Long-term immunomodulatory therapy of pediatric onset-multiple sclerosis (POMS) is based mainly on published case series and internationally agreed guidelines. Relevant studies in the Greek population are absent from the literature. The purpose of this study is to present data on the efficacy and safety of the 1st line immunomodulatory drugs in the treatment of POMS patients.The present study included 27 patients meeting the IPMSSG criteria for POMS and who are monitored at the outpatient clinic of the Multiple Sclerosis and Demyelinating Diseases Unit (MSDDU), of the 1st Neurological Department, University Hospital of Aeginition. All patients received 1st line immunomodulatory drugs as initial therapy. Clinical, laboratory, and imaging parameters of the disease were recorded before and after treatment.p = 0.002), EDSS progression  and ARR  was observed, while no changes were observed in the EDSS score, . Advanced age at treatment initiation increased the risk for drug discontinuation before 24 months of therapy , p = 0.04).Post-treatment, a significant reduction of the relapse number (mean ± SD: 2.0 ± 1.0 vs 1.2 ± 1.6, Most pediatric patients are forced to switch to either more efficacious 1st line or 2nd line drugs. Additionally, our study suggests that older age at the time of the 1st line treatment initiation, contributes to earlier drug discontinuation.The online version contains supplementary material available at 10.1007/s10072-022-06431-y. Pediatric-onset multiple sclerosis (POMS) occurs in approximately 5–10% of multiple sclerosis patients (MS) with the most common disease course being the relapsing–remitting (RRMS) , 3. ContRegardless of the great experience that has been accumulated in the treatment of pediatric MS in the last decade, the optimal therapeutic option remains uncertain. The 1st line therapies  including injectables, such as interferons and glatiramer acetate have been widely used in POMS, mainly based on data derived on retrospective and open-label studies. Moreover, the newer oral disease modifying treatments (DMTs) such as teriflunomide and dimethyl fumarate have been also established as valuable treatment options for POMS therapy –17.Current treatment guidelines for POMS are based on retrospective observational data, case series, prospective safety data, and expert’s opinion. The International Pediatric Multiple Sclerosis Study Group (IPMSSG) advised that treatment decisions should be tailored to each child and taken after discussing risks and benefits with the patient and the family , 19.In the current retrospective study, we present data from 27 POMS patients treated with 1st line DMTs, either injectables or oral, including interferons, glatiramer acetate, teriflunomide, and dimethyl fumarate, in order to describe effectiveness and safety. Additionally, we report the patients’ human leukocyte antigens (HLA) genotype, the “gold standard” for attributing genetic burden in both AOMS and POMS , 21, thuIn the current retrospective study, 27 POMS patients, of Hellenic origin, fulfilling the 2007 and/or 2013 IPMSSG criteria for POMS diagnosis  and treaA detailed clinical history was obtained, followed by thorough clinical examination. Lumbar puncture (LP), magnetic resonance imaging (MRI), and routine laboratory tests were carried out, according to the existing protocols for children and adolescents with demyelinating diseases. Anti-John Cunningham virus (JCV) antibodies’ assessment was performed in patients’ serum, at Unilabs, Copenhagen, Denmark. Cell-based assays for antibodies against aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) were performed at the Neuroimmunology Unit, Dept. of Pathophysiology, National and Kapodistrian University of Athens, Greece, with standard protocols.All POMS patients underwent thorough neurological examination, including EDSS assessment every 6 months, routine hematological tests every 6 months, and brain MRI scans  in 3 Tesla magnetic field, every 6 months. Enlargement of previously existing lesions has been also evaluated. Data regarding the number of relapses, calculating the annualized relapse rate (ARR), yearly EDSS scores and EDSS progression before and after treatment with 1st line DMTs have been collected.Relapses have been evaluated by neurologist, according to the classic definition . EDSS progression was defined as increase in EDSS score of ⩾ 1.5 points from an EDSS score of 0.0, ⩾ 1.0 point from an EDSS score of 1.0–5.5, or ⩾ 0.5 point from an EDSS score ⩾ 6.0. Patients were assessed every 24 weeks . MRI actHLA genotyping was performed at the Research Immunogenetics Laboratory of the 1st Department of Neurology at “Aeginition” University Hospital. Nineteen (19) patients and legal guardians provided written informed consent regarding immunogenetic testing. High molecular weight DNA was extracted from whole peripheral blood samples  using the DNA extraction, Maxi Kit  as per manufacturer’s instructions. HLA class II (HLA-DRB1) frequencies were determined by molecular techniques for all the specificities included in the HLA nomenclature of 2012 . The HLA-DRB1 genotyping was performed using a PCR-SSO  technique as previously described .Non-parametric tests were used for all the above evaluations, due to the irregularity of the quantitative variables. Univariate comparisons were made using Mann–Whitney and chi-square tests for quantitative and categorical variables, respectively. Multivariate and Cox regression were also performed. Statistical analyses were performed using SPSS 22 .In the current retrospective study, 27 POMS patients have been included. The mean age at disease onset was 14.5 ± 2.6 (mean ± SD) years. The mean disease duration was 3.4 ± 4.1 years, and the mean age of patients at the period starting the 1st line DMT treatment was 16.7 ± 3.1 years. Among these patients, 17 were females , and 10 were males (10/27 37%). All patients were diagnosed with RRMS. The main clinical baseline characteristics of the study population are summarized in Table Among the 27 POMS patients included in the study, 7 patients received IFN-β-1α intramuscularly (i.m.) (Avonex®) and 9 subcutaneously (s.c.) (Rebif® or Plegridy®), while 3 patients were treated with IFN-1β (Betaferon®). Three of the newer immunomodulatory agents have been administered of label, particularly: Glatiramer acetate (Copaxone®) has been administered in 3 patients, dimethyl fumarate (Tecfidera®) in 2 and Teriflunomide (Aubagio®) in 3 patients (Table rd drug administered) received a 2nd line therapy .Until the final follow-up of the study, only 2 (2/27) patients, receiving dimethyl fumarate, remained on the initial treatment, while all the other (25/27) discontinued and received another drug. In our total sample until the last follow-up of the study, 9 patients had 2 drug switches; 7 patients had 3 drug switches; while 2 patients had 4 drug switches. Up today, all patients have been switched to one or more agents approved as second-line treatment, in Greece and generally in Europe. The first drug discontinuation occurred after a mean of 29.5 ± 37 months and the second after a mean of 17 ± 13 months. The first therapeutic choice after the 1st DMT discontinuation was mainly natalizumab or fingolimod (considered as 2nd line therapies in Greece), while 2 patients switched to i.m. IFN-β-1α, 2 to glatiramer acetate, 1 to s.c. IFN-β-1α, 1 to i.m. pegylated IFN-β-1α, 2 to dimethyl fumarate and 2 to teriflunomide. All patients in the second switch , EDSS progression , ARR  was observed, but not in the EDSS score . Moreover, a significant decline-but not elimination-in the number of patients displaying MRI activity has been also observed .As shown in Fig. p = 0.02), EDSS progression , ARR  post-treatment. No difference was observed in EDSS score after interferon administration  and MRI activity  , had a significant reduction in terms of number of relapses  (data not shown). No other significant changes were observed in number of relapses, EDSS, EDSS progression, and MRI activity after interferon treatment or the other 1st line agents. Additionally, following further subgroup analysis, no differences in any clinical parameter were found between injectables and oral 1st line DMTs, , p = 0.04]. Moreover, a trend that males have approximately 8 times higher risk to discontinue therapy after 24 months of treatment was also observed   patient treated with teriflunomide had dizziness and vomiting. No adverse events (AEs) have been reported by the other 22 patients  and HLADRB1*15 (7/33), while the less representative was HLADRB1*08 (1/33) and HLADRB1*12 (1/3). Following subgroup analysis according to the presence or not of the 4 most representative HLA-DRB1 alleles  in our sample, no association was found between any of these alleles and any post-treatment clinical outcome (data not shown).Ten 10) distinct HLA-DRB1 alleles were identified  in 19 POMS patients who provided informed consent for HLA-DRB1 genotyping  showed a marginal EDSS score worsening during therapy. In general, our findings are in line with previous studies on POMS subjects, showing effectiveness in ARR reduction after receiving 1st line therapies –25.Interferons were the most common initial treatment. Specifically, i.m. and s.c. IFN-β-1α were administered in 7 and 8 POMS patients, respectively. Both showed efficacy in ARR and EDSS progression reduction but not in the number of relapses, EDSS score, and MRI activity