Glioma is one of the deadliest human cancers. Although many therapeutic strategies for glioma have been explored, these strategies are seldom used in the clinic. The challenges facing the treatment of glioma not only involve the development of chemotherapeutic drugs and immunotherapeutic agents, but also the lack of a powerful platform that could deliver these two moieties to the targeted sites. Herein, we developed chemoimmunotherapy delivery vehicles based on C6 cell membranes and DC membranes to create hybrid membrane-coated DTX nanosuspensions (DNS-[C6&DC]m).Results demonstrated successful hybrid membrane fusion and nanosuspension functionalization, and DNS-[C6&DC]m could be used for different modes of anti-glioma therapy. For drug delivery, membrane coating could be applied to target the source cancer cells via a homotypic-targeting mechanism of the C6 cell membrane. For cancer immunotherapy, biomimetic nanosuspension enabled an immune response based on the professional antigen-presenting characteristic of the dendritic cell membrane (DCm), which carry the full array of cancer cell membrane antigens and facilitate the uptake of membrane-bound tumor antigens for efficient presentation and downstream immune n.DNS-[C6&DC]m is a multifunctional biomimetic nano-drug delivery system with the potential to treat gliomas through tumor-targeted drug delivery combined with immunotherapy, thereby presenting a promising approach that may be utilized for multiple modes of cancer therapy.The online version contains supplementary material available at 10.1186/s12951-021-01110-0. Glioma is one of the deadliest human cancers, with a median survival time of less than 24 months and a 5-year survival rate of only 2–4% , 2. ThisTo date, many nanotechnology-based delivery systems (NDDSs) , 7 have New frontiers in the field of NDDS are advancing research on biomimetic nanocarriers. Among these, cell membrane-coated nanoparticles (NPs) have been widely investigated for cancer therapy, ranging from enhanced efficacy in cancer drug delivery to enhanced immunogenicity of cancer vaccines –12. For + and CD4+ T lymphocytes. Activated CD8+ T-lymphocytes (CTLs) specifically recognize tumor cells and induce tumor apoptosis m.According to a previously reported method , C6 gliog for 15 min, three times. DC membrane was added to the DiI/DiD-doped C6 membrane at DC membrane to C6 membrane protein weight ratios of 5:1, 3:1, 1:1, and 0:1, followed by sonication  to complete membrane fusion. The fluorescence spectrum of each sample was read using a plate reader  with an excitation wavelength of 500–650 nm under different mixing ratios (DCm/C6m). The fluorescence recovery of the donor (C6-DiI) at a low emission peak (nm) was utilized to indicate the increased amount of fusion.To prepare a biomimetic nanosuspension with a hybrid cell membrane as a carrier, first we verified whether it is possible to combine C6m and DCm. To conduct the Förster resonance energy transfer (FRET) study, two lipophilic dyes were employed: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt  and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate . The ultrasound fusion method was utilized for fusing the C6 and DC membranes. C6 cell membrane was stained with DiD and DiI, and with respect to C6 membrane protein, the final ratios were 0.2 and 1.26 wt% for DiI and DiD, respectively. A solution containing the C6 membrane was then added to the vial and stirred at 37 °C for 1 h. Afterward, free dye was washed away by centrifuging the membrane at 10,000×DTX-loaded/(M[C6&DC]m + MDTX-loaded), EE = MDTX-loaded/MDTX-initial. MDTX-initial is the initial mass of DTX used for DNS-[C6&DC]m preparation. M[C6&DC]m is the mass of [C6&DC]m used for DNS-[C6&DC]m preparation. MDTX-loaded is the mass of DTX loaded in [C6&DC]m, which was determined by subtracting the amount of DTX in the supernatant from MDTX-initial.DNS solution  was added to [C6&DC]m solution  and sonicated (300 W) for 10 min in an ice bath to achieve the [C6&DC]m coating. The mixture solution was centrifuged at 10,000 rpm for 5 min to remove the excess membrane, and the resulting DNS-[C6&DC]m was resuspended in deionized water for future use. DNS-[C6&DC]m was acquired by ultrasonic treatment for 5 min. The morphology was characterized using TEM, and the size and zeta potential of DNS-[C6&DC]m were measured using DLS. DNS-[C6&DC]m was stored in either 1 × PBS or PBS containing 10% FBS at 37 °C. Stability analysis was carried out by measuring the particle size and zeta potential within 72 h. The drug loading efficiency (LE) and encapsulation efficiency (EE) of DNS-[C6&DC]m were determined by HPLC  , 43. BriSDS–polyacrylamide gel electrophoresis (SDS-PAGE) was employed to examine the protein profile of DNS, DNS-C6m, DNS-DCm, and DNS-[C6&DC]m . In brieDifferent DTX formulations (1 mL) loaded in dialysis bags  were immersed in 50 mL of PBS (pH 7.4), PBS (pH 6.8) or 10% FBS with 0.5% Tween-80 (v/v), respectively. Next, the drug release system was shaken in water bath at 37 °C for 72 h under horizontal shaking at 100 rpm. 100 μL dialysis solution outside of the dialysis bag was take out at different time, and 100 μL of fresh drug-free medium was added into the dialysis solution outside of the dialysis bag. Finally, the content of DTX in solution was detected by HPLC, and the mobile phase was composed of 45% ammonium acetate solution (0.043 mol/L) and 55% acetonitrile. The column was eluted at a flow rate of 1 mL/min at 25 °C .4 cells were seeded in a 6-well culture plate and cultured for 24 h. After incubation with DiI-labeled biomimetic nanosuspensions for 15 min, the cells were washed three times with cold PBS, trypsinized, resuspended in 300 μL of PBS, and analyzed by flow cytometry .Cellular uptake of different biomimetic nanosuspensions was evaluated in bEnd.3 cells, HUVECs, and C6 glioma cells, and the cells were seeded in a 6-well culture plate at a density of 0.5 × 105 cells/well. After incubation at 37 °C for 24 h, different cell membrane-coated biomimetic nanosuspensions were stained with DiI and incubated with the above-listed cells at a concentration of 2 μg/mL for 15 min, after which they were counter-stained with 4′,6-diamidino-2-phenylinodole  for 5 min. After incubation, the cells were fixed with a 4% paraformaldehyde solution. Finally, they were fixed with 50% glycerin. Cell uptake was observed using confocal laser scanning microscopy . For quantitative analysis, 2 × 103 cells/well. DiI-DNS-C6 DC]m was then added to the cell culture medium for 0.5 h. The uptake of different cells was observed using CLSM.Homologous targeting was verified using the uptake of biomimetic nanosuspensions via different types of cancer cells. Mouse breast cancer cells (4T1), mouse melanoma cells (B16), human hepatoma cells (HepG2), and C6 glioma cells were seeded in confocal laser dishes at a density of 5 × 105 cells/well. The integrity of the cultured monolayer model was tested by measuring the trans-endothelial electrical resistance (> 200 Ω·cm2) using a Millicell-ERS voltohmmeter (Millipore). After the cells were cultured to 100% confluence, the transendothelial electrical resistance (TEER) of the cell membrane was recorded. When the TEER was over 200 Ω.cm2, C6 cells were seeded in the lower chambers at a density of 1 × 105 cells/well. Next, free DiI, DiI-DNS-DCm, DiI-DNS-C6m, and DNS-[C6&DC]m were added to the upper chamber and cultured for 4 h. Solutions collected from the bottom chamber were assessed using a plate reader, while cell uptake was observed using CLSM. Similarly, HUVECs and C6 cells were used to establish a BBTB model to investigate the penetration ability of the preparations.An in vitro BBB model was constructed with bEnd.3 cells using a Transwell cell culture system. Briefly, bEnd.3 cells were seeded in the upper chambers of Transwell cell culture plates  at 1 × 105 bEnd.3 cells or HUVECs were seeded onto the upper Transwell chamber. When the cells were overgrown, and the TEER was over 200 Ω.cm2, the lower chamber was seeded with 2 × 103 C6 glioma cells. Next, free DTX, DNS, DNS-C6m, DNS-DCm, and DNS-[C6&DC]m at DTX concentration of 50 µg/mL, were added to the upper chamber and cultured for 48 h. The inhibition rate of C6 glioma cell proliferation in the bottom chamber was quantified using the Cell Counting Kit-8 (CCK-8) method.The inhibitory effect of DTX on glioma cells in the BBB and BBTB models in vitro was similarly measured using Transwell chambers . In brieTo assess the maturation levels of DCs after different treatments, C6m, DCm and[C6&DC]m (50 µg/mL) were incubated with DCs for 48 h. The cells were washed three times with PBS and subsequently stained with anti-CD80-PE, and anti-CD86-APC antibodies (Abcam) for 30 min at 4 °C. After being washed with cold PBS, the cellular fluorescence was detected by flow cytometry. All groups were analyzed in triplicate. Furthermore, to assess the activation levels of T lymphocytes, T cells were treated as above mentioned. After cocultured for 48 h, the T lymphocytes were washed three times with PBS and subsequently stained with anti-CD4-FITC and anti-CD8-PE antibodies (Abcam) for 30 min at 4 °C. After being washed with cold PBS for thrice, the cellular fluorescence was detected by flow cytometry.5 cells/well. After 12 h, C6m, DCm, and [C6&DC]m (50 μg/mL) were added to the upper layer of the chamber. In addition, LPS-treated cells were used as a positive control, while blank medium-treated cells were used as the model group. The suspensions of RAW264.7 cells culture media after stimulation were collected after a 48-h incubation period. The collected samples were tested with mouse IL-6, IFN-γ, and TNF-α ELISA kits, according to the manufacturer’s instructions. The inhibitory effect of cytokines on C6 glioma cells was quantified using the CCK-8 assay.To further evaluate whether the treatment in this study can induce immune response from macrophages, C6 glioma cells were seeded in the lower layer of the Transwell chamber, and RAW264.7 cells were seeded in the upper layer of the chamber at a density of 2 × 103 cells/well. After incubation for 24 h, different DTX formulations  and free PTX were added at concentrations ranging from 1 to 1,000 μg/mL in 100 μL of medium, and the plates were incubated at 37 °C in a 5% CO2 atmospheric