolerance, lipid metabolism disorders, and depression-like symptoms in DD mice. Meanwhile, JTW could alleviate the inflammatory status, suppress the microglia activation, and improve hippocampal neuron apoptosis in DD mice. The dual effects of JTW might be associated with the activation of the cAMP/PKA/CREB pathway. Berberine (Ber) was identified for the in vitro experiment, it could reverse the apoptosis of HT-22 cells and up-regulate the depression-related neurotransmitter levels, and the effects of Ber were related to the activation of the cAMP/PKA/CREB pathway as well. CONCLUSION: JTW could exert both hypoglycemic and antidepressant effects through activating the cAMP/PKA/CREB signaling pathway, its active component, Ber, could improve the damage to HT-22 cells induced by glucose combined with CORT via the activation of the cAMP/PKA/CREB pathway. Ber may be one of the effective components of the dual effects of JTW.

Stool-softening effect and action mechanism of free anthraquinones extracted from Rheum palmatum L. on water deficit-induced constipation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese herbal medicine, rhubarb is said to remove accumulation with purgation, clearing heat, and discharging fire. Modern pharmacology has shown that rhubarb extract has a purgative effect when given to experimental animals in an appropriate dose. However, the active components and their mechanism of action are still not clearly defined. AIM OF THE STUDY: The current research aimed to evaluate the synergistic stool-softening effects and explore the action mechanism of rhubarb free anthraquinones (RhA) and their monomers on constipation in rats. MATERIALS AND METHODS: A rat model of water deficit-induced constipation was established to induce constipation, and these rats were treated with RhA and its monomers. ELISA, histopathology, immunohistochemistry, qPCR and Western blotting based on network pharmacology and molecular docking were conducted to explore the possible mechanism of action of RhA and its monomers. RESULTS: RhA, aloe-emodin, rhein, and chrysophanol showed stool-softening activity, and the combination of aloe-emodin and rhein had the strongest softening effect on faecal pellets. Aloe-emodin, rhein, and chrysophanol significantly increased the serum levels of vasoactive intestinal peptide (VIP), motilin (MTL), and substance P (SP), upregulated the expression of VIP, cyclase-associated protein 1 (CAP1), protein kinase A (PKA), cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 3 (AQP3), aquaporin 4 (AQP4), and aquaporin 8 (AQP8), decreased the expression of epithelial sodium channel (ENaC) and Na+/H+ exchanger 3 (NHE3), and reduced the colonic tissue concentration of Na+-K+-ATPase in the constipated rats. Osmolality of colonic fluid in model rats treated by RhA, aloe-emodin, rhein, and chrysophanol was increased. CONCLUSION: Aloe-emodin, rhein, and chrysophanol were the stool-softening components of the RhA extract, and there were certain drug-interactions between the components. RhA upregulated VIP expression, activated the cyclic adenosine monophosphate protein kinase A (cAMP/PKA) pathway, and further stimulated CFTR expression while inhibiting NHE3 and ENaC expression, resulting in a hypertonic state in the colonic lumen. Water transport could then be driven by an osmotic gradient, which in turn led to the upregulation of AQP3, AQP4, and AQP8 expression. In addition, RhA likely improved gastrointestinal motility by increasing serum VIP, SP, and MTL concentrations, thus promoting faecal excretion.

Phosphodiesterase 4 activity uniquely regulates ciliary cAMP-dependent 3T3-L1 adipogenesis.

Recent evidence indicates that the presence of a primary cilium (PC), and of selective cAMP signaling within this smallest of organelles, promotes adipogenic differentiation of 3T3-L1 preadipocytes incubated in media supplemented with either a natural (docosahexaenoic acid, DHA), or a synthetic (TUG-891), free fatty acid receptor 4 (FFAR4) agonist. Indeed, in this earlier work, activation of ciliary FFAR4 in 3T3-L1 cells was correlated with selective increases in PC cAMP and adipogenesis in these cells. However, this study was silent on the role of local PC cAMP phosphodiesterases (PDEs)-mediated events in regulating these adipogenic responses and on the identity of cAMP PDEs that could regulate the "pool" of ciliary cAMP accessed by FFAR4 agonists. In this context, we have identified the PDEs expressed by 3T3-L1 preadipocytes and showed that of these, only PDE4 inhibition promotes FFAR4-mediated adipogenesis. We propose that this work will identify more selective therapeutic targets through which to control adipogenesis, and perhaps the differentiation of other stem cells in which ciliary cAMP is critical.