elimination. Similarly, previous observational studies conducted in POMS patients have reported that i.m. IFN-β-1α led to a significant reduction of the ARR, from 1.9 pre-treatment to 0.4  and s.c.Despite the efficacy of the 1st line DMTs, most of our patients have switched to a different drug approximately after 2.5 years of treatment with the first therapy. Intramuscular IFN-β-1α was the most frequent therapy initially given, due to administration once per week. However, i.m. IFN-β-1α was also the drug which has been discontinued earlier compared to the others. In the first switch, most patients escalated to a 2nd line treatment , while in the second switch all patients escalated to a 2nd line drug . These findings are indicative of the need to treat these patients with higher potency agents. In accordance, a previous study reports that 22 of 46 patients treated with 22 mg s.c. IFN-β-1α had to escalate to higher dose (44 mg) . MoreoveThree patients were treated with glatiramer acetate. Small cohort studies conducted in Italian POMS patients investigating the effectiveness of glatiramer acetate reported a significant reduction in relapse rate from 3.1 before treatment initiation to 0.2 during the follow-up , 32. HowTeriflunomide has been administered in 3 patients, while 2 have been treated with dimethyl fumarate. Recent published data from the TERIKIDS trial  support the efficacy of teriflunomide to reduce the number of new or enlarged T2 lesions by 55% and the number of gadolinium-enhancing lesions by 75%, compared to placebo. Our patients showed no elimination of MRI activity and a trend for reduction in ARR and number of relapses after teriflunomide administration, but a worsening in EDSS and EDSS progression. Additionally, a recent study reporting real-word effectiveness data suggested that initial treatment of POMS patients with newer DMTs including dimethyl-fumarate and teriflunomide led to better disease activity control, compared to injectables (interferons and glatiramer acetate), showing greater effectiveness of the newer therapies .The safety and efficacy of dimethyl fumarate in POMS treatment have been recently studied in a small, single-arm, open-label trial  and its extension study . Treatment with dimethyl fumarate resulted in a significant radiological improvement as well as a reduction of the ARR from 1.5 for the year before entry into the study to 0.8 during the 24-week study period . The twoThe main finding of our study is that POMS patients starting 1st line DMTs in more advanced age display increased risk for treatment discontinuation before 24 months of therapy, suggesting that younger patients could be better responders. This data are in line with a recent Italian retrospective study reporting that starting therapy before 12 years of age could lead to a more favorable outcome . InteresRegarding safety, all therapies were generally well tolerated, and no serious AEs have been registered. Three patients treated with i.m. IFN-β-1α showed flu-like symptoms, while 1 patient treated with teriflunomide had dizziness and vomiting. Hepatoxicity or pancreatitis, previously reported after glatiramer acetate  and teriHLADRB1*15, while 2 patients carried the HLADRB1*03 allele. The predisposing role of HLADRB1*15 in POMS has been well appreciated, supporting a genetic similarity with AOMS [HLA-DRB1*03 has been associated with increased disease risk in the Hellenic POMS population, as well as with higher number of relapses and lesion burden at the thoracic spinal cord level [Regarding HLA genotyping, 7 patients were carriers of ith AOMS , while Hrd level .HLA-DRB1*04, HLA-DRB1*08, HLA-DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, and HLA-DRB1*16 [HLA-DRB1*16 allele, while those with disease onset 16–19 years old presented with a lower HLA-DRB1*11 allele frequency compared both to the pediatric- and the adult-onset MS group [The rest of our patients carried at least one of the protective alleles -DRB1*16 . AccordiMS group .HLA-DRB1*04 allele and the HLA-A*03-B*44-DRB1*04 haplotype are better responders to IFN-β treatment [HLA-DRB1*04:01 and HLA-DRB1*07:01 alleles has been linked to the development of interferon-neutralizing antibodies and poor response to treatment [HLA-DRB1*04 allele developed flu-like symptoms.Previous studies conducted in RRMS patients suggest that patients carrying the reatment , while treatment . RegardiOur study presents several limitations including the relatively small cohort and the limited number of patients treated with the newer 1st line DMTs glatiramer acetate, teriflunomide, and dimethyl fumarate. Additionally, given that the present study is retrospective, the patients have been followed-up for different periods. Larger scale prospective studies in the Hellenic MS population are currently underway and hopefully will shed more light also on possible immunogenetic/phenotypic correlations.In conclusion, our study supports that despite the effectiveness of the 1st line treatments in the reduction of relapse number, ARR and EDSS progression, they fail to reduce EDSS score and eliminate MRI activity. Therefore, most pediatric patients with MS are forced to switch to either more efficacious first-line or second-line drugs. Of interest, our study suggests that the more advanced patients’ age at the moment of the 1st line treatment initiation and possibly the male gender contribute to earlier drug discontinuation.Supplementary information.Supplementary file1 (DOCX 36 KB)Supplementary file2 (DOCX 15 KB)Below is the link to the electronic supplementary material.


The survival of free fat grafts is dependent primarily on adipose-derived stem cells (ADSCs); however, ADSCs are susceptible to oxidative stress in the recipient area. Astaxanthin (Axt) is a natural xanthophyll carotenoid with potent antioxidant properties and numerous clinical applications. To date, the therapeutic potential of Axt in fat grafting has not been explored. The purpose of this study is to investigate the effects of Axt on oxidatively stressed ADSCs. An oxidative model of ADSCs was developed to simulate the host’s microenvironment. Oxidative insult decreased the protein levels of Cyclin D1, type I collagen alpha 1 (COL1A1), and type II collagen alpha 1 (COL2A1), while increasing the expression of cleaved Caspase 3 and secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in ADSCs. Axt pre-treatment significantly reduced oxidative stress, increased the synthesis of an adipose extracellular matrix, alleviated inflammation, and restored the impaired adipogenic potential in the present model. Furthermore, Axt immensely activated the NF-E2-related factor 2 (Nrf2) pathway, and ML385, an inhibitor of Nrf2, could negate Axt’s protective effects. Additionally, Axt alleviated apoptosis by inhibiting bcl-2-associated X protein (BAX)/Caspase 3 signaling and improving the mitochondrial membrane potential (MMP), which could also be abolished by ML385. Our results suggest that Axt may exert its cytoprotective effect on ADSCs through the Nrf2 signaling pathway and could be therapeutic in fat grafting. Autologous fat grafting (AFG) involves harvesting fat tissue from a defined donor site and injecting it into the desired area to augment its appearance . AFG hasHowever, owing to hypoxia, oxidative stress, and mechanical injuries, the main concern in the application of AFG is its inconsistent and low graft take rate. Lv et al. demonstrated that the absorption rate after facial fat grafting varied from 17 to 74% and the pooled retention was 47% . The lowAs a mediator of cellular response during an oxidative stress condition, the NF-E2-related factor 2 (Nrf2) signaling system plays an influential role in maintaining cellular homeostasis . Under bAstaxanthin, a form of carotenoid referred to as xanthophyll, is a substance distributed widely in seafood such as salmon, crabs, or shrimp. Since being separated from lobsters in 1938 by Kuhn, R. et al., Axt has exhibited anti-oxidative, anti-inflammatory, anti-tumor, and cardioprotective properties . Axt is To determine the efficacy of Axt in promoting ADSC survival in vitro, Axt was evaluated in a cellular oxidative model for its effect on adipogenic competency, extracellular matrix (ECM) synthesis, apoptosis, and adipogenic potential. Further investigation was conducted into the regulatory role of Nrf2 signaling in the cytoprotective effect of Axt.p < 0.05 for 2 μM, p < 0.001 for 5 μM, p < 0.0001 for 10 μM, p < 0.001 for 20 μM, vs. the 0 μM; 48 h: p < 0.05 for 2 μM, p < 0.0001 for 5 μM, p < 0.0001 for 10 μM, p < 0.001 for 20 μM, vs. the 0 μM). Axt administration (10 μM) raised the expression of Ki67 and Cyclin D1 protein, implying that Axt could improve the proliferation of ADSCs (ADSCs were characterized by multilineage differentiation and a flow cytometry assay . Cell coof ADSCs B. Therefp < 0.05). The Annexin V/PI staining assay was performed to quantitatively analyze the apoptosis of ADSCs. As shown in p < 0.05, A Transwell cell migration assay was conducted to investigate the effect of Axt on migration. As illustrated in p < 0.05 for 5 μM, p < 0.001 for 10 μM, vs. the 0 μM; p < 0.01 for 5 μM, p < 0.0001 for 10 μM, vs. the 0 μM; Axt is a potent antioxidant and the transcription factor Nrf2 has been reported to affect the redox balance in ADSCs. The expression levels of Nrf2/Keap1 pathway proteins were determined in ADSCs. When Axt was administered at 2 μM, 5 