Naja annulifera envenomation. We found that N. annulifera venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. N. annulifera venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, N. annulifera venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. N. annulifera venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms.Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement’s contribution to the physiopathogenesis of  Acting via various cell types and receptors, these products stimulate a number of inflammatory events, inducing mast cell degranulation  in vitro, leading to anaphylatoxin generation and soluble Terminal Complement Complex (sTCC) assembly. These events are in part associated with the action of snake venom metalloproteinases (SVMP) and snake venom serine proteinases (SVSP) on central complement components and regulators  and Interleukin 6 (IL-6), neutrophilia, monocytosis, and pulmonary damage  was purchased from LATOXAN Laboratory . The lyophilized venom was reconstituted in sterile saline at 5 mg/mL and stored at -80°C until use. The protein and endotoxin contents were quantified by using a BCA assay protein kit (Pierce) and PYROGENT™ Plus Gel Clot LAL Assay , respectively, according to manufacturers’ recommendations. The endotoxin was present in the venom at a level below the assay’s sensitivity (< 0.125 EU/mL).PMX205 and P32 peptides were synthesized as previously described , 39. Cp4BALB/c male mice (18–22 g) were obtained from the Center for Animal Breeding of Butantan Institute. All procedures involving animals were carried out in accordance with the ethical principles for animal research adopted by the Brazilian Society of Animal Science and the National Brazilian Legislation n˚.11.794/08. The protocols used in the present study were approved by the Institutional Animal Care and Use Committee of the Butantan Institute . Experiments conducted with human samples were approved by the Human Research Ethics Committee of the Municipal Health Secretary of São Paulo. Blood samples were obtained from healthy donors after informed consent .g, and the normal human serum (NHS) was collected and stored at -80°C until use.Blood from normal sheep and rabbits was collected in an equal volume of Alsever solution  and maintained at 4°C. Human blood samples obtained from healthy donors were collected without anticoagulant and allowed to clot at 4°C for 4 h. The blood was then centrifuged at 400 x N. annulifera venom (50 µg) or sterile saline and incubated for 30 min at 37°C. The anaphylatoxins and sTCC in these samples were then quantified using BD™ CBA Human Anaphylatoxin (BD Biosciences) and MicroVue SC5b-9 Plus Enzyme Immunoassay (Quidel) Kits, respectively. AP, CP, and LP activity assays were performed as described (NHS samples (200 µL) were treated with crude escribed , 42.N. annulifera venom (2.5 µg) for 30 min at 37°C in the presence or absence of 1,10-phenanthroline  (10 mM) or phenylmethylsulfonyl fluoride (PMSF) (10 mM) SVMP and SVSP inhibitors, respectively. The reactions were stopped by adding Ethylenediamine Tetraacetic Acid (EDTA) (15 mM), and the mixtures were subjected to electrophoresis on 10% Sodium Dodecyl Sulfate Polyacrylamide Gels (SDS-PAGE)   were incubated with DS-PAGE)  under reDS-PAGE) . In addi®, Colgene, USA), a thrombin-inhibitor anticoagulant, which does not interfere with complement activity. The whole-blood samples were treated with increasing concentrations of N. annulifera venom, ranging from 3.125 to 100 µg/mL, or with sterile saline and then incubated for 30 or 60 min at 37°C. Under these conditions, N. annulifera venom induces the production of inflammatory mediators but does not promote coagulation; however, at higher concentrations (50 and 100 µg/mL), N. annulifera venom is highly hemolytic. Considering that free hemoglobin may be highly inflammatory .The ex-vivo human whole-blood model described by Mollness and colleagues  was usedammatory  and thatN. annulifera venom, human whole blood was pretreated with either the compstatin analog Cp40  , DMSO (SB290157), or 5% glucose (PMX205), for 5 min at room temperature. In addition, N. annulifera venom samples were incubated with 1,10 Phe  or vehicle (ethanol) for 15 min at room temperature. N. annulifera venom was then added to the blood samples, and the mixtures were incubated at 37°C for 30, 60, or 120 min under continuous agitation. Finally, the tubes were centrifuged at 405 × g, for 10 min at 4°C, the plasma was collected, and the samples were stored at -80°C for further quantification of inflammatory mediators.To assess complement’s role in the inflammatory events promoted by , 20 µM) , SB29015, 20 µM) , PMX205 , 20 µM)  or P32   inhibitoN. annulifera venom to induce moderate or severe envenomation illness, respectively  according to the manufacturer’s instructions. The results were expressed as AP, CP, or LP activity [%].BALB/c mice (n=6/group) were injected by the intraperitoneal (i.p.) route with a sublethal (56.5 µg) or lethal (94.2 µg) dose of ectively . As contMice were treated by the subcutaneous (s.c.) route with PMX205 (C5aR1 antagonist) or vehicle (5% glucose solution) at 1 or 2 mg/kg b.wt. at 24 h or 1 h before the induction of local and systemic reactions.N. annulifera venom (BALB/c mice (n=6/group) were each injected with 10 μg of ra venom , dissolvra venom . Mice weN. annulifera venom dose . The tissue samples were examined under a light microscope for the presence of cellular/tissue changes, and a histopathological score was determined.After euthanasia, the lungs were extracted from the mice injected with the lethal 4), prostaglandin E2 (PGE2), and thromboxane B2 (TXB2) were quantified by the LTB4 Enzyme-Linked Immunosorbent Assay (ELISA), PGE2 Monoclonal, and TXB2 ELISA kits, all from Cayman Chemical. All assays were performed according to the manufacturer’s recommendations.Human chemokines were detected by using a BD™ CBA Human Chemokine kit (BD Biosciences). The BD™ CBA Mouse Inflammation kit (BD Biosciences) was used to detect systemic chemokines and interleukins in the mouse plasma. Keratinocyte Chemokine (KC)/C-X-C motif chemokine ligand 1(CXCL1) was detected by using the LEGENDplex™ Mouse Anti-Virus response panel (Biolegend). Mouse myeloperoxidase (MPO) was quantified by using a MPO Mouse ELISA kit (HycultBiotech). leukotriene B4 . Differences were considered significant when p ≤ 0.05.Statistical analysis was performed using Student’s N. annulifera venom able to interact with the human complement system. To determine whether N. annulifera venom could interfere with the complement activity, NHS samples were incubated with the venom and then submitted to hemolytic (AP and CP) and C4b (LP) ELISA assays  for 30 or 60 min at 37°C, and the production of inflammatory mediators was quantified. We found that N. annulifera venom triggered anaphylatoxin generation and sTCC assembly (4 (LTB4), prostaglandin E2 (PGE2), and thromboxane A2 (TXA2) (N. annulifera venom induced the production of the chemokines C-C motif chemokine ligand 2 (CCL2), CCL5 and C-X-C motif chemokine ligand 8 (CXCL8)  by the antagonist SB290157  resulted4, PGE2 (C5a receptor 2 (C5aR2), a second receptor that binds C5a, has been described to act as an immune dampener to C5aR1 and TLR-4, regulating the production of inflammatory mediators . To testN. annulifera venom triggers complement activation by the extrinsic route and that the modulation of the C5a-C5aR1 axis interferes with the immunopathology associated with envenomation in vitro, we chose PMX205 for evaluating complement’s contribution to local and systemic reactions in murine models of envenomation.Given that N. annulifera venom can induce systemic inflammatory reactions in mice  induced a rapid-onset edema in the hind paws of the mice, reaching a maximum peak at 20 min after injection  at 24 h and 1 h before N. annulifera venom injection also promoted KC (murine CXCL8 homolog) chemokine release, which was accompanied by a large neutrophil infiltration, as demonstrated by MPO quantification, that occurred in a C5a-C5aR1 axis-dependent manner, since receptor blockage reduced these inflammatory events , which induces a clinical condition in mice characterized by apathy, bending of the column, a rough hair coat, dyspnea, and difficulty ambulating. This dose also promotes changes in some systemic parameters, including a decrease in circulating lymphocytes and an increase in neutrophils. It also promotes increases in IL-6 and CCL2 plasma levels. All the reactions induced by this dose reached a peak 1 h after venom inoculation, and the values returned to normal by 24 h  , with diffuse alveolar damage (DAD) featured by alveolar collapse, septal inflammation, and a thickening of the alveolar septum  . This trin vitro and in vivo approaches, we have determined that N. annulifera venom induces reduction in complement pathways activity, anaphylatoxins generation and sTCC assembly. These reactions were accompanied by lipid mediators release and production of chemokines and interleukins. C5a-C5aR1 axis signaling is the driver of these effects, since its modulation prevents local and systemic reactions induced by the venom and protects mice against an extensive ALI.Current understanding indicates that snake venoms trigger complement activation in humans –36, but N. annulifera venom induced complement activation in the whole-blood model, with consequent release of pro-inflammatory mediators that included LTB4, PGE2, TXA2, CCL2, and CXCL8. These findings are in line with the increased plasma levels of inflammatory markers observed in an in vivo model of envenomation, indicating that human envenomation by N. annulifera leads to uncontrolled inflammatory reactions and consequent development of ALI/ARDS (acute respiratory distress syndrome), a known cause of respiratory arrest and death, since patients with this inflammatory condition present augmented plasma and pulmonary levels of these pro-inflammatory mediators   ; however spleen) , electro spleen) , pain (7 spleen) , and hem spleen) , 79, 80.50 injection in mice produced a severe envenomation state, with a significant reduction of the complement activity, leukocytosis, neutrophilia, monocytosis, and strong systemic production of IL-6, CCL2, and TNF-α. Furthermore, this venom dose promoted extensive ALI and death. These changes induced in mice by injection of a lethal N. annulifera venom dose, coupled with the in vitro results we obtained from the human whole-blood model, suggest that the physiopathogenesis of envenomation by N. annulifera is similar to that occurring in sepsis and ALI/ARDS  and autoimmune conditions . Thus, considering that eculizumab is an FDA-approved medicine that is already used in the clinic and that N. annulifera venom induces a pathology mainly mediated by C5a, eculizumab may prove useful as a complementary treatment for this envenomation. In addition, PMX205 and Avacopan (CCX168), which are C5aR1 antagonists are also under clinical development for amyotrophic lateral sclerosis . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Butantan Institute .Conceived and designed the experiments: FS and DVT. Performed the experiments: FS and IV-B. Analyzed the data: FS, BC and DVT. Contributed with reagents/materials/analysis tools: DVT, BC, JL, ES, TMW. Wrote the paper: FS and DVT. All authors contributed to the article and approved the submitted version.This work was supported by São Paulo Research Foundation (FAPESP) funding to the Centre of Toxins, Immune Response and Cell Signalling (CeTICS) [grant 2013/07467-1]. DVT is a recipient of the CNPq Research Productivity Fellowship (grant 301358/2017-6).JL is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors for therapeutic purposes. JL is inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. JL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals 7W/POT-4/APL-1 and PEGylated derivatives such as APL-2/pegcetacoplan).The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Personalized drug cocktails have been widely implemented in cancer treatment, due to their merits of using a drug synergistic combination rather than a single effector. Through the incorporation of a cell microarray chip, the usage of both cell amount and reagent can be optimized and effectively reduced in cost. Herein, we present a laser direct writing strategy to fabricate a microcrater-arrayed (µCA) chip incorporated with an automatic liquid handling platform. Each cell droplet with a critical volume of 200 nL containing 100 SK-N-DZ or MCF-7 cells was utilized. The drug synergy screening took less than 30 s for a total of 81 tests. The results show that the potent drug prediction of the µCA chip is more accurate than the conventional 96-well plate assay, which are all compared using zebrafish DiI-labelled tumor xenograft models. Taken together, these findings may impact high-throughput drug screening and personalized drug medicine.Purpose: The aim of this study was to develop a rapid and automatic drug screening platform using microcrater-arrayed (µCA) cell chips. Methods: The µCA chip was fabricated using a laser direct writing technique. The fabrication time required for one 9 × 9 microarray wax chip was as quick as 1 min. On a nanodroplet handling platform, the chip was pre-coated with anti-cancer drugs, including cyclophosphamide, cisplatin, doxorubicin, oncovin, etoposide, and 5-fluorouracil, and their associated mixtures. Cell droplets containing 100 SK-N-DZ or MCF-7 cells were then loaded onto the chip. Cell viability was examined directly through a chemiluminescence assay on the chip using the CellTiter-Glo assay. Results: The time needed for the drug screening assay was demonstrated to be less than 30 s for a total of 81 tests. The prediction of optimal drug synergy from the µCA chip was found by matching it to that of the zebrafish MCF-7 tumor xenograft model, instead of the conventional 96-well plate assay. In addition, the critical reagent volume and cell number for each µCA chip test were 200 nL and 100 cells, respectively, which were significantly lower than 100 µL and 4000 cells, which were achieved using the 96-well assay. Conclusion: Our study for the µCA chip platform could improve the high-throughput drug synergy screening targeting the applications of tumor cell biology.  Cell-based microarray chips have been widely utilized to explain the gaps in limited cell sources, critical reagent volumes, and high-throughput and high-content drug screening ,3,4,5,6.The screening potential of optimal drug combinations for high-throughput and high-content drug screening has been widely studied. For example, the adoption of a feedback control system (FSC) could speed the screening up to 10,000 times more than conventional drug screening assays ,12. LargMicrofluidic technology has attracted numerous potential applications in anti-cancer drug screening, due to its compact size, extremely low dose use, and ready integration with high-throughput analysis setups ,18. For Prior to launching a new drug, mouse animal models have been widely adopted for the pre-clinical trials because of their biological relevance to humans, ability to mimic disease responses in patients, and the great potential for transgenic analysis compared with human trials . PreviouIn recent decades, zebrafish xenografts have been demonstrated to be an attractive alternative to mice. The zebrafish model presents a rapid assay platform, suitable for cellular resolution and drug target identification, and the ability to perform a vast number of transplants ,29,30. ITo address the technology gaps described above, herein we present a microcrater-arrayed cell chip (µCA chip) for facilitating the high-throughput screening of optimal drug combinations. The chip was fabricated using a configurable laser directing writing on a molten wax membrane to perform the arrayed microcrater wells. The array chip was fabricated 60 times (1 min for one 9 × 9 array chip) faster than our previously developed nanodroplet platform (1 h for one array chip) . We demoParaStamp technique [w/w base to cure, SYLGARD 184; Dow Corning, Midland, MI, USA) was performed at 70 °C for 30 s. Afterwards, the PDMS sheet absorbed with liquid-phase paraffin wax was peeled off and immediately stamped onto the examined substrates for another 30 s at room temperature. A glass slide  was used as the substrate. The waxy film coated onto substrates with a thickness of approximately 1 µm, after peeling off the PDMS stamper. The procedures for coating the waxy film on the substrate were partially modified from our previously developed echnique . First, 2 laser engraver . The maximum energy density of the laser was stated to be 3250 J/mm2. Parameters adopted for patterning circular shapes were set as “drill hole” modes, and the drilling time was set from 0.0005 s to 0.3 s with 4% power and a 10 mm/s moving velocity. The corresponding energy densities applied could be evaluated as 0.065 to 39 J/mm2. For patterning with line shapes, parameters were set as “cutting” modes, and the moving velocities were set from 5 mm/s to 70 mm/s with 4% power and an identical length of 10 mm. The energy densities were then evaluated as 260 to 18.6 J/mm2 accordingly. The activity of laser writing on the examined substrates was performed using a CO2, and the measured diameter of the patterned circles was approximately 900 µm. The center-to-center distance between nearby patterns was 2.2 mm. Neuroblastoma SK-N-DZ  and breast cancer MCF-7  cells were used for the proof-of-concept demonstration with the anti-cancer drugs cyclophosphamide , cisplatin , doxorubicin , oncovin (Onc), etoposide , and 5-fluorouracil . SK-N-DZ cells were maintained in Dulbecco’s modified Eagle medium , supplemented with 10% FBS and 1% P/S. MCF-7 cells were maintained in α-minimum essential medium , supplemented with 10% FBS and 1% P/S. A 9 × 9 array with circular patterns was first performed on a waxy film-coated glass slide by using the laser writing technique described above. The parameter was set as “drill hole” mode, and the drilling time was set as 0.2 s with a 4% laser power and a 10 mm/s moving velocity. This resulted in an energy density of 26 J/mm50 values were fitted from a four-parameter logistic equation:Y is the normalized cell viability; X is the drug concentration; and maxY and minY are the upper and lower plateaus of the curve of dose–response, respectively. maxY was set as the control output, and minY was set as 0. HillSlope defined the steepness of a drug response profile. For drug screening, first, all drug droplets with designed concentrations were dispensed onto the corresponding wells carried by a programmable liquid dispenser . Drugs were initially diluted in deionized (DI) water, and the volume of each droplet was set to 200 nL. The chip was then placed at room temperature for 10 min to dehydrate the drug droplets. Subsequently, cell droplets with a nL volume of 200 (100 cells per droplet) were dispensed into the well and then incubated for 1 day. Live cells were assessed by staining with CellTiter-Glo  and detected using a luminescent image analyzer . Cell viability with corresponding luminescent intensity was evaluated using a Fiji imaging macro software and normalized to the untreated cells. The ICFor drug evaluation performed in standard 96-well plates, media containing cancer cells with a density of 4000 cells per well were tested. The concentrated drug solutions were then added to the corresponding wells, leading to the equivalent drug concentrations compared to those used in the cell microarray assay. The final volume was 100 µL per well. A plate reader was used to determine the cell viability following the MTT  assay . Zebrafish (wild-type Danio rerio) were handled  according to standard protocols as well as NTU regulations of the Animal Care and Use Committee. Zebrafish embryos were obtained from mating adults under standard mating conditions. Prior to transplantation with MCF-7 cells (300 cells in 0.01 µL of cell culture medium), 48 h post fertilization (hpf) zebrafish embryos were anesthetized  and then transferred to a 0.1% agarose-coated dish for cell injection. DiI-labeled cells in 10 nL of cell suspension were injected into the yolk sac of the anesthetized embryos at room temperature, after which the xenografts were kept at 34 °C until the end of experiment. Cell tracker DiI dye  is a fluorescent dye (excitation at 553 nm and emission at 570 nm) available for monitoring cell movement or location during long-term observation and was well suited for the zebrafish models. Injected embryos were transferred to a 96-well plate (one embryo/well) containing drug diluted in 200 μL of E3 media (without methylene blue) 1-day post injection (dpi). Following this, the zebrafish xenografts were randomly distributed into the drug treatment groups designed for 2 days. The detection of fluorescence intensity (corresponding to the tumor growth rate) was performed at 1 dpi and 3 dpi using a fluorescence microscope using identical acquisition settings. The images were analyzed using Fiji ImageJ software. At the end of the experiments, embryos were euthanized by tricaine overdose. Acute toxicity testing on zebrafish embryos was performed following the Organization for Economic Co-operation and Development (OECD) guidelines for the fish embryo toxicity (FET) test on embryos. We used 48 h post fecundation (hpf) zebrafish embryos to perform the test. The embryos were treated with the designed drugs for 96 h. The tested concentrations were as follows: 10 (33.2 μM), 20 (66.4 μM), 30 (99.6 μM), 50 (166 μM), and 100 (332 μM) μg/mL for Cis; 1000 (7760 μM), 2000 , 3000 , 5000 , and 10000  μg/mL for 5-Fu; 100 (383 μM), 200 (766 μM), 400 (1532 μM), 500 (1915 μM), and 1000 (3830 μM) μg/mL for Cyp; 5 (8.5 μM), 10 (17 μM), 20 (34 μM), 50 (85 μM), and 