condition for 48 h. After incubation for 48 h, 20 μL of CCK-8 solution was added to each well, and the cells were cultured for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader.The antitumor activities of DNS, DNS-C6m, DNS-DCm, and DNS-[C6&DC]m were measured using a standard CCK-8 assay. C6 glioma cells were seeded in a 96-well plate at a density of 5 × 105 cells/well and cultured for 12 h. The cells were then treated with different DTX-loaded formulations  or free drugs for 48 h. Following 48 h of incubation, the cells were collected, washed three times with cold PBS, suspended in 300 μL of binding buffer, and stained with Annexin V-FITC/PI. Finally, the cells were analyzed using flow cytometry . Non-treated cells were used as negative controls, and the experiment was repeated three times.Annexin V-FITC/PI dual staining was used to analyze apoptosis. C6 glioma cells were seeded in a 6-well plate at 1 × 106 were injected into the right striatum  of ICR mice. Using 1,1′-dioctadecyl- 3,3,3′,3′-tetramethylindotricarbocyanine iodide  as the fluorescent probe, the mice were intravenously injected with free DiR, DiR-DNS-C6m, DiR-DNS-DCm, or DiR-DNS-[C6&DC]m at day 15 post-inoculation (n = 3). At 2, 12, and 24 h, the biodistribution of free DiR and DiR-labeled DTX formulations fluorescence in each group was monitored by an in vivo spectrum imaging system  using excitation and emission wavelengths of 748 and 780 nm, respectively. For in vitro fluorescence imaging, mice in the DiR-labeled DTX formulations group were sacrificed at 12 h, and their brain tissues and major organs  were collected and visualized using similar imaging parameters. The fluorescence intensity of DiR at each time point was quantitatively evaluated using the ImageJ software.An intracranial glioma-bearing mouse model was established according to previously described procedures . After aFor tumor distribution analysis, using DiI (0.5 mg/kg) as the fluorescent probe, the glioma-bearing mice were intravenously injected with free DiI, DiI-DNS-C6m, DiI-DNS-DCm, and DiI-DNS-[C6&DC]m. After treatment for 4 h, brain tissues were removed and fixed with 4% paraformaldehyde in the dark for 24 h. DAPI, which was observed at an excitation wavelength of 358 nm and emission wavelength of 461 nm, was used to stain the nucleus. Finally, the distribution of nanosuspensions in each brain tissue was observed using an inverted fluorescence microscope.To evaluate the immune response in vivo, the glioma-bearing ICR mice were divided into six experimental groups and immunized three times at intervals of 1 week by intravenous injection with PBS (Model), DNS, DNS-C6m, DNS-DCm, [C6&DC]m, and DNS-[C6&DC]m at an equal amount of DTX (20 mg/kg). After 14 days, the posterior ocular venous plexus blood, which was isolated from mice, was centrifuged  to obtain serum. The different cytokines  in serum were then quantitatively analyzed. Briefly, serum was diluted three times with the standard diluent. Cytokines were detected using an ELISA kit according to the manufacturer’s protocol. Thereafter, the spleen and draining lymph nodes were dissected and collected. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into thin sections. Immunofluorescence and immunohistochemical (IHC) staining were conducted on both lymphoid and spleen tissues to determine the levels of CD4 and CD8 receptors and TNF-α, as well as to observe the activation of the immune system of glioma-bearing mice under different treatments.C6 glioma-bearing ICR mice were randomly divided into six treatment groups (six mice/group): PBS, DTX, DNS, DNS-C6m, DNS-DCm, and DNS-[C6&DC]m. The mice were intravenously injected with the corresponding DTX formulations at a DTX dosage of 20 mg/kg every 2 days Fig. A. After We investigated the in vivo biological safety of biomimetic nanosuspensions. Normal male mice were randomly divided into six groups and intravenously injected with 200 μL of normal PBS, free DTX, DNS, DNS-C6m, DNS-DCm, and DNS-[C6&DC]m. Fifteen days later, all mice were euthanized, and blood cells and serum biochemical indexes were examined. Further, the heart, liver, spleen, lung, kidney, and brain were removed for histology via H&E staining.All experiments were conducted in triplicate and expressed as mean ± standard deviation. Statistical significance was analyzed using SPSS 19.0 software . One-way analysis of variance with a post-hoc Tukey’s test was used to determine significant differences between datasets. Statistical significance levels were set at *p < 0.05, ** p < 0.01, and *** p < 0.001, ns, not significant, as indicated in Additional file The process of preparing DNS is illustrated in Fig. To verify that the extracted C6 cell membrane did not contain tumor nucleus-related genetic material, laser confocal microscopy was used to observe the separation of the nuclear membrane from C6 glioma cells. The nucleus was collected via centrifugation, and a portion of the cell membrane was precipitated. The cell nucleus was removed, the cell membrane was collected, and the nuclear membrane was separated Fig. C, D.As the successful fusion of DCm and C6m was crucial for the drug and antigen delivery of DNS-[C6&DCm], cell fusion was first investigated. To test the fusion of the DC and C6 membranes, a C6 membrane was doped with two different dyes that constituted a FRET pair . It was , the agglomerations of the biomimetic nanosystems in PBS and PBS with 10% FBS were evaluated within 72 h at 37 °C to mimic the stability at in vivo conditions. Compared with DNS, DNS-[C6&DC]m maintained good dimensional stability and a negative zeta potential in neutral PBS or PBS containing 10% FBS . The results showed that the original protein compositions of DCm and C6m were retained in DNS-[C6&DC]m  and stimulating factors  , 57. We ups Fig. : Fig. S7+ T lymphocytes (CTLs) and CD4 + helper T cells play a vital role in regulating the immune response m are inherited from two parent cell lines, hybrid membranes could confer a continuous source of tumor antigens, which can be presented for activating macrophages to release cytokines, including IL-6, TNF-α, and IFN-γ , 61. TheP < 0.001), indicating that [C6&DC]m could effectively stimulate macrophages to release cytokines . TNF-α and IFN-γ can bind to their corresponding receptors and then move into the cell. Following uptake by target cell lysosome, cytokines facilitate the reduction of lysosomal stability, resulting in the leakage of various enzymes, which could lead to the lysis of C6 cells. Furthermore, IL-6 can change the glucose metabolism of glioma cells, lower intracellular pH, and cause cancer cell death, further indicating that the fusion of C6m and DCm is necessary for stimulating cells to produce an immune response.To monitor the effect of released cytokines, the inhibitory effect of cytokines on C6 cells was quantified using the CCK-8 assay. As shown in Additional file 50 of 7.78 μg/mL, which was much lower than that of DTX (29.29 μg/mL). The apoptosis analysis via flow cytometry revealed that DNS-[C6&DC]m induced the highest apoptotic rate of 54.26 ± 2.32% (early and late apoptosis) in C6 cells, whereas the rates were 29.52 ± 4.143%, 29.31 ± 2.181%, 19.30 ± 5.021%, and 12.85 ± 2.820% for DNS-C6m, DNS-DCm, DNS, and [C6&DC]m, respectively . As shown in Fig. &DCm was We also collected the hearts, livers, spleens, lungs, kidneys, and brains of mice treated with different DiR-labeled DTX formulations and investigated their distribution in each organ Fig. D. StrongThe brain tissue of mice injected with DiI-labeled biomimetic nanosuspensions was sectioned. As shown in Fig. + CTLs are the main force that kills cancer cells in our immune design, and CD4+ T cells can help CTLs proliferate and increase their toxicity towards tumors [+ and CD4+ T cells from the spleen in the [C6&DC]m and DNS-[C6&DC]m groups increased significantly. In comparison, a much smaller increase was observed in the DNS-C6m- and DNS-DCm-treated groups. Although DNS-C6m contains tumor antigens from C6m, its efficacy in T cell activation seemed to be similar or even lower than that of DNS, which fully proves that DNS-[C6&DC]m relies on the antigen presentation effect of DCm to induce an immune response from the body. In the fusion process, DNS-[C6&DC]m can retain the tumor antigens of tumor cells, and mature DCs stimulated by tumor-associated antigens can promote the proliferation and activation of CD8+ and CD4+ T lymphocytes. Remarkably, a similar phenomenon was observed in the lymph nodes , which proved that DNS-[C6&DC]m relies on the antigen presentation effect of DCm to induce an immune response. The delivery of antigens to macrophages affects the microenvironment of the immune system, which may potentially lead to immunogenic cell damage. IHC was used to observe TNF-α expression within the lymph nodes and tumor sites  X-ray diffraction patterns of different components in the DNS. (B) Scanning of different components in the DNS using differential scanning calorimetry. (C) FTIR spectra of different components in DNS. Fig. S2. Zeta potential of different preparations, as measured using the DLS. Fig. S3. Quantification of total proteins on DNS-[C6&DC]m by BCA assay after incubating different amount of [C6&DC]m to DNS at different membrane-to-DNS weight ratios (w/w). Fig. S4. (A) The membrane protein of the biomimetic nanosuspensions was determined via SDS-PAGE. (B) The protein contents of cancer cell membranes and biomimetic nanosuspensions were determined using the BCA kit. (C) Gray value of membrane-specific proteins ICAM, CD44 (D), MHC I(E), and CD80 (F). Fig. S5 Release profile of free DTX, DNS, and DNS-[C6&DC]m in (A) PBS at pH 7.4, (B) PBS at pH 6.8, or (C)10% FBS at 37 °C. Error bars: mean ± SD (n = 3). Fig. S6 Cell uptake of the biomimetic nanosuspensions by different cancer cells was investigated using CLSM. The intracellular uptake of DNS-[C6&DC]m in B16 (A), HepG2 (B), 4T1 (C), and C6 glioma (D) cells. The nuclei were stained with Hoechst 33258 (blue), and the DNS-[C6&DC]m were labeled with DiI (red) (40× magnification). Fig. S7. The percentage of DCs maturation. . Fig. S8. The inhibitory effect of cytokines on C6 glioma cells. . Fig. S9. The inhibitory effect of different DTX-loaded formulations on C6 glioma cells. Error bars: mean ± SD (n = 6).


