O-β-D-glucuronide (M4), jatrorrhizine-3-O-sulfate (M5), thalfendine-10-O-β-D-glucuronide (M6), berberrubine-9-O-β-D-glucuronide (M7), demethyleneberberine-2-O-sulfate (M8) and demethyleneberberine-2-O-β-D-glucuronide (M9) in rats. An accurate and reliable LC-MS/MS method was developed and validated for the determination of berberine and its nine metabolites in rat biosamples. Pharmacokinetic profiles of berberine and its nine metabolites were obtained after a single intravenous administration (4.0 mg/kg) and oral administration  of berberine in rats. For excretion study, rats were intragastrically administered a single dose of 48.2 mg/kg berberine. Our results showed that berberine could be metabolized rapidly and all the nine metabolites could be detected in vivo. The absolute bioavailability of berberine was 0.37 ± 0.11%. As for the AUC0–48 h values, phase II metabolites were much higher than those of phase I metabolites, suggesting that phase II metabolites were the major metabolites exist in blood circulation. 18.6% of the berberine was excreted in feces as berberrubine (M1). The total recovery of berberine and its nine metabolites from urine, bile and feces was 41.2%. This is the first systematic study about the pharmacokinetics and excretion of berberine and its nine metabolites, which will be beneficial for both better understanding the clinical effects and further development of berberine.Berberine, a well-known alkaloid, has been proved to possess various pharmacological activities. Previous studies demonstrated that berberine could be extensively metabolized and the metabolites also contributed to its therapeutic effects. However, as for berberine’s metabolites, especially phase II metabolites, pharmacokinetics and excretion studies were rarely reported. The objective of this study was to thoroughly investigate the pharmacokinetic and excretion profiles of berberine and its nine metabolites, namely, berberrubine (M1), demethyleneberberine (M2), jatrorrhizine (M3), jatrorrhizine-3- Coptis chinensis Franch. (Coptis japonica (Thunb.) Makino  Makino , and Phe Schneid . It has  Schneid , hypolip Schneid , anti-in Schneid , immunos Schneid  and anti Schneid  activiti Schneid  and also Schneid .O-β-D-glucuronide showed excellent glucose-lowering effect , jatrorrhizine-3-O-sulfate (M5), thalfendine-10-O-β-D-glucuronide (M6), berberrubine-9-O-β-D-glucuronide (M7), demethyleneberberine-2-O-sulfate (M8) and demethyleneberberine-2-O-β-D-glucuronide (M9) were carried out in rats. This study will help to better understand the in vivo disposition of berberine.According to our previous study, berberine could be metabolized into 97 metabolites in rats . Even therrubine . Hence, errubine , namely,O-sulfate, demethyleneberberine-2-O-β-D-glucuronide, berberrubine-9-O-β-D-glucuronide, thalifendine-10-O-β-D-glucuronide, jatrorrhizine-3-O-β-D-glucuronide, and jatrorrhizine-3-O-sulfate  were isolated and prepared in our laboratory (Berberine sulfate and berberine chloride (99.5% purity) were purchased from the Northeast General Pharmaceutical Factory . Jatrorrhizine chloride (98% purity) were obtained from Vientiane Tianjin Hengyuan Technology Co., Ltd. . Berberubine and demethyleneberberine (98% purity) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. . Tetrahydropalmatine purchased from Tianjin Shilan Technology Co., Ltd. was used as internal standard (IS). Methanol and acetonitrile  were of HPLC grade. Ammonium acetate and formic acid were purchased from Sigma-Aldrich Co. Ltd. . Ultrapure water  was used throughout the study. Demethyleneberberine-2-boratory . Other cSprague-Dawley rats  were obtained from the Beijing Military Medical Science Academy. All animals were maintained under controlled conditions  and fasted (free access to water) for at least 12 h prior to drug administration. Animal experiments were reviewed and approved by the Animal Research Ethics Committee of the Tianjin University of Traditional Chinese Medicine prior to the study.n = 6). Berberine chloride (suspended in 0.5% CMC-Na) was administered intragastrically at a single dose of 48.2, 120, and 240 mg/kg to groups one to three, respectively. The fourth group was intravenously administered with a single dose of berberine sulfate  at 4.0 mg/kg. The selection of the dosages was based on the clinical dosages of berberine used in humans , an abdominal incision was made and the bile duct was cannulated with tube to collect bile. A single 48.2 mg/kg dose of berberine chloride was orally administered to rats and bile samples were collected during intervals of 0–2, 2–4, 4–8, 8–12, 12–24, and 24–36 h post-dose. Samples were stored at −80 °C refrigerator until analysis.For excretion study, six rats were administered orally with a single dose of berberine chloride (suspended in 0.5% CMC-Na) at 48.2 mg/kg. After dosing, rats were housed in individual metabolism cages which allowed the separate collection of urine and feces. Urine samples were collected during intervals of 0–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–60, 60–72, and 72–84 h post-dosing and fecal samples were collected at 0–84 h post-dosing. After the collection of urine and fecal samples, the rats were allowed a washout period of 7 days and then employed in the biliary excretion study. After the rats were anesthetized with 20% urethane (w/v) for 30 min. Then, fecal sample extracts were centrifuged at 3,000 rpm for 10 min and an aliquot of 50 μL supernatant was transferred to a new tube and treated in the same way as the urine samples. All samples were vortexed for 2 min before LC-MS/MS analysis and the sample injection volume was 2 μL. For the determination of berberine, M1, M2, and M3, acetonitrile was used as precipitant, and for the determination of M4 to M9, methanol was used as precipitant.For plasma, urine and bile samples, similar sample preparation procedure was employed. An aliquot of 50 μL each sample was spiked with 200 μL of precipitant containing 5.00 ng/mL of IS. After vortexed for 2 min, the mixture was centrifuged at 14,000 rpm for 10 min. 150 μL of supernatant was transferred to a new tube and diluted with 50 μL (for plasma sample) or 100 μL (for urine and bile sample) of distilled water. Fecal samples were freeze-dried, weighted, pulverized into powder and ultrasonically extracted with methanol . Thus, dilution procedure was conducted. 