Hypothalamic NPFFR2 attenuates central insulin signaling and its knockout diminishes metabolic dysfunction in mouse models of diabetes mellitus.

BACKGROUND: The hypothalamus is a crucial brain region that mediates the effects of insulin and leptin signals on peripheral metabolic functions. Previous research has shown that insulin signals in the hypothalamus act via multiple neuronal circuits and anabolic/catabolic pathways that converge on the vagus nerve and sympathetic fibers to coordinate energy metabolism in peripheral organs. Additionally, neuropeptide FF (NPFF) has been identified as a regulator of feeding behaviors and energy homeostasis in the hypothalamus, but the mechanisms underlying its involvement in metabolic control remain unclear. This study aims to explore the underlying mechanisms of NPFF in modulating metabolic disorders. METHODS: In this study, we investigated the physiological role of NPFF in insulin-related energy homeostasis and metabolic health. First, we evaluated the effects of NPFF and its receptors on central insulin signaling using mouse hypothalamic cell lines and Npffr2-overexpressing mice. To further explore the effects of NPFFR2 on insulin-related metabolic disorders, such as diabetes mellitus, we used Npffr2-deleted mice in combination with the streptozotocin (STZ)-induced type 1 diabetes and high-fat diet/STZ-induced type 2 diabetic mouse models. The impacts of central NPFFR2 were demonstrated specifically through Npffr2 overexpression in the hypothalamic arcuate nucleus, which subsequently induced type 2 diabetes. RESULTS: We found that stimulating NPFFR2 in the hypothalamus blocked hypothalamic insulin activity. Npffr2 deletion improved central and peripheral metabolic symptoms in both mouse models of diabetes mellitus, exerting effects on central and systemic insulin resistance, feeding behaviors, glucose and insulin intolerance, lipid metabolism, liver steatosis, and inflammation of white adipose tissues. The overexpression of ARC Npffr2 augmented the metabolic dysregulation in the mouse model of type 2 diabetes. CONCLUSIONS: Our findings demonstrate that hypothalamic NPFFR2 negatively regulates insulin signaling in the central nervous system and plays an important role in maintaining systemic metabolic health, thereby providing valuable insights for potential clinical interventions targeting these health challenges.

Quantitative Assessment of Water Quality Improvement by Reducing External Loadings at Lake Erhai, Southwest China.

To quantitatively evaluate the effects on water quality improvement caused by reducing external loadings entering Lake Erhai through inflow rivers, a one-dimensional hydrodynamic and ecological model (DYRESM-CAEDYM) was set up to simulate the water quality and water level variations. The calibrated and validated model was used to conduct six scenarios for evaluating the water quality responses to different amounts of external loading reduction at Lake Erhai. The results show (1) the total nitrogen (TN) concentration of Lake Erhai will be higher than 0.5 mg/L without any watershed pollution control during April-November 2025, which cannot meet Grade II standard of the China Surface Water Environmental Quality Standards (GB3838-2002). (2) External loading reductions can significantly reduce the concentrations of nutrients and Chla at Lake Erhai. The effects of water quality improvement will be proportional to the reduction rate of external loading reductions. (3) Internal release might be an important source of pollution It needs to be seriously considered as well as external loading for mitigating the eutrophication at Lake Erhai in the future.

Lactate Dehydrogenase-Inhibitors Isolated from Ethyl Acetate Extract of Selaginella doederleinii by Using a Rapid Screening Method with Enzyme-Immobilized Magnetic Nanoparticles.

BACKGROUND: Lactate dehydrogenase (LDH) is one of the important enzyme systems for glycolysis and gluconeogenesis. It can catalyze the reduction and oxidation reaction between propionic acid and L-lactic acid, which is usually overexpressed in cancer cells. Therefore, inhibiting the activity of LDH is a promising way for the treatment of cancer. In this study, an effective method based on ligand fishing and ultra performance liquid chromatography-mass spectrum (UPLC-MS) was established to screen and identify active ingredients from Selaginella doederleinii with potential inhibitory activity for LDH. METHODS: Firstly, LDH was immobilized on the magnetic nanoparticles (MNPs), three immobilization parameters including LDH concentration, immobilization time and pH were optimized by single factor and response surface methodology for maximum (max) immobilization yield. Then, a mixed model of galloflavin and chlorogenic acid (inhibitors and non-inhibitors of LDH) was used to verify the specificity of immobilized LDH ligand fishing, and the conditions of ligand fishing were further optimized. Finally, combined with UPLC-MS, immobilized LDH was used to simultaneously screen and identify potential LDH inhibitors from the ethyl acetate extract of Selaginella doederleinii. RESULTS: The prepared fishing material was comprehensively characterized by scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and fourier transform infrared spectrometer (FT-IR). The optimal immobilization conditions were obtained as LDH concentration of 0.7 mg/mL, pH value of 4.5, and immobilization time of 3.5 h. Under these conditions, the max immobilization yield was (3.79 ± 0.08) × 103 U/g. The specificity analysis showed that immobilized LDH could recognize and capture ligands, and the optimal ligand fishing conditions included that the incubation time was 30 min, the elution time was 20 min, and the concentration of methanol as eluent was 80%. Finally, two LDH inhibitors, amentoflavone and robustaflavone, were screened by immobilized LDH from the ethyl acetate extract of Selaginella doederleinii. CONCLUSIONS: The study provided a meaningful evidence for discovering the bioactive constituents in ethyl acetate extract of Selaginella doederleinii related to cancer treatment, and this ligand fishing method was feasible for screening enzyme inhibitors from similar complex mixtures.