μM, and 10 μM, there was a significant increase in total Nrf2/Keap 1 in a dose-dependent manner , COL1A1 , COL2A1 , and peroxisome proliferator-activated receptor-γ (PPAR-γ) , while increasing the protein level of cleaved Caspase 3 . In the present study, a 200 μM H2O2 treatment inhibited cell proliferation, decreased extracellular matrix secretion, induced apoptosis, and impaired adipogenic potential in ADSCs, and therefore this treatment was utilized in subsequent research.Hterature . To explp < 0.05 for 5 μM, p < 0.01 for 10 μM, vs. the 0 μM) and COL2A1  in a dose-dependent manner. Our results indicated that Axt could reverse the adipogenic potential loss of ADSCs by upregulating PPAR-γ  and subsequent downstream genes including HO-1 , NQO1 , and GPX4 . To determine the protective effects of Nrf2, 4 μM ML385 was used to obviate the function of Nrf2 , COL2A1 , and PPAR-γ . Moreover, the data in p < 0.05 for Axt + ML385, vs. the Axt), MDA level , SOD activity , IL-6 level , and TNF-α secretion . These findings support the hypothesis that Nrf2 protects ADSCs against the imbalance of cellular redox.Oxidative stress is detrimental to ADSCs’ survival and differentiation. The cytoprotective effect of Axt is achieved through the enhancement of Nrf2 signaling. This study examines the role of Nrf2 signaling in oxidatively stressed ADSCs. As displayed in  of Nrf2 B. The rep < 0.001 for Axt, vs. the vehicle), and this effect could be reversed by 4 μM ML385 . Moreover, we also detected the classic BAX/Caspase 3 signaling and demonstrated that Axt inhibited the expression of BAX  and cleaved Caspase 3  in a dose-dependent manner , and ML385 (4 μM) could cancel this effect . These results indicate that Axt could downregulate BAX/Caspase 3 signaling to resist mitochondrial injuries via Nrf2 signaling.As illustrated in t manner B. Since t manner C. Axt . Senescence inhibits cell proliferation, decreases the differentiation potential, raises oxidative stress, and reduces the paracrine activity of ADSCs ,48,49. TAstaxanthin and ML385 were obtained from MCE  and were dissolved in 0.1% dimethyl sulfoxide . Hydrogen peroxide was supplied by Aladdin . Phosphate-buffered saline (PBS), fetal bovine serum (FBS), bovine serum albumin (BSA), Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture, and Minimum Essential Medium-Alpha (α-MEM) were obtained from Gibco .w/v) type I collagenase . Upon centrifugation at 400× g for 5 min, the supernatant was discarded, and the precipitation was resuspended in α-MEM. The cell suspension was filtered through a 70 μm nylon filter and then cultured in complete α-MEM (containing 10% FBS and 1% PSN) on a 10 cm culture plate. ADSCs were cultured at 37 °C in a humidified atmosphere of 5% CO2, and the media were changed every three days. At 80% confluence, the cells were defined as passage 1 and re-plated. Passages 2–3 were used for the following experiments. ADSCs were characterized by multilineage differentiation  and analysis of the expression of CD73, CD90, CD105, CD34, CD11b, CD19, and CD45  using a BD Biosciences flow cytometer and Flow Jo software .Patients  from Plastic Surgery Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College (CAMS & PUMC) were recruited to provide healthy adipose tissues following selective abdominal liposuction. All donors signed informed consent forms. After being washed twice with PBS, fat tissues were digested in a shaker at 37 °C for 45 min with the same volume of 0.2%  in accordance with the manufacturer’s protocol. In brief, cells were seeded on a 96-well plate at a concentration of 5000 per well, and then 10 μL CCK-8 solution was added to each well. After 1.5 h of incubation at 37 °C without exposure to light, the absorbance was measured using a microplate reader  at 450 nm. The experiment was repeated five times.ADSCs were seeded in a glass bottom dish. Following treatment, the cells were washed twice with cold PBS before being fixed in 4% paraformaldehyde for 20 min. The dishes were incubated with PBS solution containing 0.1% triton-X and 5% BSA for 60 min at room temperature to permeabilize and block nonspecific binding sites. After discarding the solution, cells were incubated with primary antibodies overnight at 4 °C. Primary antibodies against Ki67 , Cyclin D1 , and Nrf2  were utilized. After being washed with PBS 3 times, the dishes were incubated with fluorescent conjugate secondary antibodies for 1 h at room temperature without light. Anti-Mouse secondary conjugated with Alexa Fluor 488  and Anti-Rabbit secondary conjugated with Alexa Fluor 647  were used. Cell nuclei were stained with DAPI  for 10 min, and images were obtained using a BX53 upright fluorescence microscope .4 cells in 200 µL of the serum-free α-MEM and seeded in the upper compartment of the chamber. Then, 600 µL of complete α-MEM containing vehicle or AXT was added to the lower chamber. After incubation for another 36 h, the chambers were removed, and cotton swabs were used to remove the cells on the upper surface of the membrane. ADSCs that had migrated into the microporous membrane were washed twice with PBS, fixed with methanol solution for 30 min, and stained with 0.1% crystal violet  for 20 min. Finally, a microscope  was used to observe the cells.A Transwell assay  was performed to assess the migration capacity of ADSCs. Passage 2–3 cells were collected and resuspended at a density of 6 × 10g for 10 min at 4 °C. The supernatants were collected after centrifugation. Nuclear and cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit . Protein concentrations were assessed using a BCA assay kit . A sample of 20 μg protein was separated by SDS-PAGE (4–20%), and the bands were transferred to a PVDF membrane . After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies against Nrf2 , Keap 1 , PCNA , β-Actin , Cyclin D1 , Caspase 3 , COL1A1 , COL2A1 , PPAR-γ , HO-1 , NQO1 , GPX4 , and BAX  were employed in the experiments. The membrane was then washed 3 times and incubated with the appropriate secondary antibodies for 1 h at room temperature. The secondary antibodies used in the present study are as follows: HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L)  and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) . The protein bands were visualized using BeyoECL Plus detection reagent , and band images were obtained via the Chemi-Doc Imaging System . Semi-quantification of the bands was performed with ImageJ software .After washing twice with cold PBS, the cells were lysed in RIPA lysis buffer  containing Protease Phosphatase Inhibitor Cocktail . Afterward, the cells were scraped and centrifuged at 12,000× Cell apoptosis was analyzed by the FITC Annexin V Apoptosis Detection Kit purchased from BD Biosciences , and the procedures followed the manufacturer’s protocol. Cells were analyzed via a BD Biosciences flow cytometer , and the results were analyzed using FlowJo software .4 cells/cm2 on 6-well plates and incubated with 2 mL complete α-MEM per well. According to the manufacturer’s instructions, cell media were harvested, centrifuged, and analyzed for pro-inflammatory cytokines using the IL-6  and TNF-ELISA kits . The concentrations of cytokines were determined by measuring absorbance at 450 nm using a multimode plate reader , and each group was prepared in triplicate.The secretion of IL-6 and TNF-α in the cell media was assessed by ELISA. ADSCs were seeded at the concentration of 2 × 10Intracellular ROS detection was performed using a Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay kit . Assays were conducted according to the manufacturer’s instructions and the cells were observed under a fluorescence microscope and analyzed using Image J software .g for 10 min at 4 °C. The supernatants were collected for detection after centrifugation. Assays were performed according to the manufacturer’s protocols. Total protein concentration was determined using the BCA method.The supernatant of the cell lysate was tested for the presence of MDA and the activity of SOD using commercial kits purchased from Beyotime . After lysing ADSCs with RIPA lysis buffer, the cells were scraped and centrifuged at 12,000× The mitochondrial membrane potential was evaluated using a JC-1 detection kit . JC-1 staining was performed on cultured cell monolayers following the manufacturer’s protocols. The results were observed under a fluorescence microscope and analyzed using Image J software .t-test was utilized for the comparison of differences between two groups, and the one-way ANOVA followed by Bonferroni’s multiple comparison test was used for the comparison of more than two groups. The data were displayed as mean ± standard deviation (SD). p values < 0.05 were defined as statistical significance.In the present study, all experiments were performed in triplicate except for the CCK-8 assay, which was performed five times. The cell viability and protein expression levels of the control group were regarded as standards, and the results of other groups were calculated as the percentages of the control group. The data were analyzed using GraphPad Prism 9 . For quantitative data, the Student’s According to our results, Axt pretreatment could promote ECM synthesis, restore adipogenic potential, inhibit inflammation, and regulate the apoptosis of ADSCs under oxidative stress via the Nrf2 signal pathway. Axt may be an effective treatment for improving the survival rate of autologous fat grafts.