Moxibustion ameliorates osteoarthritis by regulating gut microbiota via impacting cAMP-related signaling pathway.

BACKGROUND: Osteoarthritis (OA) is a prevalent progressive disorder. Moxibustion has found widespread use in clinical practice for OA, while its underlying mechanism remains elusive. OBJECTIVE: To investigate whether moxibustion can ameliorate OA by influencing the metabolic processes in OA and to elucidate the specific metabolic mechanisms involved. METHODS: C57BL/6J WT mice were randomly assigned to one of three groups: the SHAM group, the ACLT group, and the ACLT+M group. In the ACLT+M group, mice underwent moxibustion treatment at acupoints Shenshu (BL23) and Zusanli (ST36) for a continuous period of 28 days, with each session lasting 20 min. We conducted a comprehensive analysis to assess the impact of moxibustion on OA, focusing on pathological changes, intestinal flora composition, and serum metabolites. RESULTS: Moxibustion treatment effectively mitigated OA-related pathological changes. Specifically, moxibustion treatment resulted in the amelioration of articular cartilage damage, synovial inflammation, subchondral bone sclerosis when compared to the ACLT group. Moreover, 16S rDNA sequencing analysis revealed that moxibustion treatment positively influenced the composition of the flora, making it more similar to that of the SHAM group. Notably, moxibustion treatment led to a reduction in the abundance of Ruminococcus and Proteobacteria in the intestine. In addition, non-targeted metabolomics analysis identified 254 significantly different metabolites between the groups. Based on KEGG pathway analysis and the observed impact of moxibustion on OA-related inflammation, moxibustion therapy is closely associated with the cAMP-related signaling pathway. CONCLUSION: Moxibustion can relieve OA by regulating intestinal flora and via impacting cAMP-related signaling pathway.

Tribuloside acts on the PDE/cAMP/PKA pathway to enhance melanogenesis, melanocyte dendricity and melanosome transport.

ETHNOPHARMACOLOGICAL RELEVANCE: Tribuloside, a natural flavonoid extracted from Chinese medicine Tribulus terrestris L., has shown potent efficacy in treating various diseases. In China, the fruits of Tribulus terrestris L. have long been utilized for relieving headache, dizziness, itchiness, and vitiligo. Water-based extract derived from Tribulus terrestris L. can enhance melanogenesis in mouse hair follicle melanocytes by elevating the expression of α-melanocyte stimulating hormone (α-MSH) and melanocortin-1 recepter (MC-1R). Nevertheless, there is a lack of information regarding the impact of tribuloside on pigmentation in both laboratory settings and living organisms. AIM OF THE STUDY: The present research aimed to examine the impact of tribuloside on pigmentation, and delve into the underlying mechanism. MATERIALS AND METHODS: Following the administration of tribuloside in human epidermal melanocytes (HEMCs), we utilized microplate reader, Masson-Fontana ammoniacal silver stain, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to measure melanin contents, dendrite lengths, melanosome counts; L-DOPA oxidation assay to indicate tyrosinase activity, Western blotting to evaluate the expression of melanogenic and associated phosphodiesterase (PDE)/cyclic adenosine monophosphate (cAMP)/cyclic-AMP dependent protein kinase A (PKA) pathway proteins. A PDE-Glo assay to verify the inhibitory effect of tribuloside on PDE was also conducted. Additionally, we examined the impact of tribuloside on the pigmentation in both zebrafish model and human skin samples. RESULTS: Tribuloside had a notable impact on the production of melanin in melanocytes, zebrafish, and human skin samples. These functions might be attributed to the inhibitory effect of tribuloside on PDE, which could increase the intracellular level of cAMP to stimulate the phosphorylation of cAMP-response element binding (CREB). Once activated, it induced microphthalmia-associated transcription factor (MITF) expression and increased the expression of tyrosinase, Rab27a and cell division cycle protein 42 (Cdc42), ultimately facilitating melanogenesis, melanocyte dendricity, and melanin transport. CONCLUSION: Tribuloside acts on the PDE/cAMP/PKA pathway to enhance melanogenesis, melanocyte dendricity, and melanosome transport; meanwhile, tribuloside does not have any toxic effects on cells and may be introduced into clinical prescriptions to promote pigmentation.