10 μL of this plasma sample was diluted with 140 μL blank plasma and 50 μL of the diluted plasma sample was transferred to a new tube and treated followed the sample processing as mentioned above. This dilution procedure was validated with spiked samples at concentrations of 300, 750, and 1,350 ng/mL in quadruplicate. Additionally, the plasma concentration of M7 for several samples also exceeded the highest concentration. These samples were repeatedly analyzed using 25 μL of sample diluted with 25 μL blank plasma. This dilution procedure was also validated with spiked samples at concentrations of 500, 700, and 900 ng/mL in quadruplicate. The dilution factors for berberine and M7 were 15 and 2, respectively.TM I-Class series UPLC system and an AB SCIEX 5500 QTRAP triple quadrupole mass spectrometer operated in positive ESI mode. Multiple reaction monitoring (MRM) mode was applied for quantitation of berberine and its metabolites. The instrument parameters included an ion spray voltage of 5.5 kV, temperature of 500 °C, curtain gas of 30 psi and collision gas of 10 psi. The LC-MS/MS system consisted of a Waters ACQUITYv/v, eluent A) and acetonitrile (eluent B) with gradient elution. Due to the fact that the polarity of phase II metabolites (M4–M9) was much greater than that of berberine and its phase I metabolites (M1–M3), two sets of mobile phase gradient elution programs were applied in this study. For the quantitative determination of berberine, M1, M2 and M3, gradient elution program one was used: 0–0.5 min, 28% B; 0.5–2.3 min, 28–62% B; 2.3–2.5 min, 62% B; 2.5–2.6 min, 62–95% B; 2.6–3.8 min, 95% B; 3.8–3.9 min, 95–28% B; 3.9–5.0 min, 28% B, and the switching valve directed the flow eluting between 0.5 and 3.0 min into the mass spectrometer and the remainder to a waste container. For the quantitative determination of M4 to M9, gradient elution program two was used: 0–4.5 min, 10–14% B; 4.5–5.5 min, 14–50% B; 5.5–6.0 min, 50–95% B; 6.0–7.0 min, 95% B; 7.0–7.1 min, 95–10% B; 7.1–8.0 min, 10% B, and the switching valve directed the flow eluting between 0.5 and 7.0 min into the mass spectrometer and the remainder to a waste container.Chromatographic separation was accomplished by using an ACQUITY UPLC BEH-C18  with an ACQUITY UPLC BEH-C18 guard cartridge  at room temperature with a flow rate of 0.3 mL/min. The mobile phase was composed of 2 mM ammonium formate/formic acid , time for peak plasma concentration (Tmax), elimination half-life (t1/2), apparent volume of distribution (Vz/F), apparent clearance (CLz/F) and mean residence time (MRT), were calculated based on a non-compartmental model with DAS 2.0 software  and compared using one-way analysis of variance. The AUC was calculated by the trapezoidal rule and Cmax and Tmax were obtained directly from the plasma profiles. The elimination rate constant (ke) was calculated from the slope of the terminal phase of the plasma concentration-time plot and t1/2 was calculated as 0.693/ke. Vz/F was calculated using the relationship, Vz/F = dose/ke·AUC0–∞. CLz/F was calculated from the relationship, CLz/F = dose/AUC0–∞. All pharmacokinetic and excretion parameters were expressed as mean ± SD. Statistical comparison was performed by one-way analysis of variance and a p value <0.05 or 0.01 was considered statistically significant.Pharmacokinetic parameters, including area under plasma concentration-time cure (AUC), the peak plasma concentration (Cr) were greater than 0.9981 in all analytes. The lower limit of quantifications (LLOQs) of berberine and M1 to M3 were 0.500 ng/mL and the LLOQs of M4 to M9 were 1.00 ng/mL with both precision and accuracy were less than 20.0%. The relative standard deviation (RSD) values for intra-and inter-day analysis were less than 9.4% and the relative error (RE) values were in the range of −11.4 to 13.8%. The matrix effects of all analytes were within 92.5–109% and the extraction recovery ranged from 69.8 to 94.7%. The stability of berberine and its nine metabolites was evaluated by analyzing quality control (QC) samples at low and high concentration levels. The RSD values ranged from 0.4 to 8.5% and the RE values ranged from −13.3 to 13.5%, which indicated that all samples were stable for 4 h at room temperature, after three freeze-thaw cycles, at −80 °C for 21 days, and post-preparative QC samples stored at 10 °C for 12 h were also stable. Validation of dilution procedure was conducted with spiked samples in quadruplicates. The dilution procedure showed excellent accuracy  and precise (RSD < 9.4%). Detailed results of method validation were showed in In our study, two sets of mobile phase gradient elution were applied. The first gradient elution was used for the determination of berberine, M1, M2, M3 and the total run time was 5.0 min. The retention times of berberine, M1, M2, M3 and IS were 1.5, 1.2, 0.75, 0.97, and 1.01 min, respectively. For the determination of M4 to M9, the second gradient elution was applied and the total run time was 8.0 min. The retention times of M4 to M9, and IS were 4.4, 5.7, 4.3, 5.2, 5.7, 4.2, and 5.8 min, respectively. The LC-MS/MS method was successfully applied to a pharmacokinetics study of berberine and its nine metabolites following a single oral administration of either 48.2, 120, or 240 mg/kg berberine and a single intravenous administration of berberine at the dose of 4.0 mg/kg. Pharmacokinetic parameters are listed in 0–48 h for berberine, M1, M6, M7, M8, and M9 increased with the dose. As shown in r2 ranged from 0.8823 to 0.9994). As for M2, M4, and M5, no significant dose proportionality was observed. Additionally, Cmax values of the berberine and its metabolites was not dose-dependent. This nonlinear pharmacokinetic behavior might be caused by the saturation of metabolism enzymes or carrier-mediated systems involved in the processes of drug absorption, distribution, biotransformation, and excretion. A comparison of the AUC0–∞ values of berberine given either orally or intravenously revealed that the absolute bioavailability of berberine was only 0.37 ± 0.11%. After oral administration, AUC0–∞ values of phase II metabolites (except for M5) were higher than that of phase I metabolites, indicating that phase II metabolites were the major exposure forms of berberine in vivo. The Vz/F of berberine was found to be 12–38 L/kg, which was exceeded the total body water in rats (0.15 L/kg for a body weight of 0.25 kg), suggesting