Gas chromatography (GC) with flame ionization detection (FID) and mass spectrometry (MS) detection were used to characterize the fatty acid (FA) compositions of ten commonly consumed  pulses. Lipids from ground pulses were extracted using a classical chloroform/methanol extraction and quantified by GC-FID with structural confirmation by GC-MS. Principal component analysis (PCA) was applied to FA compositions of the pulse extracts, and the pulses clustered into three distinct groups: one rich in linolenic acid, 18:3 , one rich in 16:0, and one in which 18:1 was highest, along with predominant 18:2. These ten pulses averaged 46.1% linoleic acid (18:2), 22.7% oleic acid (18:1), 18.0% palmitic acid (16:0), and 7.6% linolenic acid (18:3). Individual values ranged widely, with 18:2 ranging from 26.0% in black beans to 48.4% in garbanzo beans. The greatest difference was in 18:3, which ranged from 2.2% in garbanzo beans to 38.8% in pinto beans. Oxo-FA were observed in all ten samples, and the distribution of oxo-FA in the samples also varied. Overall, the very different FA compositions of pulses lead to the possibility of breeding and genetic modification between pulses to produce the most desirable FA composition for nutritional benefit. Fabaceae, also called Leguminosae, which bear seeds in pods, and the seeds are known as pulses. These include beans, peas, lentils, and chickpeas. The United Nations Food and Agriculture Organization has limited the definition of pulses to include only “crops harvested solely for dry grain, thereby excluding crops harvested green for food, which are classified as vegetable crops, as well as those crops used mainly for oil extraction and leguminous crops that are used exclusively for sowing purposes” + ion under PCI-CH4 conditions and was identified by MS and RT comparison to a standard. Several TAGs containing oxygenated FA were observed by LC-MS analysis (manuscript submitted), and the presence of a total of five different oxygenated FA were confirmed by PCI-CH4 GC-FAME analysis. A suspected sixth oxygenated FA was also observed. Although the PCI-CH4 peak mass spectra did not precisely identify the structures of the oxygenated FA, they were sufficient for establishing FAME molecular masses. The molecular masses were consistent with mono-, di-, and tri-enoic 18-carbon FAME with the substitution of a hydroxyl group for a hydrogen or insertion of an oxygen atom across a site of unsaturation to form an epoxide. Thus [M + H]+ ions of 18:1, 18:2, and 18:3 FAME were observed at m/z 297, 295, and 293, respectively, but [M + H]+ ions of the corresponding oxygenated FAME were observed at m/z 313, 311, and 309.For each sample, several other peaks were observed in the FID chromatograms that were not attributable to the FA listed in 4 signal from this peak was not strong enough to confirm its identity as an oxygenated FAME, but from its position in the chromatograms, it may be a third oxo-Ln isomer. Observation of a single oxo-O isomer, two oxo-L isomers, and three oxo-Ln isomers would be consistent with the numbers of isomers expected from epoxide formation at the carbon-carbon double bond sites of O, L, and Ln, by far the most abundant 18:1, 18:2, and 18:3 isomers, respectively, in the samples.Partial FID chromatograms including the peaks for oxygenated FAME observed for lentils, black-eyed peas, and cranberry beans are shown in 4 MS data.Several other small peaks were observed in the chromatographic region shown in The 18-carbon oxo-FA distributions did not closely follow the non-oxygenated, unsaturated 18-carbon FA distributions , as shown in For all samples, the oxo-O relative amounts were smaller and the oxo-Ln relative amounts were larger than the corresponding relative amounts of non-oxygenated 18:1 and 18:3 FA, respectively. The oxo-FA compositions, relative to the corresponding non-oxygenated, unsaturated 18-carbon FA compositions, were consistent with increasing susceptibility to oxidation with increasing unsaturation ,24,25. TA more detailed look at the oxo-FA distributions is given in For the samples in which oxo-L was found, there was also variation in which isomer was more prevalent. Oxo-L1 was present at higher levels than oxo-L2 in green split peas and in baby lima beans, for example, but the reverse was true for lentils, garbanzo beans, and black-eyed peas.Although enzymatic involvement could account for why there was such variation in the oxo-FA distributions among the samples if oxidation were happening primarily during storage and handling , it coulPhaseolus vulgaris , two Phaseolus lunatus (baby lima and butter beans), and one each of Vigna unguiculata (black-eyed peas), Cicer arietinum (garbanzo beans), Pisum sativum (green split peas), and Lens culinaris (lentils).Ten ground pulse samples were provided by Dr. Raghavendhar R. Kotha , receiveOptima Grade Methanol was from Fisher Scientific . Chloroform, heptane, and isooctane were HPLC grade from Sigma-Aldrich . Water was from an in-house Millipore  Milli-Q Water Purification System. Potassium hydroxide  and potassium chloride (Fisher Scientific) were reagent grade.2 in Folch solvent. Samples were magnetically stirred, with periodic vortexing, for 30 min. They were then centrifuged at 1000× g rpm for two minutes, and the supernatants were transferred by Pasteur pipet to a second set of 50 mL glass screw top tubes. The supernatant was washed with a 0.2 × volume of 0.9% KCl. Phase separation was again aided by centrifuging, and the lower, chloroform layer was collected. The extraction and washing procedures were then repeated on the samples using 20 mL portions of Folch solvent. For each sample, the two washed chloroform portions were combined, and a 5 mL portion was taken for GC analysis, with the remainder reserved for LC-MS analysis (reported elsewhere (manuscript submitted). For the GC portion, solvent evaporation to constant mass was performed at room temperature under a stream of nitrogen gas. Residue masses for GC analysis were 8 to 10 mg except for chickpeas, for which the residue mass was 30 mg. Residues were dissolved in 1.5 mL isooctane and treated for FAME derivatization.Lipid extractions were based on the method of Folch et al. . Ground A 0.25 mL portion of 2 M KOH in methanol was added to each of the residues dissolved in isooctane. Samples were vortexed for 30 s and kept at room temperature for 30 min. The top, isooctane layers were then transferred by Pasteur pipet to GC sample vials for analysis by GC-FID and GC-MSD.Multi-component FAME standards GLC 68B (16 FAME from C14 to C24) and GLC 463 (52 FAME from C4 to C24) were obtained from Nu-Check Prep . Methyl heneicosanoate (21:0) and Methyl tricosanoate (23:0) were also obtained from Nu-Check Prep. Methyl pentacosanoate (25:0) was obtained from Santa Cruz Biotechnology, Inc. . Methyl hexacosanoate (26:0) was obtained from Matreya LLC . Standard L6031, containing the FAME of the eight geometrical isomers of 9, 12, 15-18:3, and squalene were obtained from Sigma-Aldrich. An isomerized corn oil sample, previously described  and deriTwo GC systems were used. One system was equipped with a single FID detector, and one with the effluent split between a mass selective detector (MSD) and an FID.(a)Gas Chromatograph—Agilent Technologies  6890N GC with G2613A injector, split/splitless inlet, flame ionization detector (FID), and using OpenLab CDS ChemStation Edition for GC Systems (Rev. C.01.05 [Build 35]) software.(b)GC-MS—Agilent Technologies 5975C Inert XL EI/CI MSD with 7890A GC System and G4513A injector, split/splitless inlet, G3180B two-way splitter with makeup gas, FID, and using MassHunter GC/MS Acquisition (B.07.00 SP1.1549) software.(a)GC Column—Supelco  SP-2560 (100 m × 0.25 mm × 0.2 µm film thickness).(b)Carrier Gas—Hydrogen with operation in constant flow mode at 1.3 mL/min.(c)Inlet—Temperature 250 °C; 1 µL injection; operation in split mode at 100:1 split.(d)FID—Temperature 260 °C with total hydrogen flow at 35 mL/min, air flow at 350 mL/min, and make-up gas (N2) flow at 25 mL/min.(e)Oven—Initial temperature 70 °C, temperature gradient of 7.8 °C/min to 130 °C; gradient of 3.1 °C/min to 189 °C with 2 min hold; gradient of 1.6 °C/min to 200 °C; gradient of 3.2 °C/min to 230 °C with 22 min hold.(f)Run Time—67 min.(a)GC Column—Supelco SP-2560 (100 m × 0.25 mm × 0.2 µm film thickness).(b)Carrier Gas—Helium with operation in constant flow mode at 1 mL/min.(c)Inlet—Temperature 250 °C; 1 µL injection; operation in split mode at 50:1 split.(d)2) flow at 25 mL/min.FID—Temperature 260 °C with hydrogen flow at 35 mL/min, air flow at 350 mL/min, and make-up gas (N(e)Oven—Initial temperature 70 °C, temperature gradient of 5 °C/min to 130 °C; gradient of 2 °C/min to 180 °C; gradient of 1 °C/min to 196 °C; gradient of 2 °C/min to 230 °C with 30 min hold.(f)Run Time—100 min.(g)4); scan from m/z 50 to m/z 450 at 1.9 Hz.MSD Parameters—Operation in positive chemical ionization mode with methane reagent gas  versus peak area data were transferred to an Excel spreadsheet for further analysis. FAME mass percentages were obtained directly, and these were converted to mole percentages. Molar response factors based on the FID responses of the FAME of the GLC 463 standard were applied to the mole percentages. The corrected molar percentages were used for construction of 4 MS. Oxo-FA isomers present in these samples were also separated.The FA compositions of pulses were less complex than those of samples such as dairy products and meats, so the use of highly polar 100 m capillary columns was highly beneficial for isomer separation of pulse FA. Five 16:1 isomers and three isomers each of 18:1, 18:2, and 18:3 were included in the compositions reported here, but it is suspected that several additional isomers were present at levels too low for confirmation by PCI-CHThe FA compositions presented here, determined using basic (KOH) FAME derivatization, included the contributions from TAG and PL, as well as small amounts of other lipid components, though not free FA, which are presumed to be minor. Reports on kidney beans  and peasPCA was helpful in identifying trends and grouping the ten pulse samples into clusters with related FA compositions. HCA produced the same clusters. Since FA and TAG compositions are correlated, it is perhaps not surprising that the sample clusters from PCA and HCA reported here are the same as those found for the TAG compositions of these samples (manuscript submitted), though any discrepancies as might arise from a different or larger dataset could be informative.Phaseolus vulgaris compositions were all dominated by the oxo-Ln1 isomer. The two Phaseolus lunatus samples are less similar, which may be indicative of age at harvest.The differences in the oxo-FA compositions found for these pulse samples probably reflect enzymatic or metabolic differences. The four Future Work. While the pulses examined here are commonly available ‘market class’ pulses, there are other genotypes that are being bred for a variety of purposes, including drought resistance. We will investigate how breeding for some traits affects the FA compositions. We will extend this work to a series of breeding lines of Phaseolus vulgaris to identify cultivars with fatty acid compositions substantially different from the market class samples. We will also analyze cooked pulses to be used in dietary intervention studies, to provide FA compositions of dietary components actually consumed.