Integrative pharmacology reveals the mechanisms of Erzhi pills, A traditional Chinese formulation, stimulating melanogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi pills (EZP), a traditional Chinese medicine formula prescribed for the treatment of vitiligo, has shown promising efficacy. However, the oral bioactive components and mechanisms underlying the promotion of melanogenesis by EZP remain unclear. AIM OF THE STUDY: This study aimed to investigate the pharmacological basis and mechanism of EZP in promoting melanogenesis. MATERIALS AND METHODS: UHPLC-TOF-MS analysis was used to identify absorbed phytochemicals in serum after oral administration of EZP. Network pharmacology methods were used to predict potential targets and pathways involved in the melanogenic activity of EZP, resulting in the construction of a "compound-target-pathway" network. Zebrafish and B16F10 cells were used to evaluate the effects of EZP on tyrosinase activity and melanin content. Western blot and ELISA analyses were used to validate the effects of EZP on melanogenesis-related proteins, including MITF, TYR, CREB, p-CREB, and cAMP. RESULTS: UHPLC-TOF-MS analysis identified 36 compounds derived from EZP in serum samples. Network pharmacology predictions revealed 89 target proteins associated with the identified compounds and closely related to vitiligo. GO and KEGG analyses indicated the involvement of the cAMP/PKA signaling pathway in the promotion of melanogenesis by EZP. Experimental results showed that EZP increased tyrosinase activity and melanin content in zebrafish and B16F10 cells without inducing toxicity. Western blot and ELISA results suggested that the melanogenic effect of EZP may be related to the activation of the cAMP/PKA signaling pathway. These results confirm the feasibility of combining serum pharmacological and network pharmacological approaches. CONCLUSIONS: EZP have the potential to increase tyrosinase activity and melanin content in zebrafish and cells possibly through activation of the cAMP/PKA pathway.

Waste to Medicine: Evidence from Computational Studies on the Modulatory Role of Corn Silk on the Therapeutic Targets Implicated in Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and/or defective insulin production in the human body. Although the antidiabetic action of corn silk (CS) is well-established, the understanding of the mechanism of action (MoA) behind this potential is lacking. Hence, this study aimed to elucidate the MoA in different samples (raw and three extracts: aqueous, hydro-ethanolic, and ethanolic) as a therapeutic agent for the management of T2DM using metabolomic profiling and computational techniques. Ultra-performance liquid chromatography-mass spectrometry (UP-LCMS), in silico techniques, and density functional theory were used for compound identification and to predict the MoA. A total of 110 out of the 128 identified secondary metabolites passed the Lipinski's rule of five. The Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis revealed the cAMP pathway as the hub signaling pathway, in which ADORA1, HCAR2, and GABBR1 were identified as the key target genes implicated in the pathway. Since gallicynoic acid (-48.74 kcal/mol), dodecanedioc acid (-34.53 kcal/mol), and tetradecanedioc acid (-36.80 kcal/mol) interacted well with ADORA1, HCAR2, and GABBR1, respectively, and are thermodynamically stable in their formed compatible complexes, according to the post-molecular dynamics simulation results, they are suggested as potential drug candidates for T2DM therapy via the maintenance of normal glucose homeostasis and pancreatic ß-cell function.

[Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway].