that berberine may be well distributed in the extravascular fluids. After oral administration, the Tmax values for M7 were 11–13 h, indicating that the biotransformation of berberine into M7 was processed gradually. Conversely, Tmax values for other metabolites were all less than 4.75 h and the short Tmax indicated that berberine could be rapidly metabolized into various metabolites.After intravenous administration, the plasma concentration-time curve of berberine showed a rapid decline from 963 ± 258 to 9.82 ± 2.79 ng/mL after 12 h post-dose. Within 1.5 h after intravenous administration, all the nine metabolites were formed and reached their peak plasma concentrations. After oral administration, plasma concentrations for M3 at different time points were all below the LLOQ (0.500 ng/mL), thus, no plasma concentration-time curve and pharmacokinetic parameters for M3 were obtained in this study. Plasma concentrations for M2 after 12 h were all below the LLOQ (0.500 ng/mL), thus, the plasma concentration-time curve for M2 was displayed from 0 to 12 h. Following oral administration of berberine at the dose of 48.2, 120, and 240 mg/kg, the AUCThe LC-MS/MS method was also applied for the excretion study of berberine and its nine metabolites following a single oral administration of berberine (48.2 mg/kg) to rats. Detailed excretion data of berberine and its nine metabolites was showed in The mean urinary and biliary excretion accumulative amount-time profiles and excretion rate-time profiles of berberine and its nine metabolites are showed in m/z 322.1) vs berberrubine , thalifendine-10-O-β-D-glucuronide  vs berberrubine-9-O-β-D-glucuronide , jatrorrhizine  vs columbamine (m/z 338.2) et al. Under our chromatographic condition, thalifendine and berberrubine (M1) could be separated  was used for intravenous administration. The absolute bioavailability of berberine was found to be 0.37 ± 0.11%, which was consistent with the reported results . There w0–48 h value of berberrubine (M1) was much higher than those of demethyleneberberine (M2) and jatrorrhizine (M3), indicating that berberrubine was the main phase I metabolite. Additionally, the glucuronide and sulfate conjugations of M2 and M3, could be detected with Tmax less than 4.75 h, indicating that M2 and M3 could be metabolized into phase II conjugations rapidly. However, Tmax for M7 was found to be 11.00–13.00 h, suggesting that the glucuronidation of berberrubine (M1) was processed gradually. This might be another reason that berberrubine was detected with higher AUC0–48 h. According to the AUC0–48 h values of both phase I and phase II metabolites, the metabolic levels of phase II metabolites were much higher than those of phase I metabolites, indicating that phase II metabolic pathways were the major metabolic routes. Compared with sulfation metabolites (M5 and M8), glucuronidation metabolites (M4 and M9) showed much higher values of AUC0–48 h and Cmax, suggesting that glucuronidation was the predominant metabolic pathway for berberine following oral administration.After oral administration, the AUC0–48 h) with the order from large to small was M8 > M9 > M6 > M4 > M7 > M1 > M2 > M5 > M3 when berberine was given i.v. When berberine was given i.g., the total exposure amount (AUC0–48 h) with the order from large to small was M7 > M9 > M6 > M4 > M8 > M1 > M2 > M5. These results indicated that the metabolic extent of berberine transformed to metabolites, especially glucuronidation metabolites  after oral dose was more than that after intravenous dose. Thus, in addition to metabolism in liver, the intestinal flora and microsomes also play vital roles in the metabolism of berberine. As for M7, two maximum concentrations were observed at 0.25 and 12 h, respectively. The appearance of a secondary plasma peak may be due to an enterohepatic circulation, namely, M7, which was produced in liver via the glucuronidation of berberrubine was secreted into bile and subsequently released into the small intestine, where M7 was further hydrolyzed to berberrubine by enterobacteria and reabsorbed back into circulation and subsequently glucuronidated to produce M7 in the liver. Even though the double-absorption peak phenomenon for other phase II metabolites was not so significant, it was still observed, especially when berberine was orally administrated at the dose of 120 mg/kg. The enterohepatic circulation of berberine metabolites was also reported by Zou et al.  were 2.62 and 0.356%, respectively, and the sum of these two accumulative excretions (2.976%) was much lower than the fecal excretion of berberrubine (18.6%). These results indicated that most of the berberrubine excreted in feces may be derived from the unabsorbed berberine via demethylation metabolism. Until now, whether gastrointestinal tract could participate in the metabolism of berberine is still controversial. Some results suggested that berberine was stable in gastrointestinal tract . The accumulative biliary excretions of berberrubine (M1) and berberrubine-9-al tract , howeveral tract . The exci.g. and i.v. administration were reported for the first time. The absolute bioavailability of berberine was very low due to insufficient absorption and extensive metabolism. Berberine, M1, M6, M7, M8, and M9 showed linear dynamics in dose range of 48.2–240 mg/kg. Phase II metabolites were the major circulating components in plasma. The total excretion of berberine and its metabolites in urine, bile and feces was 41.2% within 84 h. Berberine was mainly excreted in the form of berberrubine and berberrubine-9-O-β-D-glucuronide via fecal and renal excretion route, respectively. The present disposition studies of berberine in rats will be beneficial for both better understanding the clinical effects and further development of berberine. Meanwhile, due to the inter-species differences, further studies are still needed to extrapolate the pharmacokinetic findings to human.In summary, we present a comprehensive pharmacokinetics and excretion study of berberine and its nine metabolites in rats. To our knowledge, the pharmacokinetics and excretion results of berberine’s six phase II metabolites in rats after The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine.XF and KW conducted the experiments, preformed the data analysis and wrote the manuscript. SC and LD preformed the data analysis. FQ designed and supervised this