Protective effects of ginsenoside Rg1 against oxygen-glucose-deprivation-induced apoptosis in neural stem cells.

Oxygen-glucose deprivation (OGD) causes neural damages through stroke-induced ischemia. Neural stem cells (NSCs) have been shown to alleviate ischemia-induced neural damages. However, ischemia reduces NSC survival. Ginsenoside Rg1 exerts anti-inflammatory and anti-oxidative effects, and repairs brain injury-related neural damages. We aimed to investigate whether ginsenoside Rg1 could prevent NSCs from OGD insult. Using multiple techniques, we explored neuroprotective effects of ginsenoside Rg1 on OGD-insulted NSCs. 6h treatment of OGD most significantly decreased NSC viability, and 10-20µM ginsenoside Rg1 efficiently protected NSCs against OGD insult. Annexin V-FITC/propidium iodide (PI) double staining results confirmed that ginsenoside Rg1 significantly reduced the OGD-induced apoptosis in NSCs. OGD-insulted NSCs with ginsenoside Rg1 treatment displayed reduced expressions of pro-apoptotic proteins cleaved Caspase3 and Bax, and elevated expression of anti-apoptotic protein Bcl-2 than the NSCs with OGD insult. Moreover, ginsenoside Rg1 reduced OGD-induced oxidative stress, and inhibited the expression of p-p38 and p-JNK2. Ginsenoside Rg1 protects NSCs against OGD-induced cell apoptosis through regulating the expression of apoptotic signal proteins. In addition, ginsenoside Rg1 attenuates OGD-induced oxidative stress and inhibits p38/JNK2 phosphorylation in NSCs. Our study provides solid evidence for neuroprotective effects of ginsenoside Rg1 and reveals the underlying mechanisms.

[Study on pharmacokinetics of geniposide in mice administrated by xingnaojing microemulsion and mPEG2000-PLA modified xingnaojing microemulsion].

An HPLC method for the determination of geniposide concentration in mouse plasma was developed and the pharmacokinetics after intranasal administration of Xingnaojing microemulsion (XNJ-M) and mPEG2000-PLA modified Xingnaojing microemulsion (XNJ-MM) were investigated. Eighty mice were treated by XNJ-M and XNJ-MM nasally. The plasma samples were collected at different times and the drug in samples was detected by HPLC. The pharmacokinetic parameters were calculated by the software of Kinetica. The pharmacokinetic parameters of geniposide of XNJ-M were C(max) (4.36 +/- 2.69) mg x L(-1), t(max) 1 min, MRT (29.73 +/- 4.54) min, AUC (53.63 +/- 14.03) mg x L(-1) x min. The pharmacokinetic parameters of geniposide of XNJ-MM were C(max) (9.75 +/- 4.14) mg x L(-1), t(max) 1 min, MRT(22.34 +/- 2.90) min, AUC (131.87 +/- 40.13) mg x L(-1) x min. Geniposide can be absorbed into blood in a higher degree after intranasal administration with XNJ-MM compared to XNJ-M, which maybe caused by its less irritating and more absorption.

Preparation of mPEG2000-PLA-modified Xingnaojing microemulsion and evaluation in mucosal irritation.

Xingnaojing microemulsion (XNJ-M) administered intranasally is used for stroke treatment. Methoxy poly(ethylene glycol)-poly(lactide) (mPEG-PLA) is a block copolymer with surfactant-like properties and good biodegradability and reliable biological safety. In order to investigate the possibility to lower mucosa irritation with XNJ-M, XNJ-M modified by mPEG2000-PLA (XNJ-MM) was prepared. Different ratios of mPEG2000-PLA were synthesized. The structures and properties were confirmed by 1H-NMR, IR, and DSC. The hydrophile-lipophile balance (HLB) value and critical micellar concentration of copolymers were investigated. The in situ toad palate model was adopted to investigate the ciliotoxicity of the copolymers. The XNJ-M and XNJ-MM were prepared by dropping aqueous phase method. Mucosal irritation of different Xingnaojing (XNJ) preparations was studied by behavioral observations including sneezing and scratching nose. The epithelial thickness of nasal mucosa was evaluated and the secretory protein concentration was determined. The results of in situ toad palate model demonstrated that high HLB value mPEG2000-PLA basically showed no ciliotoxicity, while EL-35 had significant dose-dependent ciliotoxicity (P<0.05). The irritating effects of mPEG2000-PLA were significantly lower than EL-35 after seven days of treatment, based on the epithelial thickness of 0.429±0.100 mm and 0.700±0.035 mm, respectively (P<0.01). The epithelial thickness with XNJ-MM was lower than XNJ-M after seven days of treatment: 0.620±0.10 mm vs. 0.809±0.153 mm, respectively (P<0.05). The mPEG2000-PLA-modified microemulsion is a promising dosage form of XNJ, based on reduced irritation on nasal mucosa.