100 (170 μM) μg/mL for Eto. The embryos were examined to determine mortality and the maximum non-lethal concentration (MNLC). Each test was performed for 20 embryos. The MNLC for fish was defined as the mortality rate ≤10%. t test was used to compare data from the two groups. Tukey HSD and one-way ANOVA tests were used to compare data from more than two groups. p < 0.05 was considered to be statistically significant.Student’s 2 laser to realize laser direct writing on wax-coated substrates. A schematic of the working principle is shown in 2 of dot laser energy. With sufficient laser energy applied, an opening in the waxy film was achieved from the glass slide substrate. The fabricated patterns typically possessed a crater-like microstructure along with the boundary, with a height of 4 µm. This suggests that the microcrater resulted from the accumulation of molten wax around the boundary. A dimensional saturation of fabricating the circle or line pattern is shown in We employed a COTo further compare the patterning efficiency, that is, whether there is relatively less or no residual wax within the pattern, we compared the water contact angle (WCA) and the surface material composition using Raman spectra  with and without laser treatments a,b. The The µCA chip with microcrater well structures was fabricated by laser direct writing a. The de50 values evaluated from 50 values derived from the chip were 121.6 ± 34.9 µg/mL (403.7 ± 115.9 μM), 13,767 ± 775 µg/mL , 3195 ± 381 µg/mL , and 1592 ± 1209 µg/mL (2706 ± 2055 μM) for Cis, 5-Fu, Cyp, and Eto, respectively. The IC50 values derived from the 96-well plate were 20.3 ± 3.8 µg/mL (67.4 ± 12.6 μM), 5950 ± 2042 µg/mL , 3928 ± 240 µg/mL , and 33.0 ± 2.0 µg/mL (56.1 ± 3.4 μM) for Cis, 5-Fu, Cyp, and Eto, respectively. From the 96-well plate assay, the IC50 values of Cis, 5-Fu and Eto were significantly lower than those of the µCA chip. Instead, the Cyp-treated cells derived a higher IC50 value from the 96-well plate assay than the µCA chip. p < 0.01), whereas no significant inhibition was achieved by such a drug combination in the 96-well plate. To leverage the efficiency of comparing in vitro and in vivo assays, zebrafish tumor xenograft models were used for comparison a. Engraf50. The MNLC for Cis, 5-Fu, Cyp, and Eto were 50 µg/mL (166 μM), 3000 µg/mL , 400 µg/mL(1532 μM), and 20 µg/mL (34 μM), respectively. In future research comparing these results with human samples, we will correlate the selections with human plasma concentrations. It is well known that the drug uptake in zebrafish models may differ from that of mammals, especially through the gill cell barrier and/or intestine. Despite the above issue, many compounds have been revealed to attenuate disease in a similar way to mouse models, such as 5-Fu . In thisThe evaluation of cell viability using DiI labeling in zebrafish xenograft models has been widely used for different cancer cell models, such as melanoma cells  and leuk2 laser micromachining has been widely adopted in numerous biomedical applications, owing to its rapid and maskless prototyping potential [COotential ,36,37,38Our previous study demonstrated that the microscale cell-culture microenvironment can benefit drug screening efficacy, as compared with a 96-well plate assay and an in vivo mouse model . This reThree-dimensional (3D) cell cultures were demonstrated to be able to reflect the in vivo-like microenvironment more effectively . Our preIn this paper, the tested drugs were first dispensed onto the surfaces of microarrays and then dried, which was followed by the loading of cell suspensions. This might result in the distinction of drug impact on cells as compared with the 96-well plate assay. The re-dissolved drugs might cause lower concentrations acting on cells, even though we previously revealed that the “first-deposition” and “post-deposition” of Cis drugs in 96-well plates have no significant difference in their drug response . Many drThe other reason causing the above distinction might be due to using different assay kits, including MTT for 96-well plates, CellTiter-Glo for microchips, and CM-DiI for zebrafish xenografts. Considering all the above discussions, indeed, the chip platform’s prediction of drug treatment partially reflects the xenograft results better than the multi-well systems. Most importantly, the time required for chip fabrication by laser direct writing in this study was 60 times faster than our previous approach using wax stamping  . In addiWe have presented a microcrater-arrayed cell chip (µCA chip) to improve anti-cancer drug synergy screening. In addition, this approach was successfully demonstrated using a zebrafish tumor xenograft model. All of these outputs were provided by the developed µCA chip platform. The results show that the potent drug prediction of the µCA was more accurate than the conventional 96-well plate assay, which was compared with the zebrafish tumor xenograft models. In conclusion, our study opens a new avenue for high-throughput drug synergy screening, which is anticipated to ignite innovative applications for tumor cell biology.


Network pharmacology further revealed 33 key targets and 75 KEGG pathways as possible pathways and targets involved in WK-mediated treatment, with the CaMKII/CNCA1C/Ca2+ pathway being the most significantly affected. This finding was validated using an AC-induced rat arrhythmias model. Pretreatment with Wenxin Keli reduced the malignant arrhythmias and shortened RR, PR, and the QT interval. Wenxin Keli exerted some antiarrhythmic effects by inhibiting p-CaMKII and intracellular Ca2+ transients and overexpressing CNCA1C. Thus, suppressing SR Ca2+ release and maintaining intracellular Ca2+ balance may be the primary mechanism of Wenxin Keli against arrhythmia. In view of the significance of CaMKII and NCX identified in this experiment, we suggest that CaMKII and NCX are essential targets for treating arrhythmias.This study employed a systems pharmacology approach to identify the active compounds and action mechanisms of Wenxin Keli for arrhythmia treatment. Sixty-eight components identified  Arrhythmias are a group of conditions that cause the heart to beat irregularly . It may Codonopsis pilosula (Franch.) Nannf., Panax notoginseng (Burkill) F.H.Chen, Nardostachys jatamansi (D.Don) DC., Amber, and Polygonatum kingianum Collett & Hemsl. It is reported to be effective in arrhythmias treatment  is handy in arrhythmia treatment. TCM is widely and frequently used in China and abroad . Wenxin reatment  and is treatment . It alsoreatment . NeverthThis study employed a systems pharmacology approach to investigate the pharmacological mechanisms of WK . The potThe UPLC/Q-TOF-MS was employed to analyze aqueous solutions and serum components of WK qualitatively. The identified components were regarded as the potential active ingredients of WK.in silico approach was employed to identify the target proteins for the possible active ingredients (components identified from 2.1.1) of WK. Predictive models including the TCMSP database (http://ibts.hkbu.edu.hk/LSP/tcmsp.php), STITCH (http://stitch.embl.de/) (http://www.swisstargetprediction.ch/), Target-Prediction (http://prediction.charite.de/index.php?site=chemdoodle_search_target), and DrugBank (http://www.drugbank.ca/) were used to predict the target proteins of WK.Target Proteins of WK: TCM formulas can effectively prevent the devastating effects of complex diseases through the synergistic effects of multiple compounds and targets . Therefombl.de/) , Swiss Thttp//triad.fsm.it/cardmoc/).Target Proteins of Arrhythmia: Targets related to arrhythmia were obtained from “The gene connection for the heart” database  network based on the candidate compounds of WK and the potential targets to understand the complex interaction of compounds and their corresponding targets at a systems level.Protein-protein Interaction (PPI) Network: Networks are used to view global relationships between nodes. In this study, a PPI network was used to identify potential target proteins associated with arrhythmia based on the arrhythmia targets identified in 2.1.2 using the Cytoscape 3.8.1 Bisogenet app .Experimental approaches for determining the targets of candidate drugs are costly, labor-intensive, and time-consuming. In this study, a systems pharmacology approach was used to determine the key targets through which WK exerts an antiarrhythmic effect. The Merge tool of the Cytoscape 3.8.1 app was employed to determine the common targets of the C-T network and PPI network. They were regarded as the key targets by WK in arrhythmia treatment.https://david.ncifcrf.gov/). An incorporated arrhythmia pathway was then assembled based on the basic pathway information by picking out the closely linked pathways related to arrhythmia pathology.Signaling pathways are essential components of the system pharmacology that link receptor-ligand interactions to pharmacodynamics outputs . An incoAcetonitrile  and formic acid  were purchased from Fisher Scientific . Purified water was acquired from a Milli-Q system . Medlab RU/4C50R multi-channel physiological signal acquisition and processing system were purchased from Shanghai Tongyu Teaching Instrument Manufacturing Co., Ltd (batch No. 17060155). WK was purchased from Shandong Buchang Pharmaceutical Co., Ltd, Tianjin, China . Aconitine (AC) was sourced from Baoji Chenguang Biotechnology Co., Ltd., Shanxi, China (batch No. ha001198). Pentobarbital sodium was purchased from Baoji Chenguang Biotechnology Co., Ltd., Shanxi, China (batch No. B21882). In the same line, the p-CaMKII rabbit antibody was sourced from Abcam (ab32678), the GAPDH rabbit antibody from Hangzhou Xianzhi biology Co., Ltd, China (AB-P-R 001), and the HRP labeled Goat anti-rabbit second antibody from Wuhan bode Bioengineering Co., Ltd, China (BA1054). The cDNA synthesis kit (K1622) was purchased from ThermoFisher Scientific, United States.WK (5 g) was dissolved in 20.6 ml purified water (Nongfu Spring) and left to stand for 1 h. The solution was then filtered through four layers of gauze to filter out the dregs. The filtrate was subsequently centrifuged for 10 min at 10,000 rpm and 4°C, and 300 ml of the supernatant was stored at −20°C. The supernatant was diluted four times and filtered through a 0.22 μm filter membrane before injection for UPLC/Q-TOF-MS determination.Blood samples (0.5 ml) were collected into serum tubes from the eye sockets of rats after 0.5, 1, 1.5, and 2 h of WK administration followed by immediate centrifugation at 3,500 rpm for 15 min at 4 C. The extracted serum was then subjected to UPLC/Q-TOF-MS determination.18 column  at a column temperature of 45°C and 5 μl injection volume. The sample was eluted at a flow rate of 0.3 ml min−1 in a gradient elution program of A (0.1% formic acid: water) and B (0.1% acetonitrile: water): 0–6 min (98–78% A); 6–10 min (78–70% A); 10–14 min (70–62% A); 14–18 min (62–62% A) 