research.The project was supported by the Technology Major Project of China “Key New Drug Creation and Manufacturing Program” (2017ZX09301012-001 and 2017ZX09301005), Major State Basic Research Development Program of China (2014CB560706), and National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway.4-dicarboxylate transporters and then strengthening the capacity of fumarate export, leading to fumarate production up to 18.9 g/L. Finally, the engineered strain E. coli W3110△4-P(H)CAI(H)SC produced 22.4 g/L fumarate in a 5-L fed-batch bioreactor.Here, pathway engineering was used to construct the noncyclic glyoxylate pathway for fumarate production. To improve the transmission efficiency of intermediate metabolites, pathway optimization was conducted by fluctuating gene expression levels to identify potential bottlenecks and then remove them, resulting in a large increase in fumarate production from 8.7 to 16.2 g/L. To further enhance its transmission efficiency of targeted metabolites, transporter engineering was used by screening the CIn this study, we offered rational metabolic engineering and flux optimization strategies for efficient production of fumarate. These strategies have great potential in developing efficient microbial cell factories for production of high-value added chemicals. Based on this oxidative TCA cycle, Escherichia coli CWF812 was able to produce 28.2 g/L with its productivity 0.448 g/L/h by deleting the iclR, fumABC, arcA, and ptsG genes and overexpressing the native ppc gene [Fumarate is a key intermediate in the tricarboxylic acid cycle (TCA) to link carbon and nitrogen metabolism, which has a variety of applications in many fields, such as food, pharmaceutical, bioplastic, and chemical industries . Recentlppc gene . As for ppc gene , only feTwo of the challenges in metabolically engineering the noncyclic glyoxylate cycle for fumarate production are how to identify and remove its potential bottlenecks and how to engineer and improve its transmission efficiency. Both challenges may benefit from the development of systems biology and synthetic biology. To identify and remove potential bottlenecks, several strategies have been developed, such as dynamic pathway analysis , X-omic Escherichia coli is a well-established model microbe for industrial application, and it possesses many advantages. Many strategies in metabolic engineering and synthetic biology can be efficiently applied for genetic manipulation in E. coli. In addition, simple salt medium or cheap medium can be used for cell growth and the biosynthesis of high-value chemicals. Moreover, E. coli is particularly suitable for the production of carboxylic acids, such as lactate [E. coli is an attractive candidate for microbial engineering of C4-dicarboxylic acids production, due to the fact that it can provide a large amount of precursors (pyruvate or α-ketoglutarate) for the biosynthesis of C4-dicarboxylic acids such as fumarate [E. coli has a great potential in engineering the transmission efficiency of synthetic pathway to achieve high-level production of fumarate. lactate , pyruvat lactate , and α-k lactate . Thus, Efumarate , succinafumarate , and malfumarate . Based oEscherichia coli W3110 was used as a host strain to rewire the noncyclic glyoxylate pathway for fumarate production CAI(H)SC, produced 22.4 g/L fumarate.In this study, Escherichia coli W3110△4 was constructed to produce pyruvate, L-malate, and α-ketoglutarate by deleting many genes including ldhA, pflB, poxB, pta, ackA, frdBC, and fumABC  [EcCS) [EcACN) [EcICL) [EcSDH) [E. coli W3110△4  , citrate) [EcCS) , aconita [EcACN) , isocitr [EcICL) , and suc [EcSDH)  were sel0△4 Fig. a. By oveely Fig. c. In addted Fig. d. Based 4-0 Fig. b. In add4-0 Fig. b. These To identify the potential bottlenecks in the noncyclic glyoxylate pathway, the expression level of individual enzymes was varied at different levels, while the remaining enzymes were all maintained at a fixed level. Based on this, we analyzed the effect of expression levels of every pathway enzyme on fumarate production, and the potential bottlenecks possibly showed a large variation range in fumarate production.AfPYC, EcCS, EcACN, EcICL, and EcSDH were introduced into E. coli W3110△4 to analyze its effects on fumarate production. When AfPYC expression was changed from low to high level, fumarate production was increased from 6.1 g/L to 12.5 g/L with variation range 103.6%  with RBS10, medium level (M) with RBS09, and low level (L) with RBS03 . Then, mAfPYC and EcICL expression were optimized in E. coli W3110△4-PCAIS at three levels  CAI(H)S still accumulated 3.5 g/L pyruvate and 4.1 g/L α-ketoglutarate CAI(H)S. The concentration of intracellular fumarate in strain E. coli W3110△4-P(H)CAI(H)S was increased by 128.6% compared with that of E. coli W3110△4-0 CAI(H)S showed a 21.2% increase in DCW compared with that of strain E. coli W3110△4-PCAIS  was obtained with strain E. coli W3110△4-P(H)CAI(H)SC by overexpressing dcuC in strain E. coli W3110△4-P(H)CAI(H)S CAI(H)S CAI(H)SC were reduced by 17.5% and 43.8% compared to that of strain E. coli W3110△4-P(H)CAI(H)S, which was similar to that of the control strain E. coli W3110△4-0, respectively CAI(H)S, respectively CAI(H)SC was tested in a 5-L fed-batch bioreactor. In this process, fumarate was accumulated gradually, and the maximal concentration of fumarate was up to 22.4 g/L at 60 h, which was 18.6% higher than that in shake flasks CAI(H)SC has great potential for efficient production of fumarate in fermentation.Fumarate production with the optimized strain 4-dicarboxylate transporter [E. coli E2 by combining the oxidative TCA cycle and the glyoxylate cycle partially, fumarate production showed a large increase up to 41.5 g/L [E. coli W3110△4-P(H)CAI(H)SC in our study was lower than that of E. coli EF02 (pSCppc) in previous study, we offered rational metabolic engineering and flux optimization strategies for efficient production of fumarate. These strategies have great potential in developing efficient microbial cell factories for production of high-value added chemicals.Metabolic engineering is conducted to rewire the complete noncyclic glyoxylate pathway for fumarate production. Recently, five metabolic engineering strategies have been developed to enhance production of fumarate: reconstructing synthetic pathway, such as the reductive TCA cycle , the oxinsporter . These rE. coli CWF4NS (pSynPC39) produced 25.5 g/L fumarate with its productivity of 0.35 g/L/h. These results showed that fumarate production could be improved by engineering the partial noncyclic glyoxylate pathway. However, fumarate production might be further increased by constructing and optimizing the complete noncyclic glyoxylate pathway to reinforce its transmission efficiency. In our study, pathway optimization was successfully applied to identify and remove the potential bottlenecks in the complete noncyclic glyoxylate pathway, and thus improve its transmission efficiency. The optimized strain E. coli W3110△4-P(H)CAI(H)SC was able to produce 22.4 g/L fumarate with its productivity of 0.37 g/L/h. These results indicated that pathway optimization could significantly increase the transmission efficiency of intermediate metabolites. This strategy not only could improve the transmission efficiency of intermediate metabolites, but also could reduce the loss of carbon flux. The increased transmission efficiency of biosynthetic pathway for fumarate production is possibly due to the balanced substrate channeling: (i) the local concentration of intermediates can appropriately meet the need of pathway enzymes by fine-tuning gene expression to balance biosynthetic pathway; (ii) the carbon flux of intermediates is efficiently concentrated on producing fumarate by preventing the competing pathways to reduce carbon loss; (iii) the feedback inhibition of intermediates is successfully circumvented by converting these inhibitors into another valid intermediate rapidly.Pathway optimization represents one significant step in identifying and removing the potential bottlenecks to improve the transmission efficiency of biosynthetic pathway. To improve the transmission efficiency of intermediate metabolites, the partial noncyclic glyoxylate pathway was constructed by replacing the native PEP-dependent PTSG system with the PEP-independent galactose translocation system, overexpressing phosphoenolpyruvate carboxylase (PPC) and acetyl-CoA synthase, and deleting malate dehydrogenase, fumarate reductase, and fumarase . The finE. coli ABCDIA [E. coli A-dcuB-Ec only produced 9.42 g/L fumarate with its productivity 0.19 g/L/h [E. coli strain harboring the complete noncyclic glyoxylate pathway for fumarate production. Based on this, there is enough intracellular fumarate to be transported outside the cell by dcuBC, and the productivity of fumarate was increased to 0.37 g/L/h. These results indicated that transporter engineering could largely increase the transmission efficiency of targeted metabolites. This strategy not only could prevent fumarate accumulation in the intracellular space, but also could alleviate the toxicity of intracellular fumarate. The improved transmission efficiency of biosynthetic pathway for fumarate production is also possibly due to the fact that dcuBC are efficient for exporting fumarate to prevent self-poisoning and reduce feedback inhibition, thus ultimately realizing maximum production of fumarate.Transporter engineering is another useful step in enhancing the transmission efficiency of biosynthetic pathway. Transporters can be rationally regulated to recognize and transport targeted metabolites between inside and outside the cell, which has shown great promise in reducing toxicity and increasing productivity . Recent i ABCDIA , in whici ABCDIA . The res19 g/L/h , possibl4-dicarboxylate transporters. Based on these strategies, fumarate production with strain E. coli W3110△4-P(H)CAI(H)SC was increased from 3.2 to 22.4 g/L. These strategies have great application potential in developing efficient microbial cell factories for production of high-value added chemicals.In this study, the noncyclic glyoxylate pathway was successfully constructed for fumarate synthesis. The transmission efficiency of intermediate metabolites was enhanced by optimizing the expression levels of pathway enzymes. Further, the transmission efficiency of targeted metabolites was improved by strengthening the CEscherichia coli W3110△ldhA△poxB△pflB△pta-ackA△frdBC△fumB△fumAC (E. coli W3110△4) was applied as host strain for overexpressing key genes [E. coli strains for fumarate production were all derived from E. coli W3110△4. E. coli JM109 and plasmid pETM6R1 [ey genes . The eng pETM6R1  were useAspergillus flavus  was amplified by PCR using plasmids pTrcHisA-AfPYC as template [EcCS, b0720), aconitase  and isocitrate lyase  genes from E. coli were amplified from plasmids pET28a-EcCS, pET28a-EcACN, and pET28a-EcICL, respectively [E. coli W3110 [Gibson Assembly was applied for constructing key plasmids according to the protocol of Gibson Assembly Cloning Kit (NEB), respectively. Pyruvate carboxylase gene from template . Citrateectively , the C4-li W3110 .LB medium used for seed culture: 5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl. Ampicillin (100 mg/mL) was added to LB medium appropriately when needed.4Cl, 1 g/L citrate, 3 g/L NaHCO3, 7.52 g/L Na2HPO4-2H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, 0.246 g/L MgSO4, 0.044 g/L CaCl2, 1 μg/L biotin, 1 μg/L thiamin, and 1 mL trace element solution . Ampicillin (100 mg/mL) and IPTG (0.4 mmol/L) were added appropriately when needed.Modified M9 minimal medium used for fermentation: 50 g/L glucose, 10 g/L yeast extract, 0.5 g/L NH600 = 0.5. This cell culture was buffered by 30 g/L CaCO3, and fermented at 37 °C for 60 h with rotation at 200 rpm. 50 g/L glucose was fed at 24 h.The seed culture was cultivated at 37 °C for 12 h with rotation at 200 rpm in a 250-mL flask containing 25 mL LB medium. After that, the broth was centrifuged to discard supernatant liquid, and then fresh M9 medium was used to suspend the pellet. Next, the cell suspension was spread equally across 500-mL flasks with 50 mL fresh M9 medium with an initial biomass OD600 = 0.5 at 37 °C for 60 h. Agitation speed and aeration rate were controlled at 200 rpm and 1.0 vvm, respectively. Culture pH was controlled at 7.0 using 20% (w/v) Na2CO3. 50 g/L glucose was fed at 24 h.Fermentation was conducted in a 5-L fed-batch bioreactor containing 2.5 L M9 medium with an initial biomass OD600) was assayed by a spectrophotometer (1OD600 = 0.33 g/L DCW (Dry Cell Weight)). Glucose concentration was quantified by a biosensor SBA-90 [The optical density at 600 nm (ODr SBA-90 . The conr SBA-90 .Intracellular metabolites were extracted by freezing-thawing in methanol . The intidnT) was used as reference gene.Total RNA was extracted by the RNAprep pure Kit (TIANGEN), and reverse transcription was conducted for cDNA synthesis as described in the protocol of Reverse Transcription Kit (Takara). Real-time quantitative PCR was carried out according to . L-IdonaPyruvate carboxylase (PYC) was assayed as reported by . CitrateAdditional file 1: Table S1. Strains and plasmids used in this study.