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1ß, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

Paeoniflorin Alleviates Cisplatin-Induced Diminished Ovarian Reserve by Restoring the Function of Ovarian Granulosa Cells via Activating FSHR/cAMP/PKA/CREB Signaling Pathway.

Paeoniflorin (PAE) is the main active compound of Radix Paeoniae Rubra (a valuable traditional Chinese medicine and a dietary supplement) and exerts beneficial effects on female reproductive function. However, the actions of PAE on diminished ovarian reserve (DOR, a very common ovarian function disorder) are still unclear. Herein, our study investigated the effect and potential mechanism of PAE on DOR by using cisplatin-induced DOR mice and functional impairment of estradiol (E2) synthesis of ovarian granulosa-like KGN cells. Our data show that PAE improved the estrous cycle, ovarian index, and serum hormones levels, including E2, and the number of antral follicles and corpora lutea in DOR mice. Further mechanism results reveal that PAE promoted aromatase expression (the key rate-limiting enzyme for E2 synthesis) and upregulated the FSHR/cAMP/PKA/CREB signaling pathway in the ovaries. Subsequently, PAE improved the levels of E2 and aromatase and activated the FSHR/cAMP/PKA/CREB signaling pathway in KGN cells, while these improving actions were inhibited by the siRNA-FSHR and FSHR antagonist treatments. In sum, PAE restored the function of E2 synthesis in ovarian granulosa cells to improve DOR by activating the FSHR/cAMP/PKA/CREB signaling pathway, which exhibited a new clue for the development of effective therapeutic agents for the treatment of DOR.

Paeonol ameliorates hippocampal neuronal damage by inhibiting GRM5/GABBR2/ß-arrestin2 and activating the cAMP-PKA signaling pathway in premenstrual irritability rats.

Premenstrual dysphoric disorder (PMDD) is a periodic psychiatric disorder with high prevalence in women of childbearing age, seriously affecting patients' work and life. Currently, the international first-line drugs for PMDD have low efficiency and increased side effects. Paeonol, a major component of the traditional Chinese medicine Cortex Moutan, has been applied in treating PMDD in China with satisfactory results, but the therapeutic mechanism is not fully understood. This study aims to evaluate the therapeutic effects and pharmacological mechanisms of paeonol on the main psychiatric symptoms and hippocampal damage in PMDD. We established a premenstrual irritability rat model by the resident-intruder paradigm and performed elevated plus maze and social interactions. And we employed the HE and Nissl staining techniques to observe the therapeutic effect of paeonol on hippocampal damage in PMDD rats. Subsequently, Elisa, qRT-PCR Array, Western Blotting, and cell models were utilized to elucidate the underlying molecular mechanisms through which paeonol intervenes in treating PMDD. In this study, we demonstrated the therapeutic effects of paeonol on irritability, anxiety, and social withdrawal behaviors in rats. In addition, we found that paeonol significantly reduced the serum corticosterone (CORT) level, improved hippocampal morphological structure and neuron number, and reduced hippocampal neuron apoptosis in PMDD rats. Paeonol reduced GRM5, GABBR2, ß-arrestin2, and GRK3 expression levels in hippocampal brain regions of PMDD rats and activated the cAMP/PKA signaling pathway. Inhibitor cell experiments showed that paeonol specifically ameliorated hippocampal injury by modulating the ß-arrestin2/PDE4-cAMP/PKA signaling pathway. The present study demonstrates, for the first time, that paeonol exerts a therapeutic effect on periodic psychotic symptoms and hippocampal injury in PMDD through inhibiting GRM5/GABBR2/ß-arrestin2 and activating cAMP-PKA signaling pathway. These findings enhance our understanding of the pharmacological mechanism underlying paeonol and provide a solid scientific foundation for its future clinical application.

Acupuncture promotes tear secretion by up-regulating VIP/cAMP/PKA/AQP5 signaling in guinea pigs with aqueous tear deficiency dry eye. / 基于VIP/cAMP/PKA/AQP5信号通路探讨针刺治疗水液缺乏型干眼的作用机制.