18–22 min (62–40% A); 22–26 min (40–0% A); 26–28 min (0–0% A); 28–29 min (0–98% A) 29–30 min (98–98% A). MS detection was performed on a high definition MS (HDMS) system  with negative and positive electrospray (ESI) modes. Its sufficient sensitivity ensured that as many putative compounds as possible were identified. The optimized operating parameters were capillary voltage; 3.0 kV(Positive)/2.0 kV(Negative), source temperature; 120°C (Positive)/110°C (Negative), desolvation gas temperature; 450°C, desolvation gas (N2) flow rate; 800 L/h, collision energy (CE); 25 V, scanning time; 0.1 s, and scanning time interval; 0.02 s. Leucine-cerebral peptide solution (Waters) at a concentration of 200 pg/ml was used as the Lock-Spray calibration solution to ensure the accuracy and repeatability of the mass-to-charge ratio. The exact mass-to-charge ratios in positive and negative ion modes were [M+H]− = 556.2771 and [M-H]− = 554.2615, respectively. The data acquisition range and time were m/z 50–1000 and 0–30 min, respectively.Chromatographic separation was performed on an ACQUITY UPLC BEH CClean grade male Sprague Dawley  rats aged 6–7 weeks and weighing 220 ± 20 g were obtained from the Experimental Animal Center of the Institute of health and environmental medicine, Academy of Military Medical Sciences, PLA, [license no. SYXK(Jin) 2014-0002]. All the animal-based experiments were approved by the Animal Care Committee of the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences and performed following the relevant guidelines and regulations. Efforts were also made to minimize animal suffering. All rats were housed in the specific pathogen-free (SPF) animal laboratory of the experimental animal center of the Institute of Radiation Medicine Chinese Academy of Medical Sciences at a temperature of 23°C ± 2°C and 35% ± 5% relative humidity. The animals were acclimatized to the environment for 1 week with free access to a standard pellet diet before initiating the experiments. The rats were randomly divided into six groups, each comprising eight animals: the control (C) group, model (M) group, low-dose WK-treated (LD) group, medium-dose WK-treated (MD) group, high-dose WK-treated (HD) group, and the amiodarone (A) group. Intragastric administration of WK was once a day for 14 consecutive days in groups LD (2.41 g/kg/d), MD (4.82 g/kg/d), and HD (9.64 g/kg/d) . Rats in group A were administrated with amiodarone (40 mg/kg), while those in groups C and M were administrated with an equal volume of distilled water.The rats were intraperitoneally anesthetized with 0.6% pentobarbital 30 mg/kg an hour after the last WK administration. They were then fixed on the plank in a prone position, followed by the insertion of electrodes into the subcutaneous tissue of the limb. The ECG parameters were subsequently recorded in each group using a Medlab RU/4C50R multi-channel physiological signal acquisition and processing system via a standard limb lead II. Rats in the other five groups, except those in group C, were subsequently injected with 0.001% AC 40 µg/kg after ECG stabilization to establish the arrhythmia model. The injection was through the caudal vein at a rate of 0.2 ml/min. Equal volumes of 0.9% normal saline were injected into rats in group C. The ECG of the rats was then observed in real-time for 20 min after the heart rhythm had normalized. The parameters measured included the initial time of arrhythmia and the types, occurrence, and duration of various VAs.All animals were euthanized after ECG detection, and their hearts were immediately harvested and stored in liquid nitrogen awaiting WB and qRt-PCR analysis.The proteins were separated on a 10% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were subsequently incubated with p-CaMKII rabbit antibody at 4°C overnight and then washed thrice with Tris-buffered saline (TBS) containing Tween 20 (TBST) to wash off the excess antibodies. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. ECL visualization was then performed, followed by calculating the gray values using the Image J software . The intensity of the target proteins was normalized to that of an internal reference to determine their relative expression level.2+ pathway was validated using Real-time Quantitative PCR (RT-qPCR) analysis. The total RNA of the myocardial pieces was first isolated using the Trizol reagent , following the manufacturer’s instructions. The RNA samples (5 μg) were then reverse-transcribed to complementary DNA (cDNA) using a cDNA synthesis kit, followed by quantitative Rt-PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems). The primer pair used was CAMKⅡ forward; 5′-CAAGTTCATCGAGGTCACCAC-3′ and reverse; 5′-ATACACAGCTCTCGTCCTCTG-3′, with RNA U6 as the internal control. The miR-1 specific primer sequences for quantitative real-time PCR were used for TaqMan MicroRNA Assays . The expression levels of the miR-1 were subsequently normalized to U6 RNA expression levels and then calculated using the 2−ΔΔCt method  with a ×40 magnification, 1.3 numerical aperture oil immersion objective, and axial resolutions of 1.5 μm. Fluo-4 AM: Fluo-4AM+8μlDMSO+2μlF-127 was first prepared and then diluted 1,000 times using 1 ml DMem+1 μl Fluo-4. Cardiomyocytes were then incubated with 500–10,002 μl Fluo-4 AM  for 20 min at 37°C and recorded in normal Tyrode’s solution. Fluo-4 was excited at 488nm, followed by measurement of the fluorescence emission at 505 nm. Images were acquired in the line-scan (X-T) mode with 512 pixels  per line at a rate of 3 ms per scan. The Ca2+ transients were then analyzed using a modified version of the MATLAB program, and their fluorescence emission intensity was expressed as F/F0, where F0 was the basal fluorescence intensity level. The recording was performed at 35°C.Calcium imaging was carried out at room temperature within 6 h after isolation of cardiomyocytes from rats in the control group . Ca2+ trt-test, while those between multiple groups were analyzed using one-way analysis of variance . p < 0.05 indicated significant differences between groups.Data were expressed as the means ± SEM. Differences between the two groups were analyzed using the Student’s two-tailed in vitro and in vivo, which were regarded as the potential active components in this study  revealed 61 targets related to arrhythmia  and 429 compound-target interactions. The mean degree value (the number of targets associated with it) of the candidate compounds was 41. Twelve compounds possessed a degree larger than 14 (2-fold degree median), indicating that they regulated multiple targets to exert varying therapeutic effects. Specially, five compounds, including Atractylenolide II, Tanshinone IIA, ginsenoside Rh2, Ginsenoside Rg1, and Jatamansinol, acted on 28, 24, 21, 20, and 16 targets, respectively. The compounds were regarded as the crucial active compounds for WK because of their prime positions in the network.PPI network of arrhythmia: There were 1395 targets closely related to known arrhythmia targets. They were regarded to be indirectly related to arrhythmia.Analysis of the common targets of WK and arrhythmia revealed 33 key targets . These tp-values. 2+ of the calcium signaling pathway were thus selected as the observation index in this study based on these findings combined with other relevant literature.The pathway information of the 33 key targets revealed 75 KEGG pathways, including the calcium, oxytocin, adrenergic signaling in cardiomyocytes, adipocytokine, and dilated cardiomyopathy signaling pathways. The pathways were closely associated with arrhythmias  based on2+ current (LTCC) plays a crucial role in the ECC. In the same line, membrane depolarization in ventricular myocytes during the action potential (AP) causes synchronous activation of multiple LTCCs, consequently triggering the release of Ca2+ from the sarcoplasmic reticulum (SR). These occurrences increase the intracellular Ca2+ that initiates contraction. Ca2+ influx via the LTCC provides a multifunctional signal that triggers muscle contraction, controls AP, and regulates gene expression (2+ leakage (2+ leak in return initiates early afterdepolarizations (EADs)  is an essential aspect of arrhythmia pathophysiology. The L-type Capression . As such leakage . The dias (EADs)  and delas (EADs) .2+ pathway. The assumption is that WK suppresses CaMKII phosphorylation and CACNA1C activation and antagonizes the intracellular Ca2+ overload to avoid the occurrence of EADs and DADs. CaMKII, CACNA1C, and Ca2+ concentration were thus selected as the observation indices of this study.Therefore, we hypothesized that WK could inhibit arrhythmias by regulating the CaMKII/CACNA1C/Ca1) Mortality: There were no deaths in groups C, HD, and A during the experiments. However, two animals in group M and one animal in the groups LD and MD died. The animals died of ventricular fibrillation (VF) after pleomorphic ventricular tachycardia (Polymorphic VT). Nonetheless, the ECG recording time of the dead rats was very short and thus could not be compared with that of other rats. As such, we only analyzed the ECG of the surviving rats. All groups had different degrees of arrhythmia except group C.p > 0.05; p < 0.01; 2) The initial time and duration of arrhythmias: There were insignificant differences in the initial time and duration of arrhythmias among the five experimental groups (3) Types of arrhythmias: VT and bigeminy were observed in the five experimental groups. However, VP was observed in groups M, LD, and A, normal ECG was observed in groups M, LD, and HD, while VF was observed in group LD .4) ECG parameters: Compared with ECG parameters before the experiment, R-R, P-R, and Q-T interval did not change in groups C and M. However, they were prolonged in groups LD, MD, and A, and shortened in group HD . In the p < 0.05). Notably, group HD had a significantly lower level of p-CaMKII than group C (p < 0.01), suggesting that a high WK dose markedly inhibited the expression of CaMKII  than group C, suggesting that AC could inhibit CACNA1C to induce arrhythmia. Compared with group M, the expression of CACNA1C increased in all groups except group LD after treatment of WK and amiodarone, with group HD exhibiting the most significant increase (p < 0.01). Notably, group HD had a significantly higher level of CACNA1C than group C (p < 0.01), suggesting that a high WK dose markedly increased the expression of CACNA1C  to evaluate the time-course effects of WK on intracellular Ca2+ homeostasis. WK significantly reduced the resting Ca2+ ratio and the amplitude of Ca2+ ratio within 5 min after adding WK (p < 0.01). WK also induced a decrease in the cytoplasmic Ca2+ ratio within 5 min in a time-dependent manner  (1) Promote Naolarized . Large Nies (TA) , therebyies (TA) .2+ Overload: Abundant Na+ influx into the cytosol causes Ca2+ overload via sequential activation of electrogenic Na+-Ca2+ exchanger (NCX), thus inducing DAD and