Bamboo, a lignocellulosic feedstock, is considered as a potentially excellent raw material and evaluated for lignocellulose degradation and bioethanol production, with a focus on using physical and chemical pre-treatment. However, studies reporting the biodegradation of bamboo lignocellulose using microbes such as bacteria and fungi are scarce.Bacillus velezensis LC1 was isolated from Cyrtotrachelus buqueti, in which the symbiotic bacteria exhibited lignocellulose degradation ability and cellulase activities. We performed genome sequencing of B. velezensis LC1, which has a 3929,782-bp ring chromosome and 46.5% GC content. The total gene length was 3,502,596 bp using gene prediction, and the GC contents were 47.29% and 40.04% in the gene and intergene regions, respectively. The genome contains 4018 coding DNA sequences, and all have been assigned predicted functions. Carbohydrate-active enzyme annotation identified 136 genes annotated to CAZy families, including GH, GTs, CEs, PLs, AAs and CBMs. Genes involved in lignocellulose degradation were identified. After a 6-day treatment, the bamboo shoot cellulose degradation efficiency reached 39.32%, and the hydrolysate was subjected to ethanol fermentation with Saccharomyces cerevisiae and Escherichia coli KO11, yielding 7.2 g/L of ethanol at 96 h.In the present study, B. velezensis strains in converting lignocellulose into ethanol. B. velezensis LC1, a symbiotic bacteria, can potentially degrade bamboo lignocellulose components and further transformation to ethanol, and expand the bamboo lignocellulosic bioethanol production.These findings provide an insight for  Lignocellulose, a widely distributed, renewable and enormous biomass resource, is one of the most important raw materials for bioethanol production . Bamboo,Lignocellulose hydrolysis, especially cellulose degradation, remains a considerable challenge in lignocellulosic bioethanol production . In natuBacillus is an excellent degrader that exhibits various abilities for degrading lignocellulose biomass, including cellulose, hemicellulose and lignin [Bacillus is still unclear [B. oryzicola and B. methylotrophicus, were classified into the B. velezensis group. Recently, the complete genome and genes associated with lignocellulose degradation of several B. velezensis strains were sequenced and are enriched in the genome [Microbial degradation of lignocellulose is a green biological refining method with advantages over physical and chemical methods . The bacd lignin , 11. Fur unclear . Dunlap  unclear  reportede genome –16. HoweCyrtotrachelus buqueti that showed a bamboo lignocellulose-degrading ability [B. velezensis LC1, determined the cellulase activities and analysed the ethanol production of bamboo shoot. CAZy genes involved in degradation of lignocellulose were identified through genomic analysis. The chemical changes of the cell wall components were investigated, as well as the hydrolytic and ethanol-fermenting properties of bamboo shoots.In the present study, we isolated an endophytic bacteria from the gut of  ability  and sequC. buqueti on CMC agar. Among the five strains, PX12 exhibited the highest cellulose hydrolysis capacity, with a higher hydrolysis capacity ratio (HCR: 4.71) than PX9 (HCR: 2.41), PX10 (HCR: 1.92), PX11 (HCR: 2.12) or PX13 (HCR: 2.56), as determined using the cellulose hydrolysis assay    method for 6 days , COG0524 (pfkb domain protein), COG2814 (Major facilitator), COG0726 , COG1263 (PTS System), COG0477 (major facilitator superfamily), COG1455 (pts system), COG1940 (ROK family) and COG2723 (beta-glucosidase) , followed by biological process (Gene number: 3025) and cellular component (Gene number: 1637) , GO:0005975 (carbohydrate metabolic processes) and GO:0016787 (hydrolase activity) , glycolysis/gluconeogenesis (ko00010), and amino and nucleotide sugar metabolism (ko00520). Forty-one genes were related to ko00500, and common enzyme endoglucanase (EC.3.2.1.4), present in ko00500, was involved in cellulose degradation  and 1 laccase were been identified , which play key roles in carbohydrates degradation, contained 44 members [The bCAN carbohydrate-active enzymes (CAZy) annotation algorithm was used to analyse CAZy annotations to identify genes involved in lignocellulose degradation. The results showed that 136 genes were identified from CAZy families and distributed into five subfamilies. In  members . AdditioBacillus that have cellulose degradation function usually belonged to the GH5 families [d-galactosidic linkages, was reported as an endo-1,4-β-galactosidase [The GH family contained various hydrolases that acted on the glycosidic bond. For example, endoglucanases EC 3.2.1.4) from families   usually has (1 → 4)-α- the end   investigation results of genes encoding endoglucanase (gene1950), beta-glucanase (gene3931), 6-phospho-beta-galactosidase (gene1280), glucan endo-1,6-β-glucosidase (gene2084), alpha-glucosidase (gene2945), acetyl xylan esterase (gene0351), xylan 1,4-beta-xylosidase (gene1870), arabinoxylan arabinofuranohydrolase (gene1955), alpha-N-arabinofuranosidase (gene2785), and arabinan endo-1,5-alpha-l-arabinosidase (gene2795) are shown in Fig. P < 0.05) in BSP cultures compared to glucose cultures. It indicated that these genes involved in BSP degradation by B. velezensis LC1.Expression profiles of selected e gene208, alpha-gB. velezensis strains contained genes encoding enzymes having lignocellulose-degrading potential [B. velezensis [B. velezensis LC1, isolated from the intestine of C. buqueti, which was reported to be a microflora with lignocellulose-degrading ability, was prepared in a bamboo shoot powder (BSP) degradation assay [B. velezensis LC1.Several previously reported genomes of otential –16. Howelezensis . B. veleon assay . We firsB. velezensis LC1, a 6-day-treated hydrolysate was prepared for ethanol fermentation using glucose-fermenting S. cerevisiae and xylose-fermenting E. coli