Migration through the extracellular matrix (ECM) is essential for cancer cells to escape the primary tumor and invade neighboring tissues with the potential for metastasis  ECM rigidity was varied from soft  to hard 22.1The HNSCC cell line SCC-61 was cultured in Dulbecco's modified Eagle's medium (ThermoFisher) containing 20% fetal bovine serum (ThermoFisher) and 0.4 μg/ml hydrocortisone (MilliporeSigma) as previously described 2.2Soft and hard PAAs were prepared from stocks of 40% acrylamide (Bio-Rad), 2% BIS-acrylamide (Bio-Rad), 10 mg/ml acrylic acid N-hydroxysuccinimide (MilliporeSigma), and 1 mg/ml fibronectin (ThermoFisher) at final concentrations of 8%/0.05%/0.1% with 200 μg/ml fibronectin and 8%/0.35%/0.1% with 215 μg/ml fibronectin, respectively, and cast on activated coverslips in 35 mm glass bottom dishes (MatTek) as previously described . Soft an2.3For traction force assays, 200 nm red fluorescent beads (ThermoFisher) were included at a final ratio of 1:125 in the PAAs to detect substrate displacements caused by cellular forces as previously described ,9. Brief2.4For invadopodia assays, PAAs were overlaid with 1% crosslinked gelatin (Polysciences) and centrifuged 50 μg/ml fluorescein isothiocyanate-labeled fibronectin to detect ECM degradation as previously described ,4,9. Bri2.5p-value < 0.05 considered statistically significant [As previously described, data were evaluated for normality using the Kolmogorov-Smirnov test, determined to be nonparametric, and compared using a Mann-Whitney test with a nificant ,4.N/A.Rachel Jerrell: Investigation, Visualization. Mitchell Leih: Formal analysis. Aron Parekh: Formal analysis, Methodology, Visualization, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


Age-related increases in chronic inflammation lead to reduced physical function via damage to muscle and joints and contribute to osteoarthritis (OA) risk. Obesity in older adults with OA further exacerbates inflammatory damage. Whether obesity reduction can lessen inflammation and improve OA is unknown; however, novel biomarkers may provide an answer. We completed a 6-mo. weight loss intervention , studying blood biomarkers of inflammation and cartilage damage along with physical function in obese older adults with  and without an OA diagnosis . Participants were aged > 60 yrs (mean = 70.2±6.0) and obese (BMI =34.6±4.7 kg/m2). At endpoint, weight loss was -6.3±4.0% and -5.8±4.1% in OA+ and OA-, respectively, with no group difference. Change scores for function for OA+ and OA- were: Short Physical Performance Battery score (+1.7±1.3 and +2.1±1.5), 8 ft up and go (-0.7±1.0 and -0.9±1.12 sec) and 6 min walk (+31.4±105.1 and +39.5±57.4 meters). All improved from baseline (p<0.05), with no group difference. Concerning blood biomarkers, there was a decrease (p<0.05) in cartilage oligomeric matrix protein (COMP: OA biomarker), indicating a potential benefit for OA. Change in COMP also differed between groups; OA- had a greater (p<0.05) reduction than OA+. Pooled results showed improved adiponectin (p<0.05), with no group difference. There were no changes for CRP, CTX-1, IL-6 and TNF-α. Our novel findings link early intervention with better reduction of OA risk and inflammation in obese older adults and also show important benefits for improved physical function regardless of OA status.


MITF) and tyrosinase expression by inhibiting cyclic adenosine monophosphate (cAMP) and cAMP response element-binding protein (CREB). Furthermore, under α-MSH stimulation, GABA-related antimelanogenic effects were mediated through the GABAA and GABAB receptors, with subsequent inhibition of Ca2+ accumulation. In B16F10 melanoma cells and zebrafish larvae, pretreatment with bicuculline, a GABAA receptor antagonist, and CGP 46381, a GABAB receptor antagonist, reversed the antimelanogenic effect of GABA following α-MSH treatment by upregulating Ca2+ accumulation. In conclusion, our results indicate that GABA inhibits α-MSH-induced melanogenesis. Hence, in addition to the health benefits of GABA in the central nervous system, it could ameliorate hyperpigmentation disorders.Gamma-aminobutyric acid (GABA) is considered the primary inhibitory neurotransmitter in the human cortex. However, whether GABA regulates melanogenesis has not been comprehensively elucidated. In this study, we reveal that GABA (20 mM) significantly inhibited α-melanocyte-stimulating hormone (α-MSH)-induced extracellular (from 354.9% ± 28.4% to 126.5% ± 16.0%) and intracellular melanin contents (from 236.7% ± 11.1% to 102.7% ± 23.1%) in B16F10 melanoma cells, without inducing cytotoxicity. In addition, α-MSH-induced hyperpigmentation in zebrafish larvae was inhibited from 246.3% ± 5.4% to 116.3% ± 3.1% at 40 mM GABA, displaying no apparent cardiotoxicity. We also clarify that the GABA-mediated antimelanogenic properties were related to the direct inhibition of microphthalmia-associated transcription factor ( MITF) ..4 cells/4 cells/mL) were treated with the indicated concentrations of GABA (0–20 mM) in the presence or absence of 500 ng/mL α-MSH for 72 h. Total protein was extracted using a PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology), and total proteins were quantified using a Bio-Rad Protein Assay Kit (Bio-Rad). Western blotting was performed to detect the expression of MITF, tyrosinase, and pCREB according to our previous study [B16F10 melanoma cells (5 × 10us study . β-Actintyrosinase activity was measured using a previously described method [4 cells/mL) were treated with 20 mM GABA in the presence or absence of 500 ng/mL α-MSH for 72 h. In a parallel experiment, bicuculline or CGP 46381 was pretreated for 2 h in the presence or absence of GABA and/or α-MSH. Then, the cells were lysed with PBS containing 1% Triton X-100 by freezing at −20 °C for 2 h and disrupted by thawing at room temperature. Total protein was quantified with Bio-Rad Protein Assay Reagents (Bio-Rad), and an equal amount of protein was mixed with 90 μL of 5 mM L-dihydroxyphenylalanine (DOPA) at 37 °C for 30 min and the absorbance at 405 nm was measured.Intracellular d method . Briefly4 cells/mL) were cultured in 3% gelatin-coated cover slips and treated with 20 mM GABA in the presence or absence of 500 ng/mL α-MSH for 72 h. The cells were fixed with 4% paraformaldehyde for 30 min and incubated with 0.1% L-DOPA in 1× PBS and 20 μM copper sulfate at 37 °C for 2 h. The images were captured using phase-contrast microscopy (MACROTECH).B16F10 melanoma cells (5 × 102+ changes were measured according to a previously described method with a slight modification [4 cells/mL) were grown in a 12-well plate and pretreated with bicuculline or CGP 46381 for 2 h prior to treatment with 20 mM GABA and/or 500 ng/mL α-MSH. Then, the cells were trypsinized and probed with a Ca2+-sensitive probe, Fluo-4 AM (1 μM) in calcium buffer , for 10 min. Kinetics scans were performed for 40 min to assess the time course of Ca2+ fluorescence changes at EX/EM 490/570 using a GloMax 96 Microplate Luminometer .Intracellular Cafication . BrieflyB16F10 melanoma cells were cultured in 8-well chamber slides and treated with 20 mM GABA or 2 mM EGTA in the presence and absence of 500 ng/mL α-MSH for 1 h. Then, the cells were washed with PBS and probed with 1 μM Fluo-4 AM in calcium buffer for 10 min. The images of the cells were captured using a CELENA S Digital Imaging System .The zebrafish was raised and handled according to standard guidelines of the Animal Care and Use Committee of Jeju National University . All methods were carried out in accordance with the approved guidelines . Eggs wewww.imagej.net, accessed on 15 June 2021) and then statistically analyzed by Sigma plot 12.0 . All data represented the mean of at least three independent experiments. Significant differences were determined using a Student t-test and an unpaired one-way analysis of variance (ANOVA) test with Bonferroni correction. Statistical significance was set at *** and ### p < 0.001, ** and ## p < 0.01, and * and # p < 0.05.All the Western blots and RT-PCR bands were quantified by ImageJ 1.50i (National Institute of Health, Manassas, VA, USA, A and GABAB receptors are associated with the antimelanogenic properties of GABA, accompanied by the inhibition of the canonical melanogenic signaling pathway and intracellular Ca2+ levels. Our findings may contribute to the potential use of GABA for the development of antimelanogenic agents. Nevertheless, whether the Ca2+ signaling pathway is strongly associated with the antimelanogenic properties of GABA should be evaluated, in detail, by targeting the GABAA and GABAB receptors.In this study, we determined that, under α-MSH stimulation, the GABA


Through the determination of apoptosis, reactive oxygen species production, and endoplasmic reticulum stress-related gene expression, we found that ERS induced by TM can be relieved by ghrelin in a concentration-dependent manner (P < 0.001). Compared with the TM group, ghrelin reduced the expression of ERS-related marker genes induced by TM. Compared with the GSK621 + TM group without ghrelin pretreatment, the mRNA expression of genes in the ghrelin pretreatment group decreased significantly (P < 0.001). The results of protein analysis showed that the levels of BIP, p-AMPK, and cleaved-caspase3 in the TM group increased significantly, while the levels decreased after ghrelin pretreatment. In group GSK621 + TM compared with group GSK621 + ghrelin+TM, ghrelin pretreatment significantly reduced the level of p-AMPK, which is consistent with the trend of the ERS-related proteins BIP and cleaved-caspase3. In conclusion, ghrelin alleviates the ERS induced by TM in a concentration-dependent manner and may or at least partly alleviate the apoptosis induced by ERS in MC3T3E1 cells by inhibiting the phosphorylation of AMPK.Ghrelin is a gastric endocrine peptide that has been found to be involved in the process of energy homeostasis and bone physiology in recent years. To explore the effects of ghrelin on endoplasmic reticulum stress (ERS) in MC3T3E1 cells and its possible mechanism, an ERS model was induced by tunicamycin (TM) in the osteoblast line MC3T3E1. TM at 1.5  As a hidden and common disease, osteoporosis is characterized by a decrease in bone mass and a change in bone microstructure. Its complications, pain and brittle fracture, seriously affect the quality of life of patients and even threaten their lives. Previous studies have shown that apoptosis induced by endoplasmic reticulum stress (ERS) in osteoblasts plays an important role . ERS is Ghrelin, the endogenous ligand of growth hormone secretagogue receptor type 1a (GHSR-1a), is composed of 28 amino acids . In receAMPK (Adenosine Monophosphate Activated Protein Kinase), a heterotrimer complex, contains a highly conserved serine/threonine kinase domain and is a key molecule in the regulation of bioenergy metabolism . PreviouBased on the ERS model of osteoblasts induced by tunicamycin (TM), one of the aims of this experiment was to pretreat osteoblasts with different concentrations of ghrelin, detect the apoptosis of osteoblasts and the content of intracellular reactive oxygen species (ROS), and observe the effect of ghrelin on ERS in a concentration-dependent manner. The other purpose was to verify the expression of ERS-related marker genes and the changes in related pathway factors in different groups at the molecular level to further explain the protective effect of ghrelin on ERS in osteoblasts and its possible related pathways.Tunicamycin (TM) was purchased from Cayman Chemical Company (USA). Ghrelin (molecular weight: 3314.8) was synthesized by Nanjing Peptide Biology Company (CN). GSK621, purchased from Cayman Chemical Company, is the agonist of AMPK.