Osteoporosis is a serious threat to human life. Guben Zenggu Granule is an empirical prescription for clinical treatment of osteoporosis. MC3T3-E1 cells are mouse osteogenic precursor cells with osteogenic differentiation, and are classic cells for studying bone metabolism and osteogenic mechanism, as well as mechanical stimulation sensitive cells. Therefore, it can be inferred that Guben Zenggu granule can repair MC3T3-E1 cells under continuous static pressure overload. This study aims to through the network of pharmacology and gene sequencing method, reveal thrift increase bone particles under the condition of continuous static pressure overload on osteogenesis mechanism of MC3T3-E1 cells. In the process of analysis, from a variety of 98 compounds was predicted in the database, a collection of 474 goals, a total of 29,164 difference between two groups of genes. Then, construction of composite targets between cells and predict targets and protein - protein interaction networks, and through the cluster analysis to further explore the relationship between the target. In addition, linkages between target proteins and cells were further identified using Gene Ontology (GO) and Pathways (KEGG Pathway). Finally, the repair effect of Guben Zenggu granule on MC3T3-E1 cells under continuous static pressure overload was verified through experiments, so as to accurately explain the pharmacodynamic mechanism of Traditional Chinese medicine. Osteoporosis (OP) is a common disease and frequently-occurring disorder of bone metabolism. The main clinical manifestations are bone loss and systemic chronic pain, and the main complications are brittle fracture . In rece2+] I concentration in bone in ovariectomized rats, thus regulating bone mineral density and stimulating biomechanical properties of bone tissue .http://tcmspw.com/tcmsp.php) (http://bionet.ncpsb.org/batman-tcm/) (https://string-db.org/)  Uniprot database (https://www.uniprot.org/) (TCMSP database (msp.php) , Batman an-tcm/) , the STRdb.org/) , Venny2.ot.org/) , CytoscaGlycine (Biofroxx), Twain 20 , Protein Marker (10-250kD), PVDF membrane (0.45 μm) (Millipore), PVDF membrane (0.22 μm) (Millipore), mouse monoclonal antibody β-actin (40KD) , rabbit polyclonal antibody NOX4 (62KD) , mouse monoclonal antibody RANKL (35KD) (Abcam), Rabbit polyclonal antibody OPG (60KD) (Abcam), rabbit polyclonal antibody ColI (129KD) (Abcam), rabbit polyclonal antibody OC (11KD) , rabbit polyclonal antibody OPN (60KD) (Abcam), mouse monoclonal antibody Runx2 (57KD) (Abcam), HRP labeling sheep fight two resistance in mice boster biological engineering co., LTD. (wuhan), HRP labeling sheep rabbit 2 resisting boster biological engineering co., Ltd. (wuhan), ECL substrate liquid Pulitzer gene technology co., Ltd. (Beijing), X-ray film , developing fixing kit .Micropipette (Eppendorf), Electrophoresis instrument power supply (Beijing Liu yi Instrument Factory), vertical electrophoresis tank (Beijing Liu yi Instrument Factory), electric rotary instrument (Beijing Liu Yi Instrument Factory), horizontal shaker , pH meter (Metter-Toledo GmbH), Electronic balance , magnetic stirrer , Enzyme labeler (Thermo) centrifuge  phosphatase inhibitor (Biyuntian), PMSF (Alding), RIPA lysis fluid (Biyuntian), BCA Protein Concentration Determination Kit, TEMED , TrISE-Base (Biofroxx), HCl , DTT (Biofroxx), SDS , Bromophenol blue , Glycerin , 30% acrylamide (Biosharp), TrisE-Base (Biofroxx), glycine (Biofroxx), SDS , Tris-base (Biofroxx), MC3T3-E1 cells  were taken out of liquid nitrogen using MEM -α + 10%FBS + 1% (Penicillin Streptomycin Solution) cell medium, and quickly put into 37°C water bath. After dissolved, transfer the cells to contain 5 ml medium in the centrifuge tube. Centrifugation was performed at 1000 RPM for 5 min at room temperature, and the supernatant was discarded. In containing 10% fetal bovine serum  the complete culture medium of cell suspension, and inoculated into a petri dish. The gently blown and mixed cells were cultured at 37°C, 5% CO2 saturation and humidity. When the cell density reached 80%, subculture, with 0.25% trypsin digestion, collect MC3T3-E1 cells after termination of digestive cells. With PBS washing cells twice, at 1500 rpm, 5 min; Complete culture medium was added, cells were blown, single cell suspension was prepared, and the culture was expanded at 37°C and 5% CO2 saturation humidity at the ratio of 1:3.1ul RNA is extracted by TRizol method and quantified by Nanodrop instrument. According to the quantitative results, 500 ng 1% agarose is used for electrophoresis detection, dscDNA is synthesized and the end is supplemented, 12.5µl A-tailing buffer is added. 12.5µl A tail buffer was added to 17.5µl DNA, and the mixture was fully mixed. After 30min at 37°C, the splice was added for PCR enrichment and Qubit was used to quantify the RNA library. Illumina HiSeq3000 was sequenced on a Start CBOT instrument, and the HiSeq 2500 was run on the machine for 11 days before the data was converted into FASTQ format.Using DESeq2 software for screening differentially expressed genes between groups, with different meet | log2FC | 1 or higher and Pvalue 0.05 or less scope of differentially expressed gene screening of the difference between the two groups. For the differentially expressed genes screened between sample groups, bidirectional hierarchical clustering of genes and samples was conducted and heat maps were used to display the clustering parameters . Mfuzz clustering method was used to classify the expression patterns into 10 groups for the sample size greater than or equal to 6.https://tcmspw.com/tcmsp.php) to retrieve the astragalus, codonopsis, angelica, epimedium, desertliving cistanche, rehmannia glutinosa, malaytea scurfpea fruit, ground beetle, dog ridge, radix linderae, antler glue ingredient, the composition of the filter is set to the OB 30% or higher, DL 0.18 or higher. For those not included in TCMSP, Batman database is used for retrieval, and Uniprot database is used for standardization and unification of target names. Will be gained by the composition by TCMSP database and Swisstargets database (http://www.swisstargetprediction.ch/) to obtain ingredients targets.In TCMSP database , molecular functions (MF) and cellular components (CC) of GO are rich in common targets for drug cell genes and are referenced in the String database. Items with correction p < 0.05 were screened by String database. Using R 3.6.3, after installing and referencing the clusterProfiler package, draw the bar and bubble charts.Common targets of drug cell genes were analyzed by KEGG pathway enrichment, and the items with Take the treated MC3T3-E1 cells in good growth condition, adjust the cell density to 5 × 104/ml with MEM-α medium, and connect them to a 96-well plate with 100 μl cell suspension per well. In the meantime, set a blank group at 37°C Cultivate overnight (add 100 μl sterile PBS to the holes around the cell wells); Treat the cells separately according to the following different groupings and cell treatment settings, each group has 3 multiple wells, cultured at 37°C for 24h; control group; MC3T3-E1 + blank serum 5%, 10%, 20%, MC3T3-E1 + low-dose serum 1%, 5%, 10%, 15%, 20%; MC3T3-E1 + medium-dose serum 1%, 5%, 10%, 15%, 20%; MC3T3-E1 + high-dose serum 1%, 5%, 10%, 15%, 20%, after cell treatment, add 10 μl MTT to each well and incubate at 37°C for 3h; aspirate the medium, add 150 μl DMSO and shake for 10min; Microplate reader detects the absorbance value OD 568.PVDF membranes were immersed in TBST  containing 5% skim milk powder and sealed with a mixer at room temperature for 2 h. A diluted with sealing fluid resistance, the PVDF membrane were soaked in a resistance to the fluid of the incubation, 4°C incubation for the night. The PVDF membrane was thoroughly rinsed by TBST 5 times, 5min/time. Put up to 3 membranes in a dish. In the process of flushing, and pay attention to whether film adhesion on board wall or membrane whether overlap. Corresponding HRP labeling 2 fight after diluted with TBST - 1:50,000, soaked the PVDF membrane in the fluid of the second antibody incubation, and incubated with the table 2 h at room temperature. Fully wash PVDF membrane with TBST 5 times, 5min/time. The enhancement solution in the ECL reagent was mixed with the stable peroxidase solution in a ratio of 1:1. The working droplets were added to the PVDF membrane and reacted for several minutes until the fluorescence band appeared. Then the excess substrate solution was absorbed with filter paper. Cover with plastic wrap, press X-ray film, put in developer solution for development, fixation solution, and rinse the film.Add 200μ L chloroform, mix thoroughly several times, and let stand at room temperature for 5 min. Centrifugation was performed at 12000rpm at 4°C for 15min, and the results were divided into three phases: upper (RNA), middle (protein) and lower (DNA). Transfer the upper water phase (about 400 μl) into another 1.5 ml EP tube, add 400 μl isopropyl alcohol, mix well, and let stand at room temperature for 10min. After centrifugation at 12000rpm at 4°C for 10min, white RNA precipitates could be seen at the bottom of the tube. Abandon the supernatant, add 1 ml without rnase 75% rnase-free 75% ethanol, vortex mixing, 4°C, 10,000 RPM, centrifugal 5 min. Repeat Step 6 once. The supernatant was discarded, the RNA was dried in air for precipitation for 