[Comparative study on rat in situ nasal absorption of geniposide of Xingnaojing nasal drop and Xingnaojing microemulsion].

Xingnaojing (XNJ) is an effective clinical drug used to treat acute stroke. Compared with injection administration, its nasal administration has better brain targeting. Therefore, through nasal administration, XNJ microemulsion could help solve the drug load of compound components of different polarities contained in large-dose and high-concentration traditional Chinese medicines, and reduce irritation to nasal mucosa In this study, the modified volume correction method and the improved rat in situ nasal perfusion model were adopted to compare the nasal absorption of geniposide contained in different XNJ preparations. The results showed that the constant absorption rate of geniposide (GE) in XNJ-D was (2.95 +/- 0.25) x 10(-3) min(-1), whereas the constant absorption rate of GE in XNJ-M was (2.16 +/- 0.21) x 10(-3) min(-1). This indicated that the rat nasal absorption of GE in different XNJ preparations complied with the first-order process and could be considered as passive absorption. GE in XNJ-D was absorbed faster than that in XNJ-M, which provided basis for the development of nasal preparations of XNJ.

[Study on tissue distribution of borneol in mice by intravenous and intranasal administrations].

OBJECTIVE: To develop a GC-FID method to determine borneol's concentration in mouse tissues, and to investigate the tissue distribution after intravenous and intranasal administrations of borneol. METHOD: Mouse brains, hearts, livers, spleens, lungs and kidneys were collected at 1, 3, 5, 10, 20, 30, 60, 90, 120 min after administration of borneol with the dose of 30.0 mg x kg(-1). The drug in tissues was extracted with ethyl acetate, and borneol's concentration detected by GC, with octadecane as the internal standard. RESULT: The calibration curve showed a good linear relationship. Extraction recoveries, inter-day and intra-day precisions and stability were in conformity with the analytical requirements of biological samples. Borneol was mainly distributed in most tissues, more in heart, brain and kidney, and less in liver, spleen and lung. CONCLUSION: The established GC-FID method is applicable for content determination of borneol in tissues. After intravenous and intranasal administrations in mice, borneol is mainly distributed in abundant blood-supply tissues. After intranasal administration, brain tissues showed the highest target coefficient and target effectiveness.

[Transdermal behavior of effective constituents in xuanbi gel plaster].

OBJECTIVE: To study the transdermal absorbability of gentiopicroside, naringin and protosappanin B contained in Xuanbi gel plaster. METHOD: The Franz diffusing cells method was adopted for the in vitro model of rat belly skins. Three indexes, gentiopicroside, naringin and protosappanin B, residued in the accept liquid, skins and plaster were determined by HPLC. RESULT: The penetration rates of gentiopicroside, naringin and protosappanin B were respectively 3.47, 1.59, 2.13 microg x cm(-2) x h(-1). After 24 h, their penetration rates were 25.42%, 11.73%, 17.78%, respectively. The residual quantities of gentiopieroside, naringin and protosappanin B in skin were 0.231, 0.593, 0.568 microg x cm(-2), ith the retention rates of 0.027%, 0.227%, 0.475%, respectively. The amount of residue of gentiopicroside, naringin and protosappanin B in plaster were 2179, 674, 278 microg, with the retention rates of 81.36%, 81.92%, 73.83%, respectively. CONCLUSION: The in vitro transdermal behavior of Xuanbi gel plaster is close to a zero-order process. The residual quantity the retention rate in skins is much lower than the penetration rate and the residual rate in plaster.

Functional characterizations of cocaine- and amphetamine-regulated transcript mRNA expression in rat hypothalamus.

Anatomical relationships of hypothalamic cocaine- and amphetamine-regulated transcript (CART) with other hypothalamic neuropeptides were examined by using in situ hybridization histochemistry and immunohistochemistry. CART mRNA was found in various regions in rat hypothalamus, including the periventricular nucleus, paraventricular nucleus, dorsomedial nucleus, perifornical regions, lateral nucleus, and the arcuate nucleus. In the paraventricular nucleus, CART mRNA is colocalized with vasopressin and corticotropin-releasing factor-containing neurons. Moreover, either fasting or diabetes attenuates CART mRNA expression in the hypothalamus, demonstrating that CART mRNA regulation is related to the fuel availability and peripheral hormonal status. Our findings suggest that the presence of CART mRNA in specific cell groups of the hypothalamus plays a role in hypothalamic regulation of neuroendocrine functions.