α-MEM medium  containing 10% fetal bovine serum and 1% penicillin and then maintained in a humidified, 5% CO2 atmosphere at 37°C. The medium was refreshed every two or three days, and the cells were subcultured using 0.05% trypsin with 0.01% EDTA.MC3T3E1 cells  were cultured in 2 incubator at 37°C at a concentration of 5∗104 cells/ml and inoculated in a 96-well plate. After the cells in the 96-well plate were completely adhered to the wall, the culture medium with different concentrations of TM  was replaced. After 20 hours, the CCK-8 reagent was added, the absorbance at 450 nm was measured by a microplate reader, and the IC50 of cells was calculated for subsequent experiments.Cell viability was evaluated by a CCK-8  method. MC3T3E1 cells were incubated overnight in a 5% CO5 cells, and resuspended in 500 μL of binding buffer. Then, 5 μL of Annexin V-FITC was added, 5 μL PI  was added, and the mixture was incubated at room temperature in the dark for 15 min. Apoptosis was detected by flow cytometry. The upper left quadrant represents a mechanically injured cell, and the upper right quadrant is a late apoptotic cell. The lower left quadrant is a normal cell, and the lower right quadrant is an early apoptotic cell. In this experiment, the proportion of the upper right quadrant + the lower right quadrant was used as the percentage of apoptotic cells.After each group was treated with the corresponding drugs and the necessary control groups were set up, the cells were digested with 0.25% trypsin (excluding EDTA), washed twice with PBS to collect 5∗106 cells were harvested by trypsin. Then, the cells were incubated in the same volume of DCFH-DA solution  at 37°C for 0.5 h according to the manufacturer's protocol so that the probes were in full contact with the cells and washed with PBS to remove the redundant probes. Flow cytometry was used to detect the production of reactive oxygen species in osteoblasts.After each group was treated with drugs, a negative control and a positive control were established at the same time. After 24 hours of culture, the cells were washed with warm PBS, and 105 cells/well). After adhering to the wall, cells were cultured to a density of approximately 70% and then replaced with warm drug medium under different treatment conditions including GSK621 pretreatment for 3 hours, ghrelin treatment for 4 hours, and TM followed by 20 hours. Total RNA was extracted according to the instructions , and the concentration and purity of RNA were measured by a spectrophotometer. Using RNA as a template, quantitative reverse transcription was performed converting 1 μg of into cDNA. Finally, cDNA was used as a template, and specific forward primers and reverse primers of each gene were added to amplify the cDNA. The primer  sequences used in this study are presented in Cells in the logarithmic growth phase were inoculated in 6-well plates (2∗10μg) was loaded into 10% SDS–PAGE gels for electrophoresis under constant pressure. The protein was then transferred to the PVDF membrane under constant current, sealed with 8% skimmed milk powder for 1.5 hours, incubated with the primary antibody  for 12–16 hours, washed on a shaker with TBST 4 times for 10 minutes each time, and then washed with the residual secondary antibody on a shaker again with the corresponding secondary antibody . The generated protein bands were exposed and visualized on a Fusion Fx machine in a dark room using ECL luminescent solution .After inoculation, the cells were cultured in complete medium at a density of approximately 70%, and the corresponding treatments were carried out according to the groups. Western blotting was carried out according to previous research protocols, and RIPA  was used to extract the total proteins. A BCA protein assay kit  was used to determine the protein concentration, and the same amount of protein  and Tukey's multiple comparisons test. P < 0.05 (two-sided) was considered to indicate a significant difference.All experiments were repeated three times. Data analysis and chart making were carried out using GraphPad Prism 7.0 software or SPSS 22.0. Flow J 7.6.1 software was used to analyze the apoptosis and ROS data of flow cytometry. ImageJ software was used to analyze protein quantification. The general characteristics of the data are described as the mean ± SD or mean ± SEM. Two independent samples were compared by the Student's μg/mL, the cell survival rate of each group was significantly different from that of the control group. The half cytotoxicity index was approximately 1.5 μg/mL, which will be used in subsequent experiments to establish the model of endoplasmic reticulum stress in MC3T3E1 cells.In P < 0.001) after 20 hours of TM treatment, while after 4 hours of pretreatment with different concentrations of ghrelin, the apoptosis rate decreased significantly with the increase of the ghrelin concentration, and the P value of the G10−11M + TM (29.3 ± 0.64), G10−9M + TM (22.5 ± 0.26), and G10−7M + TM (15.6 ± 1.90) groups compared with the TM group were 0.003, <0.001, and <0.001, respectively. Ghrelin has a concentration-dependent protective effect on the TM-induced apoptosis of MC3T3E1 cells.In P < 0.001). The fluorescence intensities of the experimental group pretreated with different concentrations of ghrelin for 4 hours were G10−11M + TM (107.2 ± 1.5), G10−9M + TM (97.6 ± 0.9), and G10−7M + TM (77.0 ± 0.6), which were significantly different from those treated with TM alone for 20 hours, with P values of 0.002, <0.001, and <0.001, respectively. In addition, ghrelin had a concentration-dependent effect on ROS production induced by TM, and the ROS content decreased with increasing pretreatment ghrelin concentration, which is consistent with the above apoptosis results.The results of P < 0.001), but decreased significantly after ghrelin pretreatment (20.0 ± 0.49) (P < 0.001). Compared with the GSK621 + TM group, the apoptosis rate in the GSK621 + G10−7M + TM (22.9 ± 0.63) group decreased significantly (P < 0.001) (see Compared with TM (28.2 ± 0.64), the apoptosis rate of GSK621 + TM (55.5 ± 1.06) increased significantly (001) see .P < 0.001. After ghrelin pretreatment , the average fluorescence intensity of ROS decreased significantly compared with that of the TM group (P < 0.001). Compared with that in the TM + GSK621 group, the ROS content in the GSK621 + G10−7M + TM group (100.3 ± 0.37) was significantly decreased (P < 0.001) (see The intracellular ROS content in TM + GSK621 (126.5 ± 0.57) was significantly higher than that in TM (100.6 ± 0.22), 001) see .α), which were significantly higher than those in the control group (P < 0.01). Figures P < 0.05).Figures P < 0.001; TM vs. G10−7M + TM (12.17 ± 1.62), P < 0.001, and C vs. G10−7M + TM, P < 0.001. The relative expression of CHOP among groups in P < 0.001; TM vs. G10−7M + TM (16.10 ± 2.33), P < 0.001, and C vs. G10−7M + TM, P < 0.001. In α among groups is as follows: TM (9.71 ± 2.18) vs. C (1.07 ± 0.43), P < 0.001; TM vs. G10−7M + TM (6.28 ± 0.75), P = 0.007, and C vs. G10−7M + TM, P = 0.001. The expression of three endoplasmic reticulum marker genes increased after TM treatment, and this phenomenon could be improved by ghrelin pretreatment for 4 hours, although there was still a certain degree of endoplasmic reticulum stress compared with the control group. In addition, BIP, CHOP, and IRE1α were all P > 0.05 between G10−7M vs. C, with no significant difference.In P values of groups G10−7M + TM (0.75 ± 0.04) and GSK621 + TM (2.32 ± 0.16) were 0.011 and <0.001, respectively. The relative expression of CHOP in P values of groups G10−7M + TM (0.75 ± 0.07) and GSK621 + TM (1.39 ± 0.09) were 0.002 and <0.001, respectively. The relative expression of IRE1α in P values of groups G10−7M + TM (0.72 ± 0.11) and GSK621 + TM (1.59 ± 0.11) were 0.029 and 0.001, respectively. On the one hand, ghrelin alleviated the ERS induced by TM to a certain extent, while GSK621 promoted further expression of ERS-related marker genes induced by TM. In addition, the GSK621 + G10−7M + TM group had a significant decrease in the relative expression of three ERS-related genes compared with the GSK621 + TM group (P < 0.001).Taking the TM group as a control group, the relative expression of BIP in P = 0.007), p-AMPK , and cleaved-caspase3  in the TM group were significantly higher than those in the control group.−7M + TM group pretreated by ghrelin is significantly lower than that in the TM group, including BIP: TM (0.87 ± 0.05) vs. G10−7M + TM (0.33 ± 0.09), P = 0.003, p-AMPK: TM (0.80 ± 0.04) vs. G10−7M + TM (0.29 ± 0.08), P = 0.003, and cleaved-caspase3: TM (1.12 ± 0.20) vs. G10−7M + TM (0.41 ± 0.05), P = 0.008. C vs. G10−7M showed no significant differences in the expression of the three types of proteins.In −7M + TM (0.15 ± 0.08) group decreased, P = 0.046; the expression of p-AMPK (P = 0.024) and cleaved-caspase3 (P = 0.001) showed a similar trend. After pretreatment with GSK621, an agonist of AMPK, for 3 hours, the relative expression of BIP, p-AMPK, and cleaved-caspase3 increased, with P values less than 0.05. In addition, comparing the relative expression of BIP with GSK621 + TM group (−7M + TM (0.28 ± 0.08), P = 0.004. Meanwhile, comparing the relative expression of p-AMPK (−7M + TM (0.60 ± 0.08), P = 0.001, and comparing the relative expression of cleaved-caspase3 (−7M + TM (0.36 ± 0.06), P < 0.0001.In TM group , GSK621 f p-AMPK , GSK621 caspase3 , GSK621 In bone physiology, there is a dynamic balance between bone formation and bone absorption. Osteoporosis is a manifestation of the balance breaking, which is characterized by a change in bone microstructure and an increase in bone fragility , and theα (inositol-requiring enzyme 1α), PERK (protein kinase RNA-like ER kinase), and ATF6 (activating transcription factor 6) [2O2 and O2− in the endoplasmic reticulum induce cell apoptosis through lipid peroxidation and mitochondrial dysfunction and change calcium homeostasis [Endoplasmic reticulum stress, as mentioned above, can be roughly understood as the self-protective state of the body caused by the accumulation of unfolded or misfolded proteins under the action of internal and external factors, also known as the “unfolded protein response (UPR),” which can also induce autophagy and apoptosis of cells beyond a certain range . Generalactor 6) . BIP/GRPactor 6) . Moreoveactor 6) . In addiactor 6) . Studieseostasis . TherefoIn this experiment, we first observed the incidence of apoptosis and the production of ROS in the ERS model pretreated with different concentrations of ghrelin on the premise of excluding the toxicity and side effects of ghrelin itself. Previous studies have shown that there is no significant difference between the ghrelin alone treatment group and the control group , 23. ConSecond, ghrelin significantly reduced the expression of ERS-related genes induced by TM and had a protective effect on cells. In addition, there was no significant difference in the three genes between the control group and the ghrelin treated group, which indicated that ghrelin itself had no significant effect on the production of ERS, excluding the possible drug effect of ghrelin itself, but the combination of ghrelin and TM cotreatment group compared with the control group showed that ghrelin could alleviate the ERS caused by TM on MC3T3E1 cells to a certain extent, but may not completely. Nevertheless, a large number of studies have shown that ERS has a protective effect on cells to a certain extent , but thi2+ through the IP3R  channel. The increase in Ca2+ in the cytoplasm activates CaMKII , which promotes apoptosis as an upstream molecule of AMPK [β during starvation [The activation of AMPK may partly depend on the increase in intracellular calcium . When ER of AMPK . Anotherarvation . TherefoIn addition, at the protein level, we can more intuitively see that TM can activate AMPK, and ghrelin can downregulate the phosphorylation level of AMPK and the level of p-AMPK induced by AMPK agonists, which inversely proves the possible relationship between the AMPK pathway and ERS and the important role of ghrelin.Certainly, we cannot deny previous research results. It has been reported that the protective effect of ghrelin is mediated by stimulating GHSR (growth hormone secretagogue receptor) , but theIt is worth noting that acylation modification was considered to be an essential factor for the biological activity of ghrelin in the past , but in This research may provide new insight into the protective effects of ghrelin on bone by maintaining endoplasmic reticulum homeostasis and regulating of ERS. Ghrelin may alleviate endoplasmic reticulum stress apoptosis induced by tunicamycin in MC3T3E1 cells at least in part by inhibiting AMPK phosphorylation.