5–10min, and the precipitation was dissolved in 20μlDEPC water. With microspectrophotometer dissolved RNA2 mu L OD260, OD280 and OD260/OD280 value, calculate the purity and concentration of RNA. According to OD260/OD280 ratio to estimate the quality of RNA, the ratio of between 1.8 ∼ 2.0 conform to the requirements of the experiments. The total RNA concentration (μg/μ L) = OD260 × 40×10-3. Save the total RNA in - 80°C refrigerator to spare.Primer sequences used for gene detection are as follows:The climbing cell slides in the culture plate were immersed in PBS, and the slides are fixed with 4% paraformaldehyde, and normal goat serum is dripped on the slides, and the slides are sealed at room temperature; incubate in a humid box at 37°C, and add fluorescence. DAPI was added dropwise to incubate in the dark, the specimens were stained nucleus; the slides were fixed with fixing solution containing anti-fluorescence quenching agent and observed under fluorescence microscope and images were collected.The tissues were soaked in 4% paraformaldehyde and then fixed for 24 h. Micro-ct was used to detect the morphological indicators of the tissues. Skyscan1276 Micro-CT Scaner software was used for scanning, and the parameters were set as follows: Voltage: 100 KV; current: 100 µA; scanning spatial resolution: 10 µm; resolution: 4032 × 2688 pixel; rotation Angle: 0.3°; exposure: 500 ms. After scanning, use Data Viewer software for calibration, and then use CT-AN software to select the area of interest. Finally, CT Vox software was used for 3D reconstruction and analysis.Gradient alcohol is used to dehydrate the tissue. The tissue blocks must be transparent after dehydrated by alcohol. The transparent agent (xylene) can be mixed with dehydrating agent and paraffin wax at the same time. It replaces the dehydrating agent and the paraffin wax penetrates the tissue smoothly. The transparent tissue blocks were dipped in three cylinders of paraffin (60°C) successively. Embedding is to encase wax-impregnated tissue blocks in paraffin blocks. The temperature of embedding wax should be slightly higher than that of immersion wax to ensure that tissue blocks and embedded paraffin wax are completely integrated. The tissue slices were placed in a 40°C water bath after being sliced by a Leica pathological slicer. Dip anti-stripping slides into the water to scoop slice, slice was attached with the appropriate placement of the slide, at 60°C baking in the oven for 3 h. Paraffin section in xylene (20 min) ⅰ - xylene (20 min) ⅱ - xylene iii (min) 20-ⅰ anhydrous ethanol (5 min) - ethanol (5 min) ⅱ-95% alcohol (5 min) - 90% alcohol (5 min) - 80% alcohol (5 min) -70% alcohol (5min), soak in distilled water for 5min. Section into Mayer’s hematoxylin  dye for 5min, wash and soak in tap water and return to blue. With 1% water soluble eosin staining section 5 min, 30 s is washed with tap water.p < 0.05 was statistically significant.SPSS22.0 software was used for variance analysis of relevant data, and According to experimental design, using DESeq2 software is not the same screening differentially expressed genes between groups, according to | log2FC | 1 or higher and Pvalue 0.05 as differentially expressed or less range of filters, according to the results of 29,164 there were differences between two groups of genes, the expression level of 14,489, There were 14,675 down-regulated expressions, and Astragalus membranaceus, Cistanche deserticola, rehmannia glutinosa, Aconitum aconitum and Herba fularii. In the Batman database retrieval Dog‘s back, antler glue and tripelidae, received 98 potential active ingredients. Uniprot database was used to standardize and unify the target names and transform them into corresponding targets in mice. A total of 474 drug targets were screened out , while the drug-containing serum group was compared with its ratio increased (p < 0.05). The ratios of Rankl/β-actin and Nox4/β-actin in the drug-containing serum group were significantly reduced (p < 0.05). Therefore, it can be seen from the table that the intervention effect of the drug-containing serum group on sustained static pressure injury cells is more significant than that of the blank serum group. For details, see The results showed that the ratio of OC/β-actin, ColⅠ/β-actin, OPN/β-actin, OPG/β-actin in the blank serum group was higher than that in the stress model group (p < 0.05). As shown in Figure 11-12. The above results showed that Smad2 and Runx2/Cbfa1 gene expressions were inhibited in MC3T3-E1 cells under continuous static pressure, while the expression of Smad2 and Runx2/Cbfa1 gene was up-regulated by guben Zenggu granule containing serum, and the expression was normalized  of Smad2 and RUNx2 expression level is lower than normal group  is a kind of composition, ways and targets of natural medicine. In this study, cell sequencing genes were analyzed to clarify the basis and mechanism of drug action, and experimental verification was carried out.In this study, KEGG and GO results showed that Guben Zenggu granule were involved in major signaling pathways including MAPK and positive regulation of cell apoptosis on MC3T3-E1 under continuous static pressure. The negative regulation of cell apoptosis process; Negative regulation of cell proliferation; Positive regulating cell proliferation; Negative regulation of gene expression; Regulate cell proliferation pathways.MAPK signaling pathway, the bone morphogenetic protein (BMPs) signaling pathway is involved in a variety of bone metabolic processes through two typical Smad protein-dependent pathways  and atypical Smad independent signaling pathways . The former is indispensable in cell stress transduction and osteogenesis, mainly reflected in Smad’s regulation of TGF-β/BMP signaling pathway, while the latter directly affects cytoskeleton depolymerization and rearrangement, mainly reflected in Smad’s regulation of MAPK transduction signal. Runx2 is a downstream target gene of the TGF-β/BMP pathway. Runx2 and Smads activated by bone morphogenetic protein jointly induce osteoblast specific gene expression and regulate bone metabolism. The target of bone morphogenetic protein signal is Runx, and BMPs signal transduction pathway is involved in the physiological response of osteoblasts to stress stimulation, and is very important in the level of information transmission in this process. The “endpoint” of mechanical stress stimulation in osteoblasts was the up-regulation of Runx2/Cbfa1 gene expression.Continuous static pressure is a typical physical factor in the study of osteoporosis. It has been shown in the clinic that an appropriate amount of pressure can promote the therapeutic effect of OP. The reason is that cells will produce a series of physiological and biochemical reactions after stress stimulation to resist the next pressure. Stimulate. In the related studies of continuous static pressure on MC3T3-E1 cells, OC, ColⅠ, OPN, RANKL, NOX4, OPG, osteocalcin, ADAM28, TD, ALP, Runx2, Wnt1, DKK-1, and other genes and their Protein expression is the main research direction .The osteoblasts responsible for bone formation activities are differentiated from bone marrow mesenchymal stem cells. Through exploring osteopontin (OPN), osteocalcin (OC), type II collagen (Col I), and osteoprotegerin (OPG) genes The expression of protein explores the molecular biological mechanism of cellular osteogenics. The results show that Guben Zenggu Granules medicated serum can regulate the osteogenic markers of MC3T3-E1, indirectly promote the osteogenic differentiation of BMSCs, and affect other bone formation indicators of osteoblasts. It has a promoting effect and can effectively improve molecular biology and anti-oxidation. The results are correlated with previous studies , confirmThe positive regulatory signaling pathway of apoptosis is an important programmed cell death for metazoan development and internal environment stability. Apoptosis signaling pathway plays an important role in osteoclast - induced bone loss. Jijie Chai suggested that Smac/DIABLO could not only promote protein hydrolysis activation of procaspase-3, but also promote the enzyme activity of mature caspase-3 . As a taAs a targeted anti-apoptotic drug, Guben Zenggu granule may positively regulate the apoptosis process through MAPK. Negative regulation of apoptosis process; Negative regulation of cell proliferation; Positive regulating cell proliferation; Negative regulation of gene expression; Regulating cell proliferation pathway and Runx2, Smad gene protein acts on MC3T3-E1 cells to treat induction of bone resorption related diseases such as osteoporosis and fracture.Too high a dose of medicated serum will inhibit the proliferation of MC3T3-E1 cells. In the final concentration gradient, a 10% dose of medicated serum has the best effect on cell proliferation. Guben Zenggu Granules medicated serum may directly participate in inducing the generation and differentiation of OB and OC by regulating TGF-β/BMP and BMP-2/Smads signaling pathways, and regulate bone metabolism in a continuous static pressure overload environment. This may be one of the effects of Guben Zenggu Granules medicated serum through regulating bone metabolism.Guben Zenggu Granules medicated serum can promote the expression of Tubulin and actin proteins in MC3T3-E1 cells in a continuous static pressure overload environment, thereby promoting bone metabolism.