OBJECTIVES: To observe the effect of acupuncture on the ocular surface symptoms and the protein expression of vasoactive intestinal peptide (VIP) / cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) / aquaporin 5(AQP5) signaling pathway in lacrimal gland tissue of aqueous tear deficiency (ATD) type dry eye model, so as to investigate its mechanism underlying improvement of ATD. METHODS: British shorthair guinea pigs were randomly divided into blank control, model, acupuncture, sham-acupuncture and medication group, with 8 guinea pigs in each group. The ATD model was established by subcutaneous injection of scopolamine hydrobromide (0.6 mg/dose, 4 times/d for 10 days). For guinea pigs of the acupuncture group, filiform needles were inserted into bilateral "Jingming"(BL1), "Cuanzhu"(BL2), "Sizhukong"(TE23), "Taiyang"(EX-HN5), and "Tongziliao"(GB1) for 15 min. For guinea pigs of the sham-acupuncture group, a blunt filiform needle was used to repeatedly prick (not pierce) the skin of the same acupoints mentioned above. The treatment in the above two groups was conducted once daily for 14 days. The guinea pigs in the medication group received administration of sodium hyaluronate eye drops in both eyes, three times a day for 14 days. The objective tests of tear film break-up time (BUT), corneal fluorescein staining score (FLS) and phenol red thread (PRT) test were conducted before and after modeling and after the intervention. After the intervention, the lacrimal index (weight of lacrimal gland/body weight) was calculated. Histopathological changes of the lacrimal gland were observed after H.E. staining. The expression of AQP5 in the lacrimal gland were detected by immunofluorescence, and the contents of VIP and AQP5 in the lacrimal gland were measured by ELISA, the protein expression levels of VIP, cAMP, PKA, p-PKA and AQP5 in the lacrimal gland were detected by Western blot. RESULTS: In comparison with the blank control group, the PRT, BUT, lacrimal index, AQP5 immunoactivity, contents of VIP and AQP5, and protein expression levels of VIP, cAMP, PKA, p-PKA and AQP5 were significantly decreased(P<0.01, P<0.05), and FLS was obviously increased (P<0.01) in the model group . Compared to the model group, the PRT, BUT, lacrimal index, AQP5 immunoactivity, contents of VIP and AQP5, and expression levels of VIP and AQP5 in both acupuncture and medication groups, and the expression levels of cAMP, PKA, p-PKA in the acupuncture group were considerably increased (P<0.01, P<0.05), while the FLS was markedly decreased in both acupuncture and medication groups (P<0.01, P<0.05). Compared with the medication group, the acupuncture group had increased PRT (P<0.05). CONCLUSIONS: Acupuncture intervention is effective in reducing ocular surface damage and promoting tear secretion in guinea pigs with ATD, which may be related to its function in activating VIP/cAMP/PKA signaling, and promoting the expression of AQP5 in the lacrimal gland.

Acupuncture at ST36 ameliorates experimental autoimmune encephalomyelitis via affecting the function of B cells.

Acupuncture at ST36 can alleviate a variety of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), while the specific mechanism for the treatment of EAE is not clear. In this study, we found that acupuncture at ST36 can significantly increase the excitability of splenic sympathetic nerve, and promote the differentiation of peripheral B and CD4+T cells in the anti-inflammatory direction. After blocking the splenic sympathetic nerve with 6-OHDA, this anti-inflammatory effect of acupuncture is partially reversed. In addition, the results of western blot and qPCR showed that acupuncture at ST36 simultaneously activated the ß2-AR-cAMP signaling pathway in the splenic B and CD4+T cells, and this activation was more significant in B cells. In vitro, when CD4+T cells were cultured alone, norepinephrine (NE) had no significant effect on their differentiation. While in the presence of B cells, NE significantly promotes the anti-inflammatory differentiation of B and CD4+T cells. Therefore, the above results reveal that acupuncture can relieve EAE by stimulating the sympathetic nerves of spleen, mainly through acting on B cells to mediate anti-inflammatory effects, and indirectly affecting the function of CD4+T cells.