Predicted in silico physicochemical properties of the newly synthesised hybrid (3) demonstrated a drug-like profile with suitable Absorption, Distribution, Metabolism, and Excretion (ADME) properties and low toxic liabilities. Preliminary cytotoxicity evaluation towards human prostate (DU145) and lung (A549) cancer cell lines demonstrated that 3 exerted a similar effect on cell viability to that produced by the reference drug 5-FU. Among the two tested cancer cell lines, the A549 cells were more susceptible for 3. Of note, hybrid 3 also had a significantly lower cytotoxic effect on healthy human lung epithelial cells (BEAS-2B) than 5-FU. Altogether our results served as an initial proof-of-concept to develop 5-FU/HO-1 mutual prodrugs as potential novel anticancer agents.In this work, the first mutual prodrug of 5-fluorouracil and heme oxygenase1 inhibitor (5-FU/HO-1 hybrid) has been designed, synthesised, and evaluated for its  Chemical shifts are in δ values (ppm) using tetramethylsilane (TMS) or CH3OH as the internal standard for spectra recorded in DMSO-6d or CD3OD, respectively. Coupling constants (J) are given in Hz. Signal multiplicities are characterised as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). Reactions were monitored by thin-layer chromatography (TLC), carried out on Merck plates (Kieselgel 60 F254), using UV light (λ = 254 and 366 nm) for visualisation and staining the TLC plate with iodine vapour in a closed chamber. Flash column chromatography was performed on silica gel 60, 0.040–0.063 mm . Mass spectra were recorded on a UPLC-MS/MS system consisted of a Waters ACQUITY® UPLC®  coupled to a Waters TQD mass spectrometer (electrospray ionisation mode ESI-tandem quadrupole). Chromatographic separations were carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column; 2.1 × 100 mm, and 1.7 mm particle size, equipped with Acquity UPLC BEH C18 Van Guard pre-column, 2.1 × 5 mm, and 1.7 mm particle size. The column was maintained at 40 °C and eluted under gradient conditions from 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 ml min−1. Eluent A: water/formic acid ; eluent B: acetonitrile/formic acid . Chromatograms were made using Waters eλ PDA detector. Spectra were analysed in 200–700 nm range with 1.2 nm resolution and sampling rate 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flowrate 600 L h−1, cone gas flow 100 L h−1, capillary potential 3.00 kV, cone potential 40 V. Nitrogen was used for both nebulising and drying gas. The data were obtained in a scan mode ranging from 50 to 2000 m/z in time 1.0 s intervals. Data acquisition software was MassLynx V 4.1 (Waters). The UPLC/MS purity of all the final compounds was confirmed to be 95% or higher.Reagents, solvents, and starting materials were purchased from commercial suppliers. Melting points were determined in an IA9200 Electrothermal apparatus equipped with a digital thermometer in capillary glass tubes and are uncorrected. Infrared spectra were recorded on a Perkin Elmer 281 Fourier-transform infrared (FTIR) spectrometer in KBr discs  or placing a sample droplet between two discs of pure NaCl (neat sample). Elemental analyses for C, H, N, were within ± 0.4% of theoretical values and were performed on a Carlo Erba Elemental Analyser Mod. 1108 apparatus. 1 , succinic anhydride , and triethylamine  was refluxed in dry methylene chloride (10 ml) for 6 h. The solvent was removed under reduced pressure, and the crude thus obtained was purified by column chromatography on silica gel using a mixture of ethyl acetate-methanol  as eluent to afford compound 2 , as a white pure solid: mp 64.0–66.5 °C. IR  cm−1 3126, 2930, 2738, 1738, 1574, 1428, 1371, 1155, 836. 1H NMR  δ 7.62 , 7.55–7.41 , 7.30–7.20 , 7.13 , 6.97 , 6.03 , 4.45 , 2.72–2.45 . UPLC/MS purity 98%, tR = 3.543 min. MS (ESI) m/z: 367.2 [M + H]+. Anal. Calcd. for C15H15BrN2O2: C, 49.06; H, 4.12; N, 7.63. Found: C, 48.99; H, 4.09; N, 7.60.A mixture of compound 2 , N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) , and a catalytic amount of 4-dimethylaminopryridine (DMAP) , were added under a nitrogen flow, and the mixture was then stirred at room temperature for 12 h. The solvent was removed under reduced pressure, and the crude thus obtained was purified by column chromatography on silica gel using a mixture of ethyl acetate-methanol  as eluent to afford compound 3 , as a white pure solid: mp 172.0–174.5° C. IR  cm−1 3448, 3122, 1732, 1671, 1509, 1412, 1366, 1265, 1143, 993. 1H NMR : δ 8.11 , 7.62 − 7.44 , 7.40 − 7.25 , 7.12 , 6.85 , 5.95 , 5.56 , 4.37 , 2.71–2.55 . UPLC/MS purity 99%, tR = 3.773 min. MS (ESI) m/z: 509.0 [M + H]+. Anal. Calcd. for C20H18BrFN4O6: C, 47.17; H, 3.56; N, 11.00. Found: C, 47.03; H, 3.49; N, 10.95.To a stirred suspension of 1-(hydroxymethyl)-5-FU  in a mixture solvent of dry methylene chloride-acetonitrile (2 + 2 ml), compound in vitro stability in different buffers and porcine esterase solution was performed on Shimadzu Prominence-i LC-2030C 3 D Plus equipped with RID20A. Detector chromatographic separation were carried out using Chromolith SpeedROD RP 18.5 µm, 1.6 × 50 mm, Merck. Spectra were analysed in 200–800 nm range with 1.2 nm resolution. The column was maintained at 30 °C and eluted under gradient conditions from 100% to 0% of eluent A over 3 min, at a flow rate of 5 ml min−1. Eluent A: water/trifluoroacetic acid ; eluent B: acetonitrile/ trifluoroacetic acid .The HPLC analysis of samples at various time intervals from 3 in DMSO (3.0 mg/mL) was prepared. To a test tube containing 0.9 ml of the corresponding Acetate (pH = 2.0) or PBS buffer solution , 0.1 ml of stock solution was added, and the mixture stirred and thermostated in a sand bath at 37 °C. Aliquots (0.1 ml) were withdrawn at specific time intervals and transferred to sample vials containing acetonitrile (0.9 ml). The percentage of compound remaining was followed by HPLC analysis. The retention time (tR) of compound 1, 2, and 3 were 0.74, 0.89, and 0.95 min, respectively. All the experiments were performed in triplicate.A stock solution of compound t) against time. Half-life (t1/2) was calculated according to k is the pseudo-first-order rate constant.0.001 g of lyophilised powder of esterase from the porcine liver  was reconstituted in 1.0 ml PBS buffer  to make an aqueous porcine esterase solution (5 U/mL), and then pre-thermostated at 37 °C. To a test tube containing 1.85 ml of PBS buffer, 0.15 ml from the stock solution of the test compound was added. The mixture was then stirred and thermostated in a sand bath at 37 °C, and 1.0 ml of the porcine esterase solution was added to initiate the enzymatic reaction. For the negative control reaction, the volume of porcine esterase solution was replaced by phosphate buffer. Aliquots (0.3 ml) were withdrawn at 0, 30, 60, 120, 180, and 240 min, and quenched with cold acetonitrile (0.7 ml)in vivo) forms of HO-1. Spleen (Sprague-Dawley rats) microsomal fractions were prepared according to the procedure outlined by Ryter et al.g for 20 min at 4 °C, followed by centrifugation of the supernatant at 100,000 g for 60 min at 4 °C. The 100,000 g pellet (microsomes) was resuspended in 100 mM potassium phosphate buffer, pH 7.8, containing 2 mM MgCl2 with a Potter-Elvehjem homogenising system. The rat spleen microsomal fractions were divided into equal aliquots, placed into microcentrifuge tubes, and stored at −80 °C for up to 2 months.Since the dominance of HO-1 protein in the rat spleen has been well documentedg, for 20 min at 4 °C. The supernatant was decanted and centrifuged at 100,000 g for 1 h at 4 °C to sediment the microsomes. The 100,000 g supernatant was saved and then stored in small amounts at −80 °C after its protein concentration was measured.Liver cytosol has been used as a source of BVR. Rat liver was perfused through the hepatic portal vein with cold 0.9% NaCl, then it was cut and flushed with 2 × 20 ml of ice-cold PBS to remove all of the blood. Liver tissue was homogenised in 3 volumes of a solution containing 1.15% KCl w/v and Tris buffer 20 mM, pH 7.8 on ice. Homogenates were centrifuged at 10,000 −1 for bilirubin). One unit of the enzyme was defined as the amount of enzyme catalysing the formation of 1 nmol of bilirubin/mg protein/h.The HO-1 activity was determined by measuring the bilirubin formation using the difference in absorbance at 464 to 530 nm as described by Ryter et al.2. The effect of 5-FU, compound 1 and 3 on cell viability was assessed by performing the 3–-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded into 96-well plates at a density of 7,0 × 10Experiments were performed on human prostate cancer cells , human lung cancer cells  and human bronchial epithelium cells . Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of heat-inactivated foetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin . Cells were incubated at 37 °C in a humidified atmosphere with 5% COpost hoc test. The level of significance for all statistical tests was set at p ≤ 0.05.Data are represented as mean ± standard error (SEM). One-way analysis of variance (ANOVA) was used to compare differences among groups, and statistical significance was assessed by the Tukey–Kramer H-imidazol-1-yl)ethanol (1) was prepared according to previously reported synthetic procedures,3) has been synthesised through a two-step pathway by following the reaction conditions depicted in 1 reacted with succinic anhydride under basic condition to give 4–(1-(3-bromophenyl)-2-(1H-imidazol-1-yl)ethoxy)-4-oxobutanoic acid (2). Intermediate 2 was then coupled with 1-hydroxymethyl-5-fluorouracil, which was prepared based on a known method1–(3-bromophenyl)-2-(11) and the 5-FU/HO-1 hybrid (3) were calculated and reported in The pharmacokinetic profile and adverse side effects (ADME/Tox) of a molecule are closely related to its physicochemical properties; thus, calculation of molecular descriptors appears to be a useful methodology to define drug-likeness,3), only one violation of the Lipinski’s rule of five occurred (i.e. MW > 500), while both the 5-FU and the imidazole-based derivative (1) fully comply with the rule. Similarly, one violation of Veber’s rule was observed for compound 3 (i.e. RBN >3). Notably, unlike specific descriptors, including the cLogP, HBD, and HBA, a statistically significant increase in MW and RBN values have been observed for approved oral drugs in this decade, as recently analysed by Shultz3  of 5-FU, 1, and 3 was performed. Results suggested that the newly synthesised hybrid (3) might exhibit a proper ADME profile with good absorption and sufficient distribution  tract (pH = 2.0), human plasma (pH = 7.4), and pancreatic fluid (pH = 8.0). As a result, the pH value strongly affected the chemical stability and hydrolysis rate of the hybrid 3 , the chemical stability of 3 was significantly lower than both in acid and neutral conditions, supporting the fact that a small percentage of free parent compounds might be available for absorption across the intestine. Indeed, the percentage of compound 3 remaining after 24 h equalled 94.2% at pH = 2.0, 77.3% at pH = 7.4, and 50.4% at pH = 8.0, respectively .The lowest 3 and subsequently converted into different active metabolites 1 should be efficiently regenerated. Therefore, the enzymatic stability of 3 in porcine esterase solution was investigated. The hydrolysis rate profile observed for the 5-FU/HO-1 hybrid in such conditions was in line with the pseudo-first-order kinetics model (3 in plasma mimicking solution was quicker (t1/2 = 136 min) than that in buffer solution , confirming the enzyme hydrolysis contribution. These data support the choice of the succinyl spacer as a suitable cleavable linker to release the active moieties from 3 in the right time frame compatible with the biological activity. Notably, hybrid 3 was not detected at 24 h incubation in porcine esterase solution .To exert its pharmacologic effect the 5-FU moiety must be released from cs model . Remarka50) in μM . As expected, the hydride 3 exhibited a lower inhibitory potency towards HO-1 than the parent compound 1 . Compound 2, a possible metabolite of 3 showed even lower inhibitory activity towards HO-1 (104.6 ± 5.8 μM). These results were consistent with previous structure-activity relationship (SAR) studies performed on azole-based analogs ,3 displayed a lower inhibitory potency towards the HO-1 with respect to parent derivative 1, this aspect does not represent an issue since hybrid 3 acts as a mutual prodrug by releasing the parent drugs .Inhibition activity assay for HO-1 was performed by extracting the enzyme from the rat spleen microsomal fraction. HO-1 activity was determined by measuring the formation of BR using the difference in absorbance at 464–530 nm, according to the protocol described in the experimental section. Results are expressed as enzyme inhibition activity , in which the overexpression of the HO-1 has been confirmed,1, in a 1:1 ratio, was also evaluated and used for comparison. Briefly, cell lines were treated with the tested compounds at different concentrations . Cell survival was calculated compared to untreated controls for 72 h. At the end of treatment, cell viability was evaluated using the MTT assay. Results are depicted in Compound 3 produced a significant dose-dependent effect on both cancer cell lines, similar to that of the reference drug 5-FU  towards the tested cancer cell lines  and 3 (IC50 = 1.45 ± 1.04 μM) than DU145 cells . Furthermore, to compare the cytotoxic effect against cancer vs. normal cell lines, compound 3 has been tested towards the non-tumorigenic human lung epithelial cell line (BEAS-2B) selected as a healthy cell model  developed according to the mutual prodrug approach. Assessed molecular descriptors and predicted ADME/Tox properties for 3 suggested an overall drug-like profile. In vitro studies performed on 3 revealed that it was chemically stable in acid and neutral pH conditions, while it showed a suitable enzymatic hydrolysis rate in porcine esterase solution, with approximately 50% of 3 remaining after two hours (i.e. t1/2 = 136 min). Finally, 3 had comparable cytotoxicity to the parent drug 5-FU on both DU145 and A549 cancer cell lines, with a significantly improved selective toxicity against lung cancer cells  compared to the reference drug 5-FU. To summarise, our data provided evidence to support the development of 5-FU/HO-1 mutual prodrugs as innovative potential anticancer agents. These findings warrant further studies in a broader panel of cancer cells, including 5-FU-resistant cells.Here, we described the synthesis of the first 5-FU/HO-1 hybrid (Click here for additional data file.


Tetraselmis sp. cultivation in a semi-ORS in Korea for biomass and bioactive lipid production. To maximize biomass productivity of Tetraselmis sp., f medium was selected because it resulted in a significantly higher biomass productivity (1.64 ± 0.03 g/L) and lower omega-6/omega-3 ratio (0.52/1) under laboratory conditions than f/2 medium (0.70/1). Then, we used climatic data-based building information modeling technology to construct a pilot plant of six semi-ORSs for controlling culture conditions, each with a culture volume of 40,000 L. Over 1 year, there were no significant variations in monthly biomass productivity, fatty acid composition, or the omega-6/omega-3 ratio; however, the lipid content correlated significantly with photosynthetic photon flux density. During year-round cultivation from November 2014 to October 2017, areal productivity was gradually increased by increasing medium salinity and injecting CO2 gas into the culture medium. Productivity peaked at 44.01 g/m2/d in October 2017. Throughout the trials, there were no significant differences in average lipid content, which was 14.88 ± 1.26%, 14.73 ± 2.44%, 12.81 ± 2.82%, and 13.63 ± 3.42% in 2014, 2015, 2016, and 2017, respectively. Our results demonstrated that high biomass productivity and constant lipid content can be sustainably maintained under Korean climate conditions.There is increasing demand for essential fatty acids (EFAs) from non-fish sources such as microalgae, which are considered a renewable and sustainable biomass. The open raceway system (ORS) is an affordable system for microalgae biomass cultivation for industrial applications. However, seasonal variations in weather can affect biomass productivity and the quality of microalgal biomass. The aim of this study was to determine the feasibility of year-round  Microalgae have received increasing interest as a biofuel resource in response to the rising energy crisis, depletion of fossil fuels, and climate change ,2. RecenTetraselmis (Chlorophyta), which has previously been reported to contain a substantial lipid fraction, is a potentially promising bioactive feedstock, like other microalgal species [ species ,10. Poly species ,13,14,15 species ,17. Huma species . Further species . The rat species . FA imba species ,21. This species , which w species .2 from the atmosphere, as well as inexpensive materials for construction [Large-scale microalgae culture systems are generally classified as either closed systems, which are wholly enclosed within the vessel , or open systems, which are directly exposed to the outdoor environment ,24. Micrtruction . Open potruction .Unfortunately, there are few commercially feasible ORSs for biomass production in Korea . For indArthrospira (Spirulina) maxima was mass cultivated [Tetraselmis sp. in semi-ORSs in Korea over a 3-year period.In our previous study, we constructed a microalgal culture facility with ORSs inside a glass greenhouse, and ltivated . In thisp < 0.05). Laboratory experiments were carried out for 28 days to determine the optimal culture medium concentration. Double and triple concentrations of f/2 medium (f and f*1.5 medium) led to an increase in the final biomass production of Tetraselmis sp. The highest biomass concentration was 1.64 ± 0.03 g/L in f medium on the final day, followed by 1.47 ± 0.07 and 1.24 ± 0.05 g/L in f*1.5 and f/2 media, respectively. As shown in Tetraselmis sp., culture experiments were carried out at different scales . The final biomass concentration gradually decreased with culture volume (Tetraselmis sp. (p > 0.05), with contents of 19.3 ± 0.8%, 17.0 ± 1.2%, 19.2 ± 1.5%, and 16.8 ± 2.5% in 2 L, 5 L, 200 L, and 40,000 L cultures, respectively. FA compositions differed among culture volumes; however, there was a correlation between culture volume and FA composition (p > 0.05). Omega-3 FA, omega-6 FA, MUFA, and SAFA made up 13.79−18.68%, 11.90−23.53%, 29.62−37.89%, and 30.21−36.48% of the total FAs in the four culture volumes, respectively. There was a significant difference in biomass concentration between the culture volumes from day 16 to day 28 , the composition of LA significantly increased (14.85 ± 0.07%) in the 40,000 L culture, but there were no differences in ALA, EPA, and GLA . The ratios of omega-6 to omega-3 (ω-6/ω-3) FAs were 0.73/1, 0.87/1, 1.37/1, and 1/1 in the 2 L, 5 L, 200 L, and 40,000 L cultures, respectively. Although there was no correlation between FA content and culture volume, the FA content was maintained in the 40,000 L volume. These results indicate that even though biomass productivity of Tetraselmis sp. was significantly reduced when the culture volume was scaled up, sustainable lipid production and maintenance of FA composition were feasible.Tetraselmis sp. −2s−1 (with a peak of up to 911.7 µmol photon m−2s−1 in June).2/d with an annual average of 9.65 ± 2.40 g/m2/d. Productivity peaked in February and September. However there were no significant differences in monthly AP, and AP did not correlate with water temperature or PPFD (p > 0.05). Lipid content of cells was lowest in November (9.9%) and highest in May (18.2%). Lipid content was positively correlated with PPFD over the year (p = 0.019).Fatty acid composition was divided into SAFA, MUFA, PUFA ω-3, and PUFA ω-6 . PolyunsTetraselmis sp.Tetraselmis sp. in the ORS were conducted to increase the annual average biomass productivity over the 3 years. Inter-annual AP varied from 5.64 to 44.01 g/m2/d , which led to cultivation failure and low AP in both 2014 and 2015. To further establish the feasibility of the ORS for microalgal biomass production, microalgae were cultivated in the pilot-scale ORS between November 2014 and November 2017. Several trials during the growing period of shown in . In 20152/d) than those in 2014 (11.12 ± 3.28 g/m2/d) and 2015 (9.71 ± 2.54 g/m2/d). However, cultivation levels were still low. In 2017, 5% CO2 gas was added to the pond as a carbon source. Average AP increased in 2017 to 32.14 ± 5.56 g/m2/d. Moreover, maximum AP was 30.22 g/m2/d in 2016 and 44.01 g/m2/d in 2017. In 2016, the salinity of the culture medium was adjusted from 33 to 70 psu by the addition of NaCl to prevent contamination. This successfully prevented the appearance of predators in the ORS. As expected, average AP in 2016 was significantly higher , and there was no significant effect of scaling up the culture volume.There was no significant difference in lipid content when the culture was scaled up and no correlation between culture volume and FA composition; however, the biomass productivity of ntration . This sunt light . In the nt light ,46, whicnt light ,48. In ont light ,50. In ont light , and sevnt light ,52. As aThe main disadvantages of outdoor cultivation in a seasonal climate, such as in Korea, are differences in biomass productivity and the biochemical composition of cells caused by different monthly environmental conditions ,54,55 anTetraselmis sp. [Tetraselmis sp. strain used in this study. Therefore, this strain could be used for the sustainable production of high-value biochemical compounds annually.During the 3-year pilot-scale cultivation, several factors reduced biomass production. These were contamination of ciliate predators, typhoons in summer, and restricted access to the ORS pilot plant. Contamination of cultures with predators is a major problem associated with cultivation failure . Especialmis sp. , was uselmis sp. , and it lmis sp. ,70. Whenlmis sp. ,72. Theslmis sp. . However2 gas was injected into the pond water. A previous study showed that biomass productivity of Tetraselmis suecica was increased by supplying 5% CO2 gas [2 gas significantly increased average annual AP but had no effect on the lipid content. Supplemental CO2 gas can increase biomass productivity by providing a carbon nutrient source to microalgae and by altering the pH of the culture medium [2 causes stress to microalgal cells, increasing lipid contents, decreasing biomass productivity [2 was appropriate in the pilot ORS.Access to the pilot ORS plant was limited as it was located in a thermal power plant site. To increase annual AP despite the limited culture period available due to the restricted access, 5% CO CO2 gas . In the e medium . Howeveructivity , and reductivity ,78. The In 2017, semi-ORS provided 117.31 tons/ha/year biomass yield and $8.15/kg biomass production cost. Details of capital investment (30-year lifespan) and operating costs are summarized in p < 0.001) [WT, °C) could be calculated by air temperature  from correlation equation: W =T 0.7781 × AT + 6.653. According to our 4-year climate data, and the above equation, the appropriate period of cultivation without a greenhouse is 6 months (May to Oct) in Korea. Assuming that the production period, the biomass yield was reduced to 58.66 tons/ha/year, and the production cost was estimated at $9.25/kg (scenario 1). However, we could cultivate for year-round by controlling the water temperature in semi-ORS. Furthermore, rainfall could be also prevented from entering the system by constructing the ORS in a greenhouse, as was done for the semi-ORS. Therefore, the biomass yield from ORS without a greenhouse is expected to decrease than an estimate, considering the inflow of rainfall (scenario 2). Despite of few references for comparing open- and semi-ORS, semi-ORS was estimated that more affordable and cost effective system than conventional open raceway pond.Most microalgae species are known to grow in a temperature range of 15–30 °C . In prev< 0.001) . Water tTetraselmis sp. MBEyh04Gc (KCTC 12432BP), which has a close relationship with Tetraselmis striata [3, 5 mg/L NaH2PO4, 30 mg/L Na2SiO3.9H2O, 3.15 mg/L FeCl3·6H2O, 4.36 mg/L Na2EDTA·2H2O, 0.0098 mg/L CuSO4·5H2O, 0.0063 mg/L Na2MoO4·2H2O, 0.18 mg/L MnCl2·4H2O, 0.022 mg/L ZnSO4·7H2O, and 0.01 mg/L CoCl2·6H2O), f medium, and f*1.5 medium  without vitamins. Each culture medium, based on natural seawater, was filtered through a GF/C filter  and sterilized in an autoclave  for 2 and 5 L cultures. For 200 and 40,000 L cultures, 1% (per culture volume) of a 13% sodium hypochlorite (NaOCl) solution was administered to the culture medium at night and let stand for 24 h, and then, NaOCl was neutralized with sodium thiosulfate (1 mol/L). After that, 10%  seed was inoculated into the pond .The marine green microalga  striata , was obt striata   with a fluorescent lamp. The air flow rate was 0.4 vvm (volume air per volume culture per minute) to prevent the sedimentation of Tetraselmis sp. cells during the culture periods.Laboratory cultures were conducted in 2 and 5 L flasks and 200 L transparent circular cylinders to investigate biomass production, lipid content, and FA profiles of 2 gas was mixed with air through the air pump to provide a 5% concentration. The 5% CO2 gas was bubbled into the ponds through aquaria air stones (150 mm × 30 mm × 30 mm) with a constant flow rate of 0.1 vvm on the bottom of the pond in 2 m intervals.Additionally, 40,000 L culture was conducted with paddle-wheel circulation. Pond temperature, salinity, and pH were measured using a water quality meter . The PPFD of the natural light source was measured with a digital photometer  at 0.2 m above the pond water surface. The pure COTetraselmis sp. was estimated by sampling and filtering 20 mL of culture medium using GF/C filter paper . Filter paper was dried at 60 °C in a drying oven  for 24 h to obtain dried cell weights (g/L). The biomass concentration  was calculated from the difference in dry weight before and after filtration. AP (g/ m2/ d) is the biomass productivity per unit of ground area occupied by the culture system and was calculated using the following equation:V is the volume of the culture medium in L, A is the area of the culture system in m2, and X1 and X0 are the biomass concentrations collected at t1 (sampling) and time t0 (inoculation), respectively. The biomass concentration of Tetraselmis sp. biomass was harvested using a tubular separator  at 15,750 g for 3 h. Harvested wet biomass was stored at −70 °C in a deep freezer and then lyophilized for 3 days in a freeze-dryer .LW is the weight of the crude lipid content (%) and AW is the dry cell mass sample weight. Experiments were analyzed for EFAs such as ALA, EPA, LA, GLA, and AA. First, the microalgae cultured for 14 days were centrifuged  at 15,100× g for 10 min. The pellet was then lyophilized for 3 days in a freeze-dryer . Each lyophilized cell sample was measured using FID-equipped gas chromatography  to analyze the FA composition of the microalgal cells. In GC analysis, the temperature was increased from 150 °C to 201 °C at 3 °C intervals for 10 min and maintained for 12 min. Then, the temperature was increased from 201 °C to 210 °C in 3 °C intervals to analyze fatty acid methyl esters. The standard used was pentadecanoic acid  [Total lipids were determined using the Soxhlet method . The cruMO, USA) .Tetraselmis sp. The average temperature from 2010 to 2014 was 12.3 ± 10.3 °C, exceeding 24 °C in summer and below −1 °C in the winter. The monthly mean temperatures are shown in 2 , was selected as the site for a pilot study on the production of own in 2 b. Computational fluid dynamics was adopted to simulate and optimize the mixing characteristics of the culture medium in the ORSs. The software Flow-3D (version 10.0) was used to simulate the fluid flow to determine optimal paddle-wheel rotational speeds by ARA Consulting and Technology Company. The rotational speed selected was 15 rpm, as reported in our previous study .The pond temperature was maintained between 20 and 25 °C, and the operation start temperature ranged from 21 to 23 °C in October. The boiler was operated variably based on the temperature changes , and the maximum operation was used to minimize temperature variation and energy input in winter (on 50 min and off 10 min).p < 0.05. Pearson’s correlation coefficients were computed to determine the relationship between biochemical composition parameters (p < 0.05).All statistical analyses were performed using GraphPad Prism 8.4.3 software . The mean values were compared using one-way analysis of variance followed by Tukey’s test. Statistical significance was at Tetraselmis sp. KCTC 12432BP and an appropriate concentration of culture medium, is appropriate for sustainable microalgal biomass production under Korean climate conditions. Although the biomass productivity of Tetraselmis sp. decreased compared to that of lab-scale cultures and the lipid content of cells varied with monthly PPFD, the productivity, FA composition, and ω-6/ω-3 ratio of the biomass cultured in the pilot-scale semi-ORS were maintained throughout the year. Furthermore, salinity was used to control contamination with predators, and injection of CO2 gas improved biomass productivity without affecting lipid content. Our results demonstrated that a 240,000 L semi-ORS constructed near the northernmost area of the Republic of Korea, together with the microalga


Panax notoginseng is known for its effectiveness in alleviating many age-related degenerative diseases, few studies have evaluated P. notoginseng components for efficacy or mechanisms of action in delaying cell replicative senescence. In this study, P. notoginseng oligosaccharides (PNO) were isolated using a stepwise purification procedure involving water extraction and alcohol precipitation followed by DEAE Sepharose Fast Flow column chromatography, preparative high performance liquid chromatography, and size-exclusion chromatography. Monosaccharides detected in PNO constituents included mannose, galactose, and sorbitose in relative molar proportions of 14.2:12.3:1, respectively, aligning with PNO absorption spectrum results resembling typical known spectra for sugars. In vitro, PNO treatment of replicative senescent NIH-3T3 fibroblasts significantly promoted cell vitality, inhibited SA-β-galactosidase  activity, and reduced p16 and p21 protein-level expression. Moreover, PNO treatment of senescent fibroblasts led to a lower proportion of G1 phase cells and higher proportion of S phase cells, while also inducing aging NIH-3T3 cells to migrate and synthesize collagen-I (CoL-I). Mechanistically, PNO treatment up-regulated expression of proliferating cell nuclear antigen (PCNA), cyclin E, cyclin D1, and cyclin-dependent kinase 4 (CDK4) proteins and promoted phosphorylation of MEK, p38, and ERK1/2 to trigger cell cycle progression. Additionally, PNO treatment also up-regulated protein-level expression of TGF-β1 and levels of p-Smad2/3, p-FAK, and p-Pax to trigger CoL-I synthesis and cell migration. Taken together, these findings demonstrate that oligosaccharides purified from P. notoginseng could reverse fibroblast replicative senescence by promoting fibroblast cell proliferation, migration, and CoL-I production.Dermal fibroblast replicative senescence that often occurs in aging skin is characterized by loss of cell proliferative capacity, cell cycle arrest, decreased cell elongation, and decreased synthesis of dermal extracellular matrix (ECM) components. Although  As people get older, skin undergoes endogenous aging that mainly manifests itself as thinning of skin leading to atrophic appearance, reduced elasticity, fine wrinkles, loss of underlying