KO11. Ethanol production from BSP hydrolysate using B. velezensis LC1 is illustrated in Fig. B. velezensis LC1 had a potent ability to convert lignocelluloses in bamboo shoot to ethanol.Shimokawa et al.  reportedB. velezensis LC1, was sequenced and analysed. The genome comprises a ring chromosome of 3,929,782 bp and has a GC content of 46.5%. A total of 136 CAZyme genes involved in lignocellulose degradation were annotated in the genome, and cellulose-degrading and ethanol-producing pathways were proposed. Moreover, the expression level of some CAZyme genes involved in cellulose and hemicellulose degradation, were up-regulated in bamboo shoot powder substrate. Moreover, a transcriptomic study would perform to identify the role of CAZyme genes in lignocellulose degradation as a future study. The cellulose degradation of bamboo shoot by the strain was 39.32%, and the hydrolysate was subjected to ethanol fermentation; the ethanol yield was 7.2 g/L at 96 h. This study suggests that B. velezensis LC1 could be used for bamboo lignocellulose degradation and bioconversion of lignocelluloses to ethanol.The genome of a gut symbiotic bacteria, Cyrtotrachelus buqueti specimens were sampled from the Muchuan County , China. Gut was extracted from individual insects and stored at 4 °C for isolation of bacteria. The gut was blended, homogenized and serially diluted (10−1 to 10−9), and inoculums of 10−7 to 10−9 dilution were plated on carboxymethyl cellulose (CMC) agar [MC) agar  for cellMC) agar . Hydroly16sRNA V3–V4 region was considered for amplification for bacterial identification and amplified using bacterial primers (27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGYTACCTTGTACGACTT-3′); moreover, F 5′-GCCCATATTTCCATTTCTCC-3′ and R 5′-GTGGTCGTTATGGAAATAAAGG-3′ were selected for amplification of the house-keeping gene rpoB. Thermocycling conditions were as follows: initial denaturation at 94 °C (2 min), followed by 30 cycles of denaturation at 94 °C (30 s), annealing at 55 °C (30 s) and extension at 72 °C (100 s), ultimately extending at 72 °C (2 min). The amplicons were checked by electrophoresis on a 1% agarose gel. MEGA5 was used to establish phylogenetic relationships among the obtained sequence and reference genes that were retrieved in NCBI GenBank through the neighbour-joining method.The B. velezensis LC1 was extracted according to the CTAB method based on the protocol described by Lin [Genomic DNA of d by Lin , and it d by Lin . HGAP 2.http://www.cazy.org/).Glimmer 3.02 was used to predict coding DNA sequences (CDSs). A BLAST search was then conducted for CDSs in some widely used databases: NCBI non-redundant (NR) database, Swiss-Prot, COGs, Gene Ontology (GO) and KEGGs , 49. GO,− ΔΔCT Method. 16S rRNA was used as reference gene.The primers used for qRT-PCR in this study were performed in Additional file B. velezensis LC1 was used to degrade the bamboo shoot powder (BSP), which was prepared as described by Luo et al. [B. velezensis LC1 was cultured in liquid medium at a pH of 7.2, temperature of 37 °C and 200 rpm for 6 days. The culture medium comprised BSP 10 g/L, (NH4)2SO4 2 g/L, K2HPO4 1 g/L, KH2PO4 1 g/L, MgSO4 0.2 g/L, CaCl2 0.1 g/L, FeSO4·7H2O 0.05 g/L and MnSO4·H2O 0.02 g/L. The reaction mixture was incubated at 100 °C for 30 min to terminate the reaction and centrifuged at 13,000 rpm for 10 min, and the hydrolysate-containing supernatant and deposit were collected separately. The obtained deposit was dried and weighed to determine the cellulose levels using the Van Soest method [To obtain fermentable sugar from bamboo shoots, o et al. . B. velet method . The hydt method . The BSPSaccharomyces cerevisiae, a glucose-fermenting strain, was pre-cultured in YPD at 30 °C for 24 h, and E. coli KO11, a xylose-fermenting strain, was cultured in LB at 37 °C for 24 h. S. cerevisiae (50 g/L) and E. coli KO11 (100 g/L) were prepared after centrifugation of pre-cultured cells. The initial cell concentrations were 0.33 g/L (S. cerevisiae) and 1.0 g/L (E. coli KO11) at the beginning of fermentation. 100 ml hydrolysate was used for ethanol fermentation in 250-ml serum bottles under anaerobic conditions. The fermentation was performed at 37 °C and 200 rpm for 96 h. After 48 h fermentation, ethanol production was monitored every 12 h. The ethanol concentration was determined via HPLC. All reactions were repeated three times.Additional file 1: Figure S1. Cellulolytic activities of 4 isolates, PX9, PX10, PX11, and PX13, cultured on the CMC agar plate with congo red.Additional file 2: Table S1. COG annotations of carbohydrate metabolism in genome of B. velezensis LC1.Additional file 3: Table S2. GO annotations of carbohydrate metabolism in genome of B. velezensis LC1Additional file 4: Table S3. KEGG Pathway classification of B. velezensis LC1.Additional file 5: Table S4. Annotation of dioxygenase, monooxygenase, peroxidase, laccase and oxidoreductase genes identified in the genome of B. velezensis LC1.Additional file 6: Table S5. CAZy annotation in the genome of B. velezensis LC1.Additional file 7: Table S6. Primers used for RT-qPCR analysis.


The pathological process of traumatic spinal cord injury (SCI) involves excessive activation of microglia leading to the overproduction of proinflammatory cytokines and causing neuronal injury. Sphingosine kinase 1 (Sphk1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P), plays an important role in mediating inflammation, cell proliferation, survival, and immunity.We aim to investigate the mechanism and pathway of the Sphk1-mediated neuroinflammatory response in a rodent model of SCI. Sixty Sprague-Dawley rats were randomly assigned to sham surgery, SCI, or PF543 (a specific Sphk1 inhibitor) groups. Functional outcomes included blinded hindlimb locomotor rating and inclined plane test.We discovered that Sphk1 is upregulated in injured spinal cord tissue of rats after SCI and is associated with production of S1P and subsequent NF-κB p65 activation. PF543 attenuated p65 activation, reduced inflammatory response, and relieved neuronal damage, leading to improved functional recovery. Western blot analysis confirmed that expression of S1P receptor 3 (S1PR3) and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) are activated in microglia of SCI rats and mitigated by PF543. In vitro, we demonstrated that Bay11-7085 suppressed NF-κB p65 and inhibited amplification of the