TA  Lead to CaD and TA . As a reuinidine  in clini+ Currents: AC significantly increases Ca2+ influx, inhibits outward K+ currents, prolongs repolarization, and produces DAD and reentry, resulting in arrhythmias (3) Inhibition of Outward Khythmias .2+ influx. It triggers the release of Ca2+ via the Ca2+ release channels, ryanodine receptors (RyRs) of the SR. During normal AP, early LTCC peaks trigger robust SR release followed by partial inactivation because of two processes: Ca2+-and voltage-dependent inactivation. LTCC regulates AP repolarization and is vital in developing cardiac arrhythmias such as VF, the leading cause of sudden cardiac death (2+ elevation (2+ influx through LTCC (4) Inhibition of LTCC: LTCC is activated upon membrane depolarization and Caac death . Studiesac death . The effac death . Moreovelevation . In contugh LTCC . These d2+ overload and apoptosis via the p38 signaling pathway (5) Others: AC also stimulates the vagus nerve and inhibits the sinoatrial node and conduction system, resulting in a slowed heart rate and conduction block . Moreove pathway .AC is a major bioactive diterpenoid alkaloid derived from aconitum plants. It has been used in the past as an antipyretic, analgesic, antirheumatic drug, and a neurotransmission inhibitor . However, and VF . AC-indu, and VF , decreas, and VF , and leaIn the present study, the antagonistic effects of WK on AC-induced arrhythmia were observed in rats. ECG recording experiments demonstrated that AC administration decreased the heart rate and prolonged the Q-T intervals, leading to VP, coupled rhythm, and tachycardia. These occurrences ultimately induced VF and rat mortality. These findings are consistent with those of 2+ homeostasis, resulting in excitation-contraction coupling. Ventricular AP starts with a sodium (Na+) influx through voltage-gated Na+ channels that depolarize the cell. Voltage-sensitive LTCC is activated at a certain threshold, allowing the influence of Ca2+ into the cytosol, which triggers a much larger Ca2+ release from the SR, the main intracellular Ca2+ storage organelle  and is the fundamental link between electrical and mechanical activation in the heart  that pumps Ca2+ back into the SR stores. It is also caused by Ca2+ extrusion and releases into the extracellular space through NCX, which exchanges three Na+ ions entering for one Ca2+ ion leaving the cell . SERCA2a takes up approximately 63% of cytosolic Ca2+, while NCX extrudes 37% of Ca2+ in humans  and intracellular Carganelle . Notably+ sparks . They orhe heart . The cytthe cell . The NCXn humans . The depcroscopy . In this2+ flowing into LTCC through mycoplasma voltage gating triggers SR to release large amounts of Ca2+. This myocardial contraction and blood drawing process driven by Ca2+ is called ECC  Ca2+ channel family. It is structurally homologous to voltage-gated Na+ channels and belongs to a family of six membrane-spanning voltage-gated ion channels. CACNA1C, CACNB2, and CACNA2D1 genes encode the pore-forming a-subunit (Cav1.2) and the b2 and a2d ancillary subunits that form the cardiac Ca2+ channel, which generates the LTCC  for relaxation to occur in normal conditions. This phenomenon consequently takes Ca2+ back into the SR. NCX operates in both the Ca2+ efflux and influx (or reverse) modes, depending on the internal and external concentrations of Na+ and Ca2+. INa is elevated in AC-induced myocytes, while NCX works almost exclusively in the Na+ extrusion mode, thus significantly increasing the amount of Ca2+ influx. This influx raises the cellular and SR Ca2+ content, resulting in larger Ca2+ transients. In contrast, other studies postulate that SR Ca2+ release modulates the sarcolemmal ICaL, suggesting a retrograde in communication between the SR and the sarcolemmal LTCC in cardiac ECC  (ICaL must inhibit the transportation of Cadiac ECC . MoreoveCACNA1C) . NotablyCACNA1C) . StudiesCACNA1C) .2+ overload is not achieved by increasing LTCC. However, these findings contrasted those of 2+ release channel and LTCC. In this study, WK significantly reduced intracellular Ca2+ transients, thus confirming that the increased expression of NCX and the presence of Ca2+ influx mode were the primary causes of AC-induced arrhythmia. AC also increased the expression of p-CaMKⅡ, thus promoting the release of Ca2+ from SR. The Ca2+ release of SR inhibited LTCC because of the retrograde communication between them. QRT-PCR results of CACNA1C also confirmed these findings. Similarly, laser-scanning confocal calcium imaging directly demonstrated the inhibitory effect of WK on intracellular Ca2+ concentration. Maintaining intracellular Ca2+ balance may thus be the primary mechanism of WK against arrhythmia.This study showed that AC has a significant inhibitory effect on LTCC, indicating that AC-induced intracellular Ca1) It is a good model for TCM research: The approach also detected the chemical components of WK besides the anti-arrhythmia mechanism and correlated the potential active components with their pharmacodynamic mechanisms. The research model was more systematic and complete, thus proving a good model for TCM research.2) It provides a research direction of antiarrhythmic effects of TCM: Besides the most relevant calcium signaling pathway, other signaling pathways were also detected, thus providing a good direction for subsequent studies. The pathways included the oxytocin signaling pathway, adrenergic signaling in cardiomyocytes, adipocytokine signaling pathway, and dilated cardiomyopathy associated with the metabolism of adrenaline, angiotensin, fats, glycometabolism, apoptotic factors, and other arrhythmia-related factors. These findings demonstrate that TCM treatment of diseases has a multi-target comprehensive effect, thus further proving that systems pharmacology is an effective method in TCM study.3) Construction of appropriate disease model: The present study used AC to construct a pure arrhythmia rat model instead of the toxic arrhythmia that causes myocardial infarction and heart failure. The pure arrhythmia model was more conductive to reveal the pathogenesis of arrhythmia because it did not interfere with the complex pathological mechanisms.2+ transients. CaMKII and NCX play a vital role in Ca2+ balance. As such, blocking CaMKII and NCX can be a safe and effective strategy against arrhythmias.4) New antiarrhythmic strategy: This study demonstrated that WK had therapeutic effects in AC-induced rat arrhythmias by inhibiting p-CaMKⅡ, over-expressing CNCA1C, and inhibiting intracellular Cain vivo and in vitro as the potential active components of WK. Among them, 33 key targets were screened based on their corresponding targets of WK potential active ingredients and targets of arrhythmia. These targets were regarded as the antiarrhythmic targets of WK . Moreover, AC-induced SR Ca2+ leakage through the RyR2 channel further inhibits LTCC (Carability . AC actirhythmia . The intrhythmia . There iits LTCC .2+ transients and overexpressing CNCA1C, thus suppressing SR Ca2+ release, which is the most probable mechanism of WK inhibition of the Ca2+ overload. Maintaining intracellular Ca2+ balance is, therefore, an essential mechanism of WK against arrhythmia. The antiarrhythmic mechanism of WK also suggests that TCM has a multi-target comprehensive effect against diseases. Notably, high dose WK has the best impact and should thus be highly considered during clinical treatment.However, pretreatment with WK lowers mortality, lessens malignant arrhythmias, and shortens RR, PR, and QT intervals than AC-induced rats. WK induces some antiarrhythmic effects by inhibiting p-CaMKⅡ and intracellular Ca2+ oscillations. As such, its activation is directly linked to increased APD, EADs, and arrhythmias (2+ homeostatic proteins involved in E-C are CaMKII targets (2+ to activate CaMKII, which then binds to and phosphorylates β2a to trigger EADs. Therefore, CaMKII can serve as a novel therapeutic target for arrhythmias.CaMKII activity reflects the frequency of cytosolic Cahythmias . The Ca2 targets . APD pro+ accumulation and reversal, leading to a high cytoplasmic Ca2+ influx.Similarly, NCX blocking is a promising safe and effective strategy against repolarization-dependent arrhythmias because it causes intracellular Na


Ischemic stroke is a major cause of death and disability, intensely demanding innovative and accessible therapeutic strategies. Approaches presenting a prolonged period for therapeutic intervention and new treatment administration routes are promising tools for stroke treatment. Here, we evaluated the potential neuroprotective properties of nasally administered human adipose tissue mesenchymal stem cell (hAT-MSC)-derived extracellular vesicles (EVs) obtained from healthy individuals who underwent liposuction. After a single intranasal EV (200 µg/kg) administered 24 h after a focal permanent ischemic stroke in rats, a higher number of EVs, improvement of the blood–brain barrier, and re-stabilization of vascularization were observed in the recoverable peri-infarct zone, as well as a significant decrease in infarct volume. In addition, EV treatment recovered long-term motor (front paws symmetry) and behavioral impairment (short- and long-term memory and anxiety-like behavior) induced by ischemic stroke. In line with these findings, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy for stroke. Stroke is a major cause of morbidity and mortality worldwide and affects health, economic, and social capabilities ,2,3. IscThe pathophysiology of ischemic stroke is characterized by blood flow obstruction in a restricted brain region, forming an infarct nucleus surrounded by an area known as the penumbra zone  , which iIn vivo studies using rat models of brain ischemia demonstrated that systemic treatment with mesenchymal stem cells (MSC) improves neurological outcome and alleviates the symptoms of disability in therapeutic intervention performed 12 and 24 h after an ischemic insult ,24. AlthA set of methodologies was performed, aiming to present original contributions and results to the understanding of certain components of stroke and of neuroprotective strategies of EVs. Specifically, we evaluated the short- and long-term effects on forepaws symmetry, behavioral performance, and anxiety-like behavior; short effect on EVs distribution in parenchyma brain, brain infarct volume and BBB permeability; and long-term effect on brain angiogenesis. MSC were obtained from human adipose tissue mesenchymal cells (hAT-MSC) through a minimally invasive procedure (liposuction) from healthy individuals. Additionally, intranasal EVs administration presented neuroprotective effect even when administered 24 h after stroke (200 µg/kg), this effect was effective for up to 42 days after stroke. This neuroprotective effect may be related to the high number of EVs in the peri-infarct region when compared to other brain regions. Dose curve effect of EV0 treatment on front paws symmetry. Effect of 200μg/kg EV treatment on front paws symmetry: The task was performed only on the seventh day after treatment with EV0 or EV1. ISC animals spent less time in the open arms compared to the other groups, indicating a stroke-induced anxiogenic-like effect, which was abolished by EV0 and EV1 treatment .The number of branches and total length of blood vessels were evaluated in M2 and SS brain regions through images acquired in three positions  a.Since ischemic stroke is a significant cause of morbidity and mortality , researcEVs are important mediators of communication between cells, and it has been identified that native brain cell-released EVs present tropism to injured regions ,53,54. AStimulation of angiogenesis, as observed by EV treatment, has been shown to improve neurological and motor function in animal stroke models , an effeHere, we demonstrate that intranasal hAT-MSC-derived EVs administered 24 h after stroke promoted long-term neuroprotective effects, offering a remarkably therapeutic window. Although the three EVs show protective properties, findings are not comparable since EV0, EV1, and EV2 are from different sources . Together, these findings indicate that hAT-MSC-derived EVs are a promising potential therapeutic strategy for patients with focal permanent ischemic stroke.The cells were obtained from commercial and human sources.6 cells was thawed at 37 °C and plated in a 25 cm2 flask (TPP). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS) , 100 units/mL penicillin, 100 μg/mL streptomycin , 50 mg/L gentamicin (Sigma-Aldrich), and 2.5 mg/L fungizone (Sigma-Aldrich) (pH 7.4). After 24 h, debris and non-adherent cells were gently removed [4 cells/75 cm2 (passage 2: P2). Cellular density was determined by manually counting the number of cells in each passage [Cells were obtained from POIETICS Bank Adipose-Derived Stem Cells . These cells were negative for mycoplasma, bacteria, yeast, and fungi, as reported by the supplier company. A frozen vial containing ~1 × 10 removed . When ad passage . The cel passage ,62,63.g for 5 min. The stromal vascular fraction (pellet) was resuspended in DMEM + 20% FBS (Cripion) medium and cultured in a culture flask containing 25 cm2  at 37 °C in a humidified 5% CO2 atmosphere. After 24 h, non-adherent cells were gently removed [4 cells/75 cm2 (TPP) (passage1: P1). Cells were then cultured in DMEM (Sigma-Aldrich) containing 10% FBS (Cripion), 100 units/mL penicillin (Gibco/Thermo Fisher Scientific), 100 μg/mL streptomycin (Gibco/Thermo Fisher Scientific), gentamicin 50 mg/L (Sigma-Aldrich), fungizone (2.5 mg) (Sigma-Aldrich), and L-glutamine 4 mM (Gibco/Thermo Fisher Scientific). These cells were named cell 1—patient 1 (C1) and cell 2—patient 2 (C2). C1 and C2 were expanded under the same conditions described above and used only from the 4th to 8th passage [Cells were obtained from the subcutaneous adipose tissue of two (32-year-old and 34-year-old) women who underwent abdominal liposuction at the Hospital de Clínicas in Porto Alegre, RS, Brazil. Informed consent was obtained from all subjects involved in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by Research and Graduate Group (Grupo de Pesquisa e Pós-Graduação: GPPG 2018-0374) and Research Ethics Committee (Comitê de Ética em Pesquisa CAEE: 94521618.4.0000.5327) of the Experimental Research Center at Hospital de Clínicas de Porto Alegre. Fresh adipose tissue was washed with PBS buffer, minced, and digested for 1 h in 0.1% collagenase at 37 °C. The digestion process was stopped by the addition of DMEM containing 20% FBS (Cripion). The digested suspension was filtered through a 70 µm nylon mesh cell filter to retain tissue debris. The filtered suspension was centrifuged at 400×  passage ,61. C0, g for 5 min at room temperature, and the cell pellet was re-suspended in DMEM + 10% FBS and counted in a Neubauer chamber. Briefly, the cells were incubated with antibodies at a concentration of 1:50 for 4 h at 37 °C. The cell suspensions were centrifuged at 400× g for 5 min at room temperature, and cell pellets were resuspended in 200 μL of PBS. Ten thousand events were analyzed using flow cytometry  [hAT-MSCs were centrifuged at 400× NJ, USA) . Cells i4 hAT-MSCs was placed on a slide and analyzed by immunofluorescence. Cells were maintained under culture conditions for 72 h to allow adherence to coverslips. Cells were then incubated for 4 h at 37 °C with the same antibodies used for cytometry: CD34, CD45, CD90, and CD105, at a ratio of 1:500. The negative control was prepared by incubating only the secondary antibodies, Alexa Fluor 555 and 488 (Invitrogen). Cells on coverslips were gently washed with PBS (four times) to remove excess antibodies and then fixed in 4% PFA for 2 h. Following fixation, the cells were gently washed again with PBS and then fixed with Fluoromount (Sigma-Aldrich) onto a slide for further analysis. Images were acquired using an 8-bit grayscale confocal laser scanning microscope . Approximately 10 × 15 sections with 0.7 μm thick confocal were captured parallel to the coverslip (XY sections) using a ×20 objective. Z-stack reconstruction and analysis were conducted using ImageJ software .An aliquot of 1 × 10As cultured hAT-MSCs (P4-P8) reached 80% confluence, DMEM + 10% FBS medium was replaced by DMEM FBS-free medium to avoid EV contamination with FBS-proteins and FBS-EVs, following previous protocols ,64,65,66g for 15 min, then 2000× g for 15 min, and then 10,000× g for 30 min). The supernatants were filtered through a 0.22 μm membrane. The isolation was completed by centrifugation . The supernatant was discarded, PBS was used to wash the pellet containing EVs, and the cell suspension was centrifuged at 100,000× g at 4 °C for 2 h. This centrifugation protocol (speed and time) was shown to preserve EVs in the pellet while preventing contamination with stress-related biological constituents [For EV isolation, the collected medium was centrifuged (3 times) at 4 °C:  at 25 °C.We used photon correlation spectroscopy to measure the particle size and polydispersity index (PDI). The EV suspension derived from hAT-MSCs (50 µL) at 1 mg/mL was diluted in 1 mL of PBS. All analyses were performed in triplicate using a Malvern Nano-ZS90Transmission electron microscopy (TEM) analysis, using a direct examination technique, was used to evaluate the diameter of EVs . EV suspg for 2 h at 4 °C, and the pellet was suspended with the diluent of the fluorescent kit. Filtered PKH26 (4 mM) and EVs (200 μg/mL) were mixed at a ratio of 1:1 for 5 min, followed by the addition of 5% BSA. To remove excess dye, the EVs were washed three times, and then 5 mL of PBS was added and centrifuged at 100,000× g for 2 h at 4 °C and the supernatant discarded. In the last centrifugation step, the stained EV pellet was suspended in PBS (0.5 mL). The solution was filtered through a 0.2 μm membrane filter to remove dye aggregates [EVs were labeled with the red fluorescent membrane dye PKH26 (Sigma-Aldrich). In brief, the EV-containing PBS solution was centrifuged at 100,000× gregates .Adult (90–120 days old) male Wistar rats weighing 350–400 g were maintained under controlled light (12/12 h light/dark cycle) at 22 °C ± 2 °C, with water and food ad libitum. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior recommendations for animal studies. This study was approved by the Ethics Committee for the Use of Animals at the Universidade Federal do Rio Grande do Sul (project identification: 31888). A schematic of the procedure is shown in Focal permanent ischemia and sham procedures: Anesthetized animals (under ketamine hydrochloride: 90 mg/kg i.p. and xylazine hydrochloride:10 mg/kg i.p.) were placed into a stereotaxic apparatus. After skin incision, the skull was exposed, and a craniotomy was performed by exposing the left frontoparietal cortex (+2 to −6 mm A.P. and −2 to −4 mm M.L. from the bregma). A focal permanent ischemic lesion was induced by thermocoagulation of the motor and sensorimotor pial vessels. Blood vessels were thermocoagulated by placing a hot probe near the dura mater for 2 min until a red-brown color indicated complete thermo-coagulation. Soon after, the skin was sutured, and the animals were placed on a heating pad at 37 °C until full recovery from anesthesia. Animals from the sham group were subjected to craniotomy as described above. Animals were randomly allocated to three treatment groups: sham, ischemic (ISC), and ischemic treated with EVs (ISC + EV). Our stroke model of focal cerebral permanent ischemia represents a protocol to investigate brain lesions with good reproducibility and low mortality ,50,68,692 flow rate at 0.8–1.0 L/min with isoflurane levels of 2.5–3.0%) received a single 50 µL of EVs (ISC + EVs) or 50 µL PBS , slowly administrated for 30 s in both nasal cavities, as previously reported [Intranasal EV treatment was performed 24 h after the ischemic or sham procedure. Sedated animals . Brain slices were incubated for 5 min in the dark with 1 μg/mL Hoechst 33342 solution dye (Sigma-Aldrich) to detect cell nuclei. The slices were washed with PBS (four times), and the slices were fixed with the fluoro mount (Sigma-Aldrich). Slice images for counting EVs were acquired using an 8-bit grayscale confocal laser scanning microscope (Olympus FV1000). Approximately 10–15 sections with 0.7 μm thick confocal were captured parallel to the coverslip (XY sections) using a ×60 objective. Z-stack reconstructions and analyses to count the vesicles in the brain tissue were conducted using ImageJ Free software , and background noise was removed using the “subtract background” tool. Images were converted to binary masks using the default threshold option, and vesicles were counted with the “analyze particles” tool (size = 0.05–0.90 μm). These settings were programmed into a macro and used for all the analyzed images (http://rsb.info.nih.gov/ij/ (accessed on June 2018)).The distribution of fluorescent EVs (PKH26- Sigma-Aldrich) in rat brains (naive and ISC + EV1) was analyzed 18 h after intranasal administration, as previously reported ,49. Anes2 flow rate of 0.8–1.0 Â mL/min with isoflurane levels of 2.5–3.0%), decapitated, and the brain removed. Coronal sections of the whole brain were sliced at 2 mm, and slices were immersed in 2% 2, 3, 5-Triphenyl-tetrazolium chloride (TTC) (Sigma-Aldrich) for 30 min at 37 °C. After incubation, the slices were dipped in 4% buffered paraformaldehyde (pH 7.4) for 24 h. The infarct area was evaluated as an area devoid of red staining. The infarct volume was measured using the ImageJ software [For evaluating infarct volume, naive, sham, ISC, ISC + EV0, and ISC + EV2 animals were sedated 48 h after treatment (Osoftware .http://labs.gaidi.ca/rat-brain-atlas/ (accessed on January 2020). Blood vessel parameters, such as the total length (sum of the length of segments and isolated elements) and the number of branches, were