fat, dryness, and increased sensitivity . Major pFibroblast entry into a senescent state begins with cell cycle arrest in the G1 cell phase . The inaP. notoginseng (Burkill) F. H. Chen, commonly known as “Sanqi” in China, belongs to the family Araliaceae  obtained from the Cell Resource Center of the Shanghai Institute for Biological Sciences  were used in all experiments. Cells were cultured in DMEM and newborn bovine serum (NBS) . Other reagents  were obtained commercially . Mouse SA-β-galactosidase, p16, and p21 ELISA Kits were purchased from the Beyotime Institute of Biotechnology . Mouse monoclonal antibodies against p16, p21, CoL-I, PCNA, cyclin D1, cyclin E, CDK4, FAK/p-FAK, Pax/p-Pax, MEK/p-MEK, ERK/p-ERK, p38/p-p38, TGF-β1, Smad 2/3/p-Smad 2/3, GAPDH, and β-actin were purchased from Abcam. For western blotting, luminol reagent was purchased from Santa Cruz Biotechnology . Sheep anti-rat secondary antibody and sheep anti-rabbit secondary antibody were purchased from Beijing Biosynthesis Biotechnology Co., LTD. . All other reagents were of analytical grade and were produced in China.P. notoginseng roots) was provided by the Panax Notoginseng Research Institute,  in October 2017. The voucher specimen (2213) was deposited at the Research Center of Traditional Chinese Medicine, The Affiliated Hospital to Changchun University of Chinese Medicine, Jilin Province, China. Dried P. notoginseng roots  were crushed to form a 100-mesh powder that was extracted two times with 10 L of distilled water at 100°C under reflux for 1 h. The supernatant was consolidated, concentrated, then precipitates were formed after addition of 75% ethanol and incubation overnight  followed by elution with 2 M NaCl. Each eluate was collected automatically as 4-ml fractions into tubes. The absorption curve of eluted sugars (determined at 490 nm) was determined using the phenol sulfuric acid method, then the protein absorption curve was determined using absorbance readings at 280 nm. Fractions with major sugar contents were pooled, dialyzed, then lyophilized  at 100°C for 1 h. Next, chloroform was added to extract monosaccharides and repeated for a total of at least five chloroform extractions. Extracted monosaccharides were analyzed via GC-MS using a 6890N/5973i GC/MS System  equipped with a DB-1 capillary column .The monosaccharide composition of PNO was determined using GC-MS with a slight modification . In brie−1 (PNO (10 mg) was mixed with dried potassium bromide (KBr) then the mixture was compressed and placed in an infrared spectrometer (Tensor27 type) and analyzed over a frequency range of 400–4,000 cm−1 .2 incubator . Cells in logarithmic growth phase were harvested via trypsinization, centrifuged, then adjusted to a cell density of 1 × 105 cells/ml with fresh DMEM containing 10% NBS. Next, cells were dispensed into a petri dish (10-cm diameter) for continuous culture. After 8–10 passages, cells exhibited characteristics of young cells , while cells after 45–50 passages exhibited characteristics associated with a state of replicative senescence  (NIH-3T3 cells were cultured in DMEM at 37°C with 10% NBS and 100 μg/ml streptomycin in a humidified 5% COl group) .4 cells/well) were plated in a 96-well plate for 24 h then culture medium was discarded and replaced with DMEM containing 0.4% NBS followed by incubation of cells for another 24 h. Cells were incubated with PNO at 15.63 μg/ml, 31.25 μg/ml, and 62.5 μg/ml (10 mg PNO was dissolved in DMEM containing 0.4% NBS to 1 mg/ml to create a concentrated stock solution that was sterilized by filtration through a 0.22-μm filter. The stock was then adjusted to the aforementioned concentrations by addition of sterile DMEM containing 0.4% NBS). Meanwhile, the positive control was incubated with epidermal growth factor (EGF) in the same medium for 48 h. Next, MTT was added to each well  then plates were incubated for 4 h at 37°C. Next, each well received 150 µl of DMSO and plates were shaken for 10 min and absorbance readings (570 nm) were recorded using a microplate reader, with all data collected from triplicate samples.A modified version of the MTT assay was used to test the effects of PNO on NIH-3T3 cell viability . NIH-3T34 cells/well) were placed in a six-well plate and cultured using the aforementioned culture method. Next, cells were collected, washed twice with PBS buffer, and lysed in 1% Triton X-100 during a 1-h incubation at 4°C. The supernatant was collected then the protein concentration was measured using a BCA Protein Assay Kit. Next, ELISA was used to detect effects of PNO treatment on SA-β-Gal activity in replicative senescent NIH-3T3 cells according to the manufacturer’s instructions  for various time periods, washed with PBS, and resuspended in 50 µl Annexin V-FITC binding buffer (BD Biosciences) at room temperature (RT) then 3 μl of Annexin V-FITC was added followed by incubation for 15 min. Next, 6 μL of propidium iodide (PI) was added to each well then plates were left at room temperature for an additional 5 min followed by analysis of cells using a flow cytometer . CI values were calculated using CalcuSyn 2.0 software (Biosoft).NIH-3T3 cells were incubated in 12-well plates (3 × 104 cells/well) were cultured in a six-well plate using the same culture method as mentioned above, the cells were washed twice with ice-cold PBS then incubated in 70% alcohol at 4°C for 2 h and centrifuged. After centrifugation, cell pellets were collected, suspended in PI, then incubated for 30 min at 37°C followed by determination of fluorescence intensity using flow cytometry .Flow cytometry was used to detect effects of PNO on the cell cycle . After N4 cells/well) were placed in a 12-well plate and cultured for 24 h. Then a sterile pipette tip was used to gently scratch the cell layer from top to bottom to form a uniform scratch then scratched layers were washed with PBS buffer to remove wound debris. Scraped cells were treated with PNO or EGF then the cells were cultured with serum-free medium in a CO2 incubator at 37°C for 24 and 48 h, respectively. After taking pictures of scratches using a multifunctional imager, cell migration distance was measured using Image-J Analysis software.Scratch tests were performed to assess migration ability of treated cells with a slight modification . NIH-3T3Protein preparations of cells of each experimental group were obtained by treatment of cells with lysis buffer  followed by centrifugation . Supernatants were collected then a BCA Protein Assay Kit was used to determine protein concentration. Next, proteins in each sample  were separated via electrophoresis (12% SDS-PAGE gels) followed by transfer of proteins to PVDF membranes. Next, proteins on membranes were incubated overnight at 4°C with various monoclonal antibodies . The next day, membranes were washed then incubated with secondary antibodies followed by detection of proteins using chemiluminescent reagents .post hoc test for multiple comparisons testing. Statistical evaluation was performed using GraphPad Prism, version 5.0 . p < 0.05 was considered statistically significant.All data are represented as mean ± STD of three biological replicates in this study. Statistical analysis was performed using one-way ANOVA and Tukey’s P. notoginseng roots followed by alcohol precipitation, extracted components were separated using DEAE Sepharose Fast Flow ion exchange chromatography. As shown in P. notoginseng oligosaccharide (PNO) (After water extraction of de (PNO) .−1, C–H absorption peaks at 3,000–2,800 cm−1, and pyranose absorption peaks at 1,100–800 cm−1. These results suggest that PNO has a typical absorption profile associated with sugars.Next, we determined PNO composition and residue identification. As shown in As shown in As compared with the T0 group, secreted SA-β-Gal activity of the T1 group was significantly increased. After adding various concentrations of PNO , SA-β-Gal activity decreased by 24.22 ± 0.65%, 33.95 ± 1.92%, and 39.96 ± 4.11% in the T1+PNO group, respectively, as compared to the T1 group. Treatment with EGF (50 ng/ml) led to a decrease of SA-β-Gal activity by 42.19 ± 2.74% in the T1+EGF group . DiffereAs shown in As shown in Compared with the T0 group, levels of CoL-I in the T1 group decreased. As compared with the T1 group, PNO in concentrations of 15.63, 31.25, and 62.5 μg/ml led to increased cellular expression of CoL-I in the T1+PNO group  mirrorinAs shown in As compared with the T0 group, relative protein level ratios of p-MEK/MEK, p-p38/p38, and p-ERK1/2/ERK1/2 in the T1 group were reduced, while treatment of cells with 31.25 and 62.5 μg/ml PNO promoted phosphorylation of these proteins in the T1+PNO group . Thus, PAs compared with the T0 group, p-FAK and p-Pax protein levels were significantly reduced in the T1 group. After treatment of the T1+PNO group with 31.25 and 62.5 μg/ml PNO, increases in expression levels of p-FAK and p-Pax proteins were observed as compared to corresponding T1 group levels , mirroriCompared with the T1 group, TGF-β1, p-Smad2, and p-Smad3 protein levels were significantly increased in cells treated with 31.25 and 62.5 μg/ml PNO, mirroring the effect observed for EGF-treated cells in the T1+PNO group . Thus, PP. notoginseng in a stepwise manner using ethanol precipitation, anion exchange chromatography, HPLC, and gel column chromatography. Ultimately, PNO treatment boosted cell proliferation activity, migration, and CoL-I synthesis in undergoing replicative senescence. These results suggest that this active preparation exerted an effective skin anti-aging effect that has not been reported previously for oligosaccharides. However, recently it has been reported that oligosaccharides from various sources exhibited anti-photoaging healing effects when used to treat dermal wounds  applied alone or as a cream-based formulation have been shown to accelerate wound healing  significantly diminished facial wrinkles and alleviated other symptoms of facial skin aging  and paxillin (Pax) protein activities. In non-senescent cells, Pax activation by p-FAK promotes cell migration. . ActivatP. notoginseng (PNO). PNO was shown to activate the MAPK pathway to induce synthesis of cell cycle regulatory proteins that promoted cell proliferation, cell migration and production of CoL-I. PNO was shown to alleviate replicative senescence by activating the TGF-β1/Smad signaling pathway as an underlying mechanism exploited by PNO to delay cell entry into a replicative senescence state and slow skin cellular aging-related processes (An oligosaccharide mixture that delayed replicative senescence of NIH-3T3 fibroblasts was prepared from rocesses .