inflammation cascade by S1P, reducing the release of proinflammatory TNF-α. We further confirmed that phosphorylation of p38 MAPK and activation of NF-κB p65 is inhibited by PF543 and CAY10444. p38 MAPK phosphorylation and NF-κB p65 activation were enhanced by exogenous S1P and inhibited by the specific inhibitor SB204580, ultimately indicating that the S1P/S1PR3/p38 MAPK pathway contributes to the NF-κB p65 inflammatory response.Our results demonstrate a critical role of Sphk1 in the post-traumatic SCI inflammatory cascade and present the Sphk1/S1P/S1PR3 axis as a potential target for therapeutic intervention to control neuroinflammation, relieve neuronal damage, and improve functional outcomes in SCI. Spinal cord injury (SCI) affects approximately 282,000 people in the USA, with 17,000 new cases each year . SecondaThough knowledge of the complex pathological cascades that contribute to secondary tissue damage of the CNS grows, the inflammatory response primarily mediated by macrophages in the neurological recovery process remains controversial , 5. MicrSphingosine kinases (Sphks) are the rate-limiting catalyzing enzymes that convert sphingosine into sphingosine-1-phosphate (S1P), an important lipid signal conductor known to regulate physiological processes such as cell growth, death, senescence, adhesion, migration, inflammation, and angiogenesis –12. PrevThe Sphk1/S1P/S1PR axis has been a proposed therapeutic target for cancer and inflammatory diseases . HoweverThis study identifies the role of the Sphk1/S1P mechanism and pathway in SCI. We found that microglia were activated and predominantly polarized to the M1 phenotype in the injured spinal cord tissue of rats in vivo and under LPS stimulation in vitro. In addition, we demonstrated that Sphk1 expression is upregulated in activated microglia and that inhibition of Sphk1 attenuates the inflammatory response and relieves neuronal damage facilitated by M1 microglia, ultimately reducing the downstream activation of p38 MAPK and NF-κB. Functional outcomes after traumatic SCI improved with targeted inhibition of the Sphk1/S1P pathway. This study demonstrates that the Sphk1/S1P/S1PR3 axis participates in a specific pathway that regulates neuroinflammation in traumatic SCI and opens the door to a therapeutic target for mitigating neuronal injury and improving neurological outcome in a disease process for which there are currently no targeted pharmacologic interventions.Cell counting kit-8 (CCK-8) was obtained from Dojindo . TNF-α ELISA Kit was purchased from R&D Systems . BAY11-7085 and SB203580 were purchased from Selleck Chemicals . LPS, PF543 hydrochloride, and S1P  were purchased from Sigma-Aldrich . CAY10444 was purchased from Cayman Chemical . Recombinant Human β-NGF was purchased from Peprotech (USA). Monoclonal antibodies specific for cleaved caspase-3, COX-2, phospho-p38 MAPK, phospho-NF-κB p65, NF-κB p65, and IκB were procured from Cell Signaling Technologies . Polyclonal antibodies specific for Iba-1, NeuN, iNOS, IL-6, TNF-α, Bax, and p38 MAPK and a monoclonal antibody specific for GAPDH were purchased from Proteintech . Polyclonal antibodies specific for Bcl-2 and Sphk1 and a monoclonal antibody specific for S1PR3 were purchased from Abcam . AlexaFluor 568, AlexaFluor 488 donkey anti-rabbit/mouse, and horseradish peroxidase-labeled secondary antibodies were purchased from Abcam. 4′, 6-Diamidino-2-phenylindole (DAPI) was obtained from Yeasen Biotech . For the in vitro studies, PF543 hydrochloride was dissolved in dimethyl sulfoxide (DMSO) and was diluted appropriately with cell culture medium . Bay 11-7085 (inhibitor of NF-κB) was dissolved in DMSO and was diluted into the concentration of 5 μM. SB203580 (inhibitor of P38 MAPK) was dissolved in DMSO and was diluted into the concentration of 10 μM. CAY10444 (inhibitor of S1P3) was dissolved in DMSO  and was n = 19), SCI (n = 25), and PF543 group (n = 16). All rats were housed under controlled environmental conditions. The care and use protocols of all animals were conducted according to the guidelines set forth by the Chinese National Institutes of Health. To induce SCI, the rats were anesthetized with 4% chloral hydrate (3 ml/kg) via intraperitoneal injection. Then, we performed a laminectomy at the T9 vertebra. After fully exposing the spinal cord, a vascular clip  was used to induce moderate crush injury for 1 min, as we previously published [A total of 60 adult female Sprague-Dawley rats (200–250 g) were acquired from the Charles River Laboratory Animal Company in Shanghai, China, and were randomly divided into three groups: Sham  locomotion rating scale  and the Rats were sacrificed with 8% chloral hydrate  at specific time-points after SCI and then perfused with normal saline. For the H&E staining, Nissl staining and immunofluorescence analysis, tissue segments containing the spinal cord lesion (1 cm on each side of the lesion) and spinal cord sections (0.5-cm) from each specimen were dissected out, post-fixed with 4% paraformaldehyde for 6 h, and then embedded in paraffin. Transverse sections (5 μm thick) were mounted on slides following staining. Histopathological examinations were performed by H&E and Nissl staining, according to the manufacturer’s instructions. Bright-field images were acquired using light microscopy. The transverse sections were treated with the following primary antibodies: anti-Sphk1 (1:100), anti-S1PR3 (1:100), anti-Iba-1 (1:200), anti-NeuN (1:100), and anti-cleaved caspase 3 (1:400) before being washed four times with PBS and incubated with AlexaFluor 568 and AlexaFluor 488 donkey anti-rabbit/mouse secondary antibodies for 1 h at 37 °C. Sections were then washed with PBS, incubated with DAPI for 1 min, rinsed with PBS, and finally sealed with a coverslip. All images were captured on a fluorescence microscope.2 and were passaged every 3 days.The murine HAPI microglial cell line was purchased from Otwo Biotech . The PC12 cell line was purchased from the Shanghai Institute of Cell Biology and has been differentiated in vitro by 5% horse serum and NGF (50 ng/ml) to acquire neuronal features, which were confirmed via a microscope. HAPI cells and PC12 cells cultured in DMEM medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 μg/ml streptomycin, were incubated at 37 °C in a humidified atmosphere containing 5% COTo establish the HAPI inflammation model, we added LPS (1 μg