quantified using the Angiogenesis Analyzer Plugin (Gilles Carpentier Research) ImageJ software [https://imagej.nih.gov/ij/ accessed on June 2019) [After 42 days of treatment, animals from the naive, ISC, and ISC + EV2 groups were anesthetized and received intracardiac injection of 50 mg/mL (500 µL) fluorescein isothiocyanate-dextran amine (Merck KGaA) to label blood vessels. After 5 min, rat brains were excised, immediately fixed in 4% PFA, and cut into 30 µm coronal slices in a vibratome. Images were acquired using a fluorescence microscope . The images were taken from the ipsilateral and contralateral sides in the secondary motor cortex (M2) and somatosensory (SS) regions using the following coordinates: +2.20, 0.2, and −1.88 mm A.P. to Bregma (PAXINUS online Rat Brain Atlas: software  (https:/ne 2019) ,74.g for 10 min. The concentration of the blue color was measured in the supernatant using a spectrophotometer at a wavelength of 620 nm. EB dye was expressed in micrograms per gram of brain tissue [Evans blue penetration into brain parenchyma: Naive, sham, ISC, and ISC + EV2 animals were anesthetized  48 h after treatment, and 3 mL/kg of 2% Evans blue (EB)  solution in saline was administered through the gingival artery . After 1n tissue ,76.2O + 0.1% formic acid) and solvent B (acetonitrile (ACN) as follows: A → 65% B → 35% (0–5 min), A → 70% B → 30% (5–10 min), A → 65% B → 35% (10–17 min). The flow rate was set at 0.7 mL/min. Sample preparation was performed by adding 10 µL of liquor to 40 µL of ACN and mixing in a vortex. The solution was transferred to conical vials and 10 µL was injected. Albumin stock solutions 1 mg/mL in water were stored at −20 ± 2 °C. For each day of analysis, standard solutions of albumin were prepared at 0.1, 0.5, 1, 10, 50, and 100 µg/mL.CSF albumin levels: Albumin assay was performed using high-performance liquid chromatography coupled to a fluorescence detector (HPLC-FLD). The CSF method was validated according to FDA guidelines ,78. HPLCA group of animals was submitted to the cylinder task, open field task, and novel object recognition task; another group of animals was submitted to the cylinder task and elevated plus-maze task. The cylinder task, which evaluates neurologic dysfunction (front paws symmetry), was performed before surgery; the presence of ~100% basal symmetry was used as inclusion criterion in the study.The CT can evaluate the motor symmetry of the front paws. The apparatus consists of a transparent glass cylinder 20 cm in diameter and 30 cm in height (20 raising movements were counted). All animals  were firstly submitted to this task 24 h before surgery to verify basal symmetry. This task evaluated how the rats raised their bodies in contact with their paws on the cylinder wall. The ipsilateral (to the lesion), contralateral, or both front paws preferences were counted in a blinded analysis. The asymmetry of each animal was calculated using the following formula: asymmetry =  − . The performance was recorded using ANY-Maze software version 6.3 . The asymmetry percentage was converted into a symmetry percentage . The samThis task evaluates habituation to novelty (assessing short- and long-term memory through exploratory activity) and locomotor activity ,80. The Behavioral sessions lasting 10 min were performed on days 7, 21, and 42 after EV treatment. At 90 min after the OFT session, object recognition (OR) short- and long-term memories were evaluated . The aniThis task is widely used to study anxiety-like behavior . The appp < 0.05); thus, statistical analyses of both experiments were performed using the Mann–Whitney test, and the data were reported as median and interquartile range.Brain lesion size and the number of brain vesicles showed a non-homogeneous distribution  were not performed due to differences in EVs sources . In the parametric distribution, the data were presented as mean ± SD. All analyses were performed using GraphPad Prism version 6.0 .The results of BBB integrity, angiogenesis, OFT short-term memory, and EPMT were evaluated using unpaired


Apln) gene expression in epididymal white adipose tissue was increased in HFD_Veh, but reduced to LFD_Veh levels in the HFD_RK group. Peroxisome proliferator activated receptor alpha (Ppara) gene expression was increased in the hepatic tissue of HFD_RK and HFD_RKw4 groups. Overall, our findings suggest that long term daily use of RK prevents diet-induced weight gain, normalizes high-fat diet-induced adipose Apln, and increases hepatic Ppara expression.Raspberry ketone [4-(4-hydroxyphenyl)-2-butanone] is a natural aromatic compound found in raspberries and other fruits. Raspberry ketone (RK) is synthetically produced for use as a commercial flavoring agent. In the United States and other markets, it is sold as a dietary supplement for weight control. The potential of RK to reduce or prevent excessive weight gain is unclear and could be a convergence of several different actions. This study sought to determine whether acute RK can immediately delay carbohydrate hyperglycemia and reduce gastrointestinal emptying. In addition, we explored the metabolic signature of chronic RK to prevent or remedy the metabolic effects of diet-induced weight gain. In high-fat diet -fed male mice, acute oral gavage of RK (200 mg/kg) reduced hyperglycemia from oral sucrose load (4 g/kg) at 15 min. In HFD-fed female mice, acute oral RK resulted in an increase in blood glucose at 30 min. Chronic daily oral gavage of RK (200 mg/kg) commencing with HFD access (HFD_RK) for 11 weeks resulted in less body weight gain and reduced fat mass compared with vehicle treated (HFD_Veh) and chronic RK starting 4 weeks after HFD access (HFD_RKw4) groups. Compared with a control group fed a low-fat diet  and dosed with vehicle (LFD_Veh), glucose AUC of an oral glucose tolerance test was increased with HFD_Veh, but not in HFD_RK or HFD_RKw4. Apelin ( It is txidation . Previouxidation , 2020. Txidation . Anothere levels . In vitre enzyme . Relatede enzyme , RK coule enzyme . Taken tprevented HFD-induced weight gain after 4-week coadministration of HFD and RK . All the mice were housed, four mice per cage, upon arrival and were acclimated on standard chow  for one week. They had free access to water, unless otherwise noted, and maintained on a 12-h light and 12-h dark cycle with lights on from 0500 to 1700 h. Oral gavage was performed using single-use, sterile plastic feeding tubes . The animal care protocol was approved by the Institutional Animal Care and Use Committee of Rutgers University (OLAW #A3262-01); Protocol #999900014.n = 24 per sex, 4 weeks old) was fed a high-fat diet , and six male and six female mice were fed with sucrose matched low-fat diet . The mice were fed for a period of 17 weeks and subjected to carbohydrate meal tests in the following 2 weeks with a 7-day washout between treatments. This long exposure to the HFD was utilized as a procedure to produce an alteration in blood dysregulation to facilitate the examination of in vivo α-glucosidase activity. A similar approach has been used in the examination of other natural products (α-glucosidase inhibition (n = 12) were orally gavaged with acarbose , and the other half were orally gavaged with raspberry ketone . A reference sample of RK from the lot number was deposited in a secure, climate-controlled repository  at age of 12 weeks was single housed and fed HFD for 2 weeks. The mice were fasted for 4 h but had free access to water. They were then orally gavaged a test meal containing phenol red (0.1%) and RK  or vehicle . Three RK and four vehicle mice were euthanized immediately after dosing (0 min), and the other mice were euthanized after 30 min. The stomach and small intestine were dissected with pylorus and cardia clamped. The stomach was transferred and homogenized in 10 ml.1N NaOH. After sedimentation at room temperature for 60 min, 5 ml of supernatant was mixed with 0.5 ml 33% trichloroacetic acid to precipitate any proteins and centrifuged at 3,000 rpm for 30 min. The supernatant was transferred to a new tube, and an equal volume of 0.5N NaOH was added to intensify the phenol red color. Phenol red concentration was measured by absorbance at 560 nm using a spectrophotometer. GE rate was calculated as follows: (1-absorbance at 30 min/absorbance at 0 min) × 100.Gastric emptying and small intestine transit rate were measured according to the methods of Amira and colleagues . BrieflyThe whole length of the small intestine was measured after it was dissected. The intestine was opened from the end part and a drop of 0.1 N NaOH was used to localize the end point that the test meal reached. The length between the end point of test meal and the starting part of the intestine was defined as the distance traveled by the test meal. Intestinal transit rate was calculated as the ratio of the distance traveled by the test meal to the total length of small intestine × 100.n = 8 per group, and fed either HFD or sucrose-matched LFD. All the mice also simultaneously received a daily oral gavage of vehicle or 200 mg/kg RK for 12 weeks. The four diet-compound groups were as follows: LFD_Veh, HFD_Veh, HFD_RKw4, and HFD_RK. Mice in the HFD_RKw4 group were orally gavaged with vehicle for the first 4 weeks (28 days) and switched to 200 mg/kg RK for the following 8 weeks. Mice in the other three groups were on their initial diet and treatment for over 12 weeks (84 days). All the mice had free access to water and respective diet at all times, unless otherwise noted and maintained on a 12-h light and 12-h dark cycle with lights on from 05:00 to 17:00 h. The mice were weighed daily, and food consumption was measured every week throughout the entire dosing period. Daily dosing was performed between 08:30 and 10:00 h.Thirty-two male mice  were equally separated into four groups by body weight, with Mice lean, fat, and fluid mass were measured using an EchoMRI 3-in-1  body composition analyzer. Body composition was assessed a day (Day 0) before the initiation of diet and daily oral treatment and on week 11 (Day 77).Insulin Tolerance Test (ITT) was performed on week 9. The mice were fasted for 5 h and injected intraperitoneally (IP) with 0.75 U/Kg Humulin R . Blood glucose levels were measured at 0, 15, 30, 60, 90, 120, and 180 min. The mice were returned to home cages and given food and water immediately after the ITT. The mice were not administered RK or Veh during the day of ITT. Oral dosing of compounds was resumed a day after the ITT.Oral glucose tolerance test was performed on week 10. The mice were fasted for 6 h and given a dose of glucose (2 g/kg) orally. Blood glucose levels were measured at 0, 15, 30, 60, 90, 120, and 180 min. The mice were returned to home cages and given food and water immediately after the Oral Glucose Tolerance Test (OGTT) but were not given RK or Veh. Oral dosing of compounds was resumed a day after the OGTT .All the mice were food-deprived in