Cucumis sativus L. “Jin You No. 1”) seedlings grown under high nitrate condition (HN). The results showed that MT and NO independently alleviated the inhibition of growth and photosynthesis capacity of cucumber seedlings under nitrate stress. NO was required for MT to enhance the root activity, root length, lateral root number and the accumulation of calcium, magnesium and iron in the roots of cucumber seedlings grown under nitrate stress. Consistently, the expression of adventitious rootless 1 gene (CsARL1) was modulated. Furthermore, exogenous MT induced accumulation of endogenous MT, NO, indole-3-acetic acid (IAA) and abscisic acid (ABA), mainly within 24 h after treatment, in which MT and NO were further increased at 48 h and 96 h, IAA and ABA were further increased at 16 h in the presence of SNP. In contrast, the accumulation of endogenous IAA, MT and ABA slightly decreased within 24 h, NO significantly decreased at 192 h in the presence of cPTIO. Correspondingly, the expression levels of genes involved in nitrogen metabolism (CsNR1 and CsNR2), MT metabolism , auxin carriers and response factors , ABA synthesis and catabolism  were upregulated by MT, in which CsNR1, CsNR2, CsAUX1, CsNCED3 and CsT5H were further induced in the presence of SNP in roots of cucumber seedlings. These observations indicated that NO act as a crucial factor in MT, alleviating nitrate stress through regulating the mechanism of root growth in cucumber seedlings.Melatonin (MT) and nitric oxide (NO) in plants can function cooperatively to alleviate salt stress, sodic alkaline stress and immune response, as well as adventitious root formation. The interaction of MT and NO on the nitrate stress tolerance of cucumber seedlings are not well understood. We investigated the effects of exogenous MT, NO donor (SNP) and NO scavenger (cPTIO) on the growth; photosynthesis; characteristics of root morphological; accumulation of mineral elements, endogenous NO, MT, IAA and ABA; and related genes expression in cucumber ( Applicagetables ,5. The uN-acetyl-5-methoxytryptamine) is a pleiotropic, orchestrating regulator, existing in almost all organisms [N-acetyltransferase (SNAT) [N-acetylserotonin methyltransferase (ASMT) [4 in cucumber [2O2 or enhancing activity of antioxidant enzymes and concentrations of antioxidants in cucumber seedlings [Malus species under drought stress [Melatonin  ,10 and Ne (ASMT) ,12,13. Fe (ASMT) . The MT e (ASMT) , photosye (ASMT) ,16, leafe (ASMT) , seed gee (ASMT)  and circe (ASMT) . As a poe (ASMT) . MT allecucumber . The preeedlings . MT medit stress . MT promt stress . MT enhat stress , and NO t stress .Cucumis sativus L.) is one of the primary greenhouse vegetables in China and is very sensitive to nitrate stress [3− levels in the soil severely inhibit the growth and development of cucumber, showed shorter and narrower stems and weaker root systems [CsNR and CsGOGAT) in cucumber seedlings under nitrate stress [Cucumber  expression, and NO increased IAA accumulation in tomatoes [It was demonstrated that MT promoted adventitious root or lateral root development by regulating IAA and nitric oxide (NO) signaling in tomatoes  or Arabithaliana . MT trigtomatoes . NO was tomatoes . The RNAtomatoes . Much ofPrevious research showed that NO contributed to the MT-enhanced salt stress, sodic alkaline stress, immune response, adventitious root formation, etc. However, the effects of interaction of MT and NO on the nitrate stress tolerance of cucumber seedlings are not well understood. In the present study, we investigated the effects of MT and NO on the photosynthesis capacity; pigment content; root morphological characteristics; accumulation of mineral elements; changes of endogenous NO, MT, IAA and ABA; and on related gene expression of cucumber seedlings grown under nitrate stress. The outcome of this research should help gain a deeper insight into the interaction of MT and NO in regulating the nitrate stress tolerance.3− stress cucumber seedlings, NO3− concentration in the nutrient solution was elevated up to 75 mM, and 2 μM MT and 100 μM SNP (NO donor) were applied based on high NO3−, respectively. As shown in In order to assess the effect of exogenous MT and NO in the alleviation of high NOThe accumulation of K, Ca, Mg and Fe in leaves and roots of cucumber seedlings were affected by nitrate stress, MT and SNP treatments . ContentThe exogenous MT and NO on stem, diameter, leaf and root growth, as well as biomass accumulation were investigated in this study. As shown in The root activity was significantly enhanced in the HN + MT + SNP treatment, which was significantly weakened by the HN + MT + cPTIO treatment, compared with HN and HN + MT d. Root aIn accordance with parameters, visible leaf margin chlorosis was observed in cucumber seedlings treated with HN + MT + cPTIO during the experiment period j. MoreovIn the study here, pigments content and leaf chlorophyll fluorescence of the 1st, 2nd, 3rd true leaves were separately investigated. As shown in As shown in The MT content in treatments of HN + MT, + SNP and + cPTIO were increased significantly within 16 h, as compared with HN. The MT content in HN + MT + SNP and HN + MT + cPTIO maintained a higher level than HN between 48 h and 96 h a. The IACsT5H, CsSNAT1, CsSNAT2, CsASMT1, Cs2-ODD11, s2-ODD33), two genes involved in nitrogen metabolism (CsNR1 and CsNR2), three genes involved in ABA metabolism  and four genes involved in IAA transport and root growth regulation . As shown in CsT5H was significantly down-regulated at 16 h after HN–MT–SNP treatment, as compared with HN + MT. However, CsT5H was significantly upregulated at 24 h after treatments of HN + MT, + SNP and + cPTIO, as compared with HN, in which HN + MT + SNP was significantly higher than in HN + MT. The expression levels of CsSNAT1 and CsSNAT2 were significantly higher in HN + MT + SNP treatment than in HN + MT + cPTIO treatment at 24 h. Additionally, the expression of Cs2-ODD11 and Cs2-ODD33 was upregulated by HN + MT, compared with HN, and reached the peak at 24 h. The expression of CsNR1 tended to increase within 24 h after HN + MT and HN + MT + SNP treatment, compared with HN. To compare with HN + MT, CsNR1 was significantly upregulated at 96 h after HN + MT + SNP treatment. In contrast, it was down-regulated after HN + MT + cPTIO treatment through the whole period except 16 h, as compared with HN. The expression of CsNR2 was significantly upregulated after the HN + MT + SNP treatment between 24 h and 96 h, as compared with HN + MT  that function in nitrogen metabolism were significantly induced by MT [In general, both MT and NO have been reported to play an independently important role in plant stress responses ; NO evided by MT . HoweverFIT1, FRO2 and IRT1, thereby increasing the availability of soluble Fe [Chlorophyll was easily destroyed during photosynthesis or under environmental stress . MT coulluble Fe . In the Previous research showed that MT plays a vital role in strengthening adventitious and lateral root formation and growth ,30,33,433)2 and KNO3 [Ca provides intermolecular linkages and was considered to have a crucial function in stabilizing cell walls and membranes . MT posiand KNO3 . In the Cs2-ODD11, Cs2-ODD33 and CsT5H were significantly higher in HN + MT treatment at 24 h, as compared with the HN, while CsT5H and CsSNAT2 in HN + MT + SNP was significantly higher than in HN + MT at 24 h in HN + MT + SNP  can reflect adventitious root formation [CsARF17 is a negative regulator of adventitious root formation [CsARL1 kept a higher level in HN + MT than in HN during 16 h and 48 h  were sown in sponge blocks and irrigated with “Yamazaki” nutrient solution Ca(NO3)2·4H2O 826 mg·L−1, KNO3 607 mg·L−1, NH4H2PO4 153 mg·L−1, MgSO4·7H2O 370 mg·L−1, NaFe-EDTA 