Fushenmu treatment ameliorates RyR2 with related metabolites in a zebrafish model of barium chloride induced arrhythmia.

BACKGROUND: Fushenmu (Pini Radix in Poria, FSM) is a folk parasitic herb that has been mainly used for palpitation and amnesiain in traditional Chinese medicine (TCM). Recently, as an individual herb or a component of formulations, Fushenmu exhibits therapeutic potential for the treatment of cardiac arrhythmias. Yet, how specific targets or pathways of Fushenmu inhibit arrhythmia has not yet been reported. METHODS: Here, based on clinical functional genomics, metabolomics and molecular biologic technologies, a network construction strategy was adopted to identify FSM therapeutic targets and biomarkers that might explore its functions. RESULTS: In this study, it was found that FSM recovered arrhythmia-associated heart failure in barium chloride (BaCl2) induced arrhythmic zebrafish embryos, as was evidenced by the shortened cardiac sinus venosus-bulbus arteriosus (SV-BA) distance, smaller cardiovascular bleeding areas, and reduced cardiomyocyte apoptosis. Moreover, analysis via ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-QTOF-ESI-MS/MS) components identification and network pharmacology prediction showed that 11 main active components of FSM acted on 33 candidate therapeutic targets. Metabolomic analysis also suggested that FSM could rescue 242 abnormal metabolites from arrhythmic zebrafish embryos. Further analysis based on the combination of target prediction and metabolomic results illustrated that FSM down-regulated Ryanodine Receptor 2 (RyR2) expressions, inhibited adrenaline and 3',5'-Cyclic AMP (cAMP) levels in a dose-dependent manner, which was confirmed by metabolites quantification and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. CONCLUSION: In summary, this study revealed that FSM mitigated BaCl2 induced cardiac damage caused by arrhythmia by suppressing RyR2 expressions, decreasing adrenaline and cAMP through the adrenergic signalling pathway.

Targeting beta-catenin signaling for prevention of colorectal cancer - Nutraceutical, drug, and dietary options.

Progressive up-regulation of ß-catenin signaling is very common in the transformation of colorectal epithelium to colorectal cancer (CRC). Practical measures for opposing such signaling hence have potential for preventing or slowing such transformation. cAMP/PKA activity in colon epithelium, as stimulated by COX-2-generated prostaglandins and ß2-adrenergic signaling, boosts ß-catenin activity, whereas cGMP/PKG signaling has the opposite effect. Bacterial generation of short-chain fatty acids (as supported by unrefined high-carbohydrate diets, berberine, and probiotics), dietary calcium, daily aspirin, antioxidants opposing cox-2 induction, and nicotine avoidance, can suppress cAMP production in colonic epithelium, whereas cGMP can be boosted via linaclotides, PDE5 inhibitors such as sildenafil or icariin, and likely high-dose biotin. Selective activation of estrogen receptor-ß by soy isoflavones, support of adequate vitamin D receptor activity with UV exposure or supplemental vitamin D, and inhibition of CK2 activity with flavanols such as quercetin, can also oppose ß-catenin signaling in colorectal epithelium. Secondary bile acids, the colonic production of which can be diminished by low-fat diets and berberine, can up-regulate ß-catenin activity by down-regulating farnesoid X receptor expression. Stimulation of PI3K/Akt via insulin, IGF-I, TLR4, and EGFR receptors boosts ß-catenin levels via inhibition of glycogen synthase-3ß; plant-based diets can down-regulate insulin and IGF-I levels, exercise training and leanness can keep insulin low, anthocyanins and their key metabolite ferulic acid have potential for opposing TLR4 signaling, and silibinin is a direct antagonist for EGFR. Partially hydrolyzed phytate can oppose growth factor-mediated down-regulation of ß-catenin by inhibiting Akt activation. Multifactorial strategies for safely opposing ß-catenin signaling can be complemented with measures that diminish colonic mutagenesis and DNA hypomethylation - such as avoidance of heme-rich meat and charred or processed meats, consumption of phase II-inductive foods and nutraceuticals (e.g., Crucifera), and assurance of adequate folate status.