new clean cages for 5 h (Day84). The mice were then deeply anesthetized under isoflurane mixed with oxygen and exsanguinated followed by decapitation. Blood was collected in EDTA tubes by cardiac puncture under deep isoflurane anesthesia, and 50-ul whole blood was analyzed by Element HT5 Hematology Analyzer . The remaining plasma was used for plasma biomarkers analysis.Brown adipose tissue and white adipose tissue including inguinal and epididymal adipose tissue, liver and mediobasal hypothalamus  were collected in RNase later and stored at −80°C.Plasma triglyceride, free fatty acids, cholesterol, and insulin were analyzed using a commercially available triglyceride quantification kit , free fatty acids quantification kit , HDL and LDL/VLDL cholesterol quantification kit , and mouse insulin enzyme-linked immunosorbent assay (ELISA) kit .Adipose tissue RNA was extracted using a standard TRIzol extraction combined with Machery-Nagel Nucleospin RNA extraction kit. DNase I was used to minimize any genomic DNA contamination. RNA quantity was determined using a NanoDrop ND-2000 spectrophotometer .Complementary DNA was synthesized from 200 ng of total RNA in a 20-ul reaction system containing a Superscript III  reverse transcriptase, 4 μl 5 × SS buffer, 1.25 μl 100 mM dNTP, 100 ng random hex primers , 40 U/μl Rnasin , and 100 mM dithiothreitol in diethylpyrocarbonate-treated water. Reverse transcription was conducted using the following protocol: 25°C for 5 min, 50°C for 60 min, 70°C for 15 min and then cooling to 4°C for 5 min. The cDNA was diluted 1:20 using nuclease free water and stored at −20°C. Naive tissue RNA was used for positive and negative controls (no reverse transcriptase) and processed simultaneously with the experimental samples.® Green Supermix , primers, and nuclease free water. The amplification protocols for all genes are as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 45 s, and completed with a dissociation step for melting point analysis with 60 cycles of 95°C for 10 s, 65 to 95°C (in increments of 0.5°C) for 5 s, and 95°C for 5 s.For quantitative real-time polymerase chain reaction (PCR), a CFX opus 96 real-time PCR instrument  was used for gene amplification from 4 μl of cDNA using SsoAdvancedTm Universal SYBRRelative mRNA expression was calculated using the δδ cycle threshold (CT) method. The CT for each transcript was determined by consistently setting the baseline threshold at the lowest point of the exponential curve where the slope of the curve was steepest for all plates. Geometric means of the reference genes Gapdh and Hprt were used to calculate δCT. The relative linear quantity of target molecules was calculated using the formula 2^δδCT. All gene expression data were expressed as an n-fold difference relative to the LFD_Veh group.Adra1a), adrenoceptor alpha-1B (Adra1b), adrenoceptor alpha-2B (Adra2b), adrenoceptor alpha-2C (Adra2c), arachidonate 15-lipoxygenase (Alox15), apelin (Apln), apelin receptor (Aplnr), beta-3 adrenergic receptor (b3AR), gamma-aminobutyric acid receptor subunit gamma-2 (Gabrg2), glutamate ionotropic receptor AMPA-type subunit 1 (Gria1), heme oxygenase (Hmox1), interleukin 6 (Il-6), hormone-sensitive lipase (Lipe), lipoprotein lipase (Lpl), neuropeptide Y(Npy), proopiomelanocortin (Pomc), peroxisome proliferator-activated receptor alpha (Ppara), peroxisome proliferator-activated receptor gamma (Pparg), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha (Pparg1ca), tumor necrosis factor(TNF), uncoupled protein 1 (Ucp1), and uncoupled protein 2 (Ucp2). Primers for housekeeping genes Gapdh and Hprt were synthesized by Life Technologies, Inc. , and primer sequences were as follows:Primers for the following genes were purchased from BioRad Laboratories: adrenoceptor alpha-1A . All the data were represented as mean ± standard error of the mean (SEM), and a p value less than 0.05 was considered statistically significant.For daily body weight gain, weekly food intake, glucose response to insulin, or oral glucose administration, an analysis of variance (ANOVA) with repeated measures was performed. For the other measurements, one-way ANOVA was performed. When justified, Newman Keuls p < 0.001; females: 22.2 ± 0.6 g, p < 0.001). Fasting glycemia in the LFD control male and female mice was 124 ± 5.2 and 111 ± 2.9 mg/dl, respectively. Fasting glucose levels were not significantly elevated in either the male (132 ± 4.2 mg/dl) or female (122 ± 2.4 mg/dl) HFD mice. For oral sucrose dosing in males, there were group , time , and group X time  effects. Post hoc testing revealed blood glucose levels were overall lower in acarbose (5 mg/kg) than vehicle group (p < 0.005). Blood glucose levels in the Acar group were significantly lower than in the vehicle group at 15 and 30 min (p < 0.05 for both), whereas blood glucose levels in the RK group was only significantly lower than in the vehicle-treated group at 15 min (p < 0.05)  = 4.7, p < 0.05] with Acar having lower AUC than vehicle (p < 0.05)  = 84.5, p < 0.0005] and group X time . Post hoc testing revealed that blood glucose in Acar was lower than in vehicle at 15 min (p < 0.05), and that RK dosing elevated blood glucose at 30 min compared with Acar and vehicle (p < 0.05 for both)  and female (32.1 ± 1.1 g) mice compared with the controls  . There w < 0.05) . For oraor both) . There wor both) . For oraGastric emptying rate was measured using a test meal containing phenol red. In the Veh group, the gastric emptying (GE) rate of test meal from stomach is around 43 and 30% for the male and female mice, respectively. RK did not significantly accelerate or delay test meal emptying in either male or female mice . IntestiF = 15.2, p < 0.0005], days effect , and treatment X day effect . Post hoc testing revealed that all the groups had increased body weight gain compared with LFD_Veh (p < 0.005). HFD_RK had reduced body weight gain compared with HFD_Veh (p < 0.05). On Day 25, HFD_RK had less body weight gain compared with the HFD_Veh group (p < 0.05). The difference between HFD_Veh and HFD_RK remained until the end of the study . Because RK (200 mg/kg) dosing began on day 28 for the HFD_RKw4 group, an additional analysis of body weight gain from day 29 to the end of the study was performed (last 8 weeks). There were treatment , time , and group x time  effects. Post hoc testing indicated that there was a difference between HFD_Veh and HFD_RK (p < 0.05), but that there were no group differences between HFD_Veh and HFD_RKw4 and that there were no group differences between HFD_RK and HFD RKw4  = 14.5, p < 0.0005]. Post hoc testing indicated that HFD_Veh, HFD_RKwk4, and HFD_RK had increased body weight gain compared with LFD_Veh . There was significantly less body weight gain in HFD_RK compared with HFD_Veh . The baseline body weights were 24.34 ± 0.32, 24.42 ± 0.49, 25.68 ± 0.52, and 25.03 ± 0.56 g for LFD_Veh, HFD_Veh, HFD_RKw4, and HFD_RK, respectively. Over the 11 weeks, there were treatment effect . All mice on the HFD group had higher cumulative energy intake compared with the LFD-fed mice  = 12.8, p < 0.005]. Post hoc testing indicated that all the HFD-fed groups had increased fat mass compared with the LFD vehicle group . In addition, HFD_RK had lower fat mass gain than HFD_Veh  = 16.2, p < 0.0005]. The differences in the ratio were similar to the group difference in fat mass, with all the HFD-fed groups having a higher ratio than LFD_Veh (p < 0.005), and HFD_RK had a lower ratio than HFD_Veh  = 3.3, p < 0.05] with post hoc testing, indicating that LFD_Veh had the lowest blood glucose. There was also an effect for time  but not for treatment X time  = 2.3, p = 0.09]  = 3, p < 0.05] with post hoc testing revealing lower overall blood glucose levels in LFD_Veh compared with HFD_Veh (p < 0.05). There was a time effect  but not treatment X time effect  = 3, p < 0.05] with only a difference between LFD_Veh and HFD_Veh (p < 0.05) . The post hoc testing did not reveal differences among LFD_Veh, HFD_RK, and HFD_RKw4. Blood glucose values at OGTT were 177 ± 3.5 mg/dl for LFD_Veh, 202.5 ± 3.7 mg/dl for HFD_Veh, 184.1 ± 7.9 mg/dl for HFD_RKw4, and 178 ± 4.8 mg/dl for HFD_RK.We performed ITT and OGTT tests to determine whether RK improves blood glucose response to insulin or glucose administration due to its body weight and adiposity reduction effect. On Week 9, for ITT, there was a significant effect of treatment [t X time . There w = 0.09] . On weeke effect . There w < 0.05) . There wApln) expression in epididymal adipose tissue . Post hoc testing revealed that HFD_Veh and HFD_RKw4 had increased Apln expression compared with LFD_Veh . However, HFD_RK was only different from HFD_Veh (p < 0.05)  = 2.9, p = 0.056]. Raspberry ketone tends to suppress Apln expression by around 69% when compared with the HFD control group (Ppargc1a) expression in epididymal adipose tissue . However, the post hoc analysis did not indicate statistical differences among the groups  = 5.7, p < 0.005] for peroxisome proliferator-activated receptor alpha (Ppara) expression. The post hoc analysis revealed that HFD_RKw4 and HFD_RK had increased Ppara expression compared with LFD_Veh . In addition, there was an effect  on Ppara expression in brown adipose tissue. The post hoc analysis showed that HFD vehicle had increased Ppara expression compared with LFD control (p < 0.05). Ppara expression in the RK-treated mice had no statistical difference when compared with LFD control or HFD control  . In inguol group . All thee groups . In the  control .F = 3.9, p < 0.05], and post hoc testing revealed that HFD_Veh had higher insulin levels than LFD_Veh and HFD_RKw4 (p < 0.05 for both)  = 4.2, p < 0.05] with post hoc testing revealing that HFD_RKw4 and HFD_RK had reduced LYM counts compared with LFD_Veh (p < 0.05 for both). The post hoc testing also indicated that HFD_RK had reduced LYM counts compared with HFD_Veh (p < 0.05)  = 3.4, p < 0.05]. Post hoc testing revealed an increase in NEU % in HFD_RKw4 compared with HFD_Veh  . Whole b < 0.05) . In addiin vitro studies have demonstrated that RK affects lipid metabolism by reducing lipid accumulation and promoting lipolysis and fatty acid oxidation   . Several control . In a se actions . One limLipe, a hormone-sensitive lipase that is a major enzyme that catabolizes triglycerides during lipolysis . However, plasma insulin levels were highest in the vehicle-treated HFD-fed group but not significantly different from the RK-treated HFD-fed group. The reduction of apelin expression may indicate the anti-adipogenesis effect of RK  expression. PPARα is a ligand activated transcription factor and highly expressed in the liver, brown adipose tissue, heart, and kidney  in rats ; Protocol #999900014.NB and LH designed the study. LH performed the experiments, analyzed the data, and drafted the manuscript. NB analyzed the data and drafted and edited the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.