20 mg·L−1, H3BO3 2.86 mg·L−1, MnSO4·4H2O 2.13 mg·L−1, ZnSO4·7H2O 0.22 mg·L−1, CuSO4·5H2O 0.08 mg·L−1, (NH4)6Mo7O24·4H2O 0.02 mg·L−1). Cucumber seedlings with one fully expanded true leaf were transplanted into hydroponic container. Aeration was switched on for 15 min every hour in hydroponic system. The average air temperature was 20 °C during greenhouse cultivation. After four days adaptation, nitrate nitrogen, MT, SNP, cPTIO were added into nutrient solution, constituting different treatment 2 and KNO3, (3) HN + MT, high nitrate nitrogen treatment plus 2 μmol·L−1 melatonin (MT), (4) HN + SNP, high nitrate nitrogen treatment plus 100 μmol·L−1 sodium nitroprusside .There were four treatments in experiment one: (1) CK, cucumber seedlings were cultivated with “Yamazaki” nutrient solution, (2) HN, high nitrate nitrogen treatment, NO3− concentration was upregulated to 75 mM in Yamazaki nutrient solution by increasing Ca(NO3)2 and KNO3, (2) HN + MT, high nitrate nitrogen treatment plus 2 μmol·L−1 MT, (3) HN + MT + cPTIO, high nitrate nitrogen treatment plus 2 μmol·L−1 MT and 10 μmol·L−1 2-(4-carboxyphenyl) -4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide , (4) HN + MT + SNP, high nitrate nitrogen treatment plus 2 μmol·L−1 MT and 100 μmol·L−1 SNP.The second experiment included four treatments: (1) HN, high nitrate nitrogen treatment, NOMT and cPTIO were produced by TCI, Co. Japan, SNP was produced by Damao Co., Tianjin, China. A total of 720 cucumber seedlings were cultivated in the second experiment. Three replicates for each treatment were adopted, and each replicate contained 30 seedlings. The uniform seedlings were randomly selected for parameters analysis after 16 days and 8 days treatment in experiment’s one and two, respectively.Shoot height of cucumber seedlings from stem basal to apical was measured by a ruler. Stem diameter below cotyledons was measured by a vernier caliper. Leaf area was measured by a leaf area meter . Seedlings were separated into shoot and root portions; fresh and dry weight were measured by electronic balance. Dry weight was determined after tissue was oven dried at 100 °C oven for 1 h and 75 °C oven for constant. The dried samples were used for analyzing the composition and content of mineral elements.Lateral roots were firstly removed, then cultivated in different nutrient solution treatment for 8 days, then morphological parameters  were measured by a root scanner  and analyzed by using WinRHIZO 4.1 software .−1 H2SO4 was added to stop the reaction with the root. The root was dried with filter paper and then extracted with ethyl acetate. The extract was measured at 485 nm by a UV-spectrophotometer .Root activity was determined by triphenyltetrazolium chloride (TTC) method  and expr2 concentration (Ci) and transpiration rate (Tr) was measured with a photosynthetic system  on the second true leaf between 9:00 and 11:00 at 800 μmol·m−2·s−1. Chlorophyll and carotenoid content in the first, second and third true leaf were analyzed according to our previous research [v/v) acetone. The absorbance at 663 nm, 645 nm and 440 nm were analyzed on a UV-spectrophotometer .The net photosynthetic rates (Pn), stomatal conductance (Gs), intercellular COresearch . The samChlorophyll fluorescence investigation was carried out in the first, second and third true leaf, which were dark adapted for 30 min prior to measurement. Measurements were performed by using a fluorescence imaging system-PAM . The parameters of YII indicates the effective quantum yield of photochemical energy conversion in PSII, ETR represented electron transport rate through PSII, and the NPQ provides an estimate of the heat dissipation by the leaves.2SO4 in a digester tube, boiled gently until H2SO4 decomposing into a large amount of white smoke. Then, the temperature was increased until the solution became uniform brown and black. About 10 drops of H2O2 were added after for 5 min boiling; after cooling, we added 5 drops of H2O2 repeatedly, boiling again. This was repeated 3–5 times until the reaction solution become colorless or clear, then added water to a constant volume of 100 mL. The potassium was analyzed by use of flame atomic absorption spectrophotometer  equipped with potassium hollow cathode lamp, the presser of acetylene was adjusted to 0.085–0.090 kpa, the absorption wavelength was 248.3 nm.Cucumber seedlings were separated into shoot and root two portions and dried to constant weight at 75 °C after 1 h at 105 °C for quantification of potassium, calcium, magnesium and iron. A total of 0.3 g sample was mixed with 8 mL of concentrated HA total of 2.0 g dried sample was mixed with 2.0 mL of 95% ethanol in a 30 mL porcelain crucible for pre-ash treatment. Reaction solution was heated in an electric furnace at 525 °C for 1 h, until the ash content was nearly white. Crucible was then removed to cool to room temperature and then moistened with a small amount of water, adding diluted hydrochloric acid solution to about 20 mL, boiled, then filtered and washed 3 times, dissolving the filtrate to constant volume at 100 mL. Then, the absorbance was read at 422.7 nm , 285.2 nm (magnesium) and 271.9 nm (iron) by use of flame atomic absorption spectrophotometer  fit with hollow cathode lamp; the presser of acetylene was adjusted to 0.085–0.090 kpa.Seedling root samples collected at 8 h, 16 h, 24 h, 48 h, 96 h and 192 h after treatments were rapidly frozen in liquid nitrogen and stored at −80 °C until use. Each treatment sample comprised three biological replicates. Each replicate was collected from at least six uniform seedlings. NO content was determined by using “NO kit”, . The protein quantification was determined by BCA method, and diluted to 0.896 mg/mL, then determined the MT, IAA and ABA content. Contents of melatonin (MT), indole-3-acetic acid (IAA) and abscisic acid (ABA) were determined by using the “Plant hormone ABA ELISA kit” .Seedling root samples collected at 8 h, 16 h, 24 h, 48 h and 96 h after treatment were rapidly frozen in liquid nitrogen and stored at −80 °C until use. Expression of genes involved in the biosynthesis or catabolism of NO, MT, IAA, ABA and root growth-related genes were evaluated by performing quantitative real-time PCR (qRT-PCR), as described in our previous research . Total R2O, 400 nM of forward and 400 nM of reverse primer. The amplification program was 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melting curve analyses were performed at the end of 40 cycles (95 °C for 5 s followed by a constant increase from 60 to 95 °C). Relative fold expression changes were calculated using the 2−ΔΔCt method [The qRT-PCR was conducted using the Light Cycler 480 II real-time PCR system  with one step SYBR Premix Ex Taq TM . The reactions reagent mix was 5 μL SYBR Premix Ex Taq II, 1.0 μL cDNA template, 3.2 μL ddHt method  with IQ5https://www.ncbi.nlm.nih.gov/tools/primer-blast/ accessed on 26 June 2022). The primer quality and efficiency were checked by qPCR on series of diluting cDNA. The primer sequences, Ct values and amplification efficiency and correlation coefficient for each gene are listed in the Genes were designed on the NCBI primer blast website , followed by Duncan’s multiple range tests of SPSS 21.0 at CsNR1, CsNR2, CsAUX1, CsNCED3, CsT5H) were induced by exogenous MT and NO under nitrate stress. This study provided deeper knowledge on the roles of MT in alleviating nitrate stress.In our study, MT interacted with NO to mitigate nitrate stress by increasing the number of lateral root, root elongation, root activity, and accumulation of mineral elements , as well as the endogenous MT, NO, IAA, ABA in the roots cucumber seedlings. Correspondingly, the transcription levels of genes involved in the metabolism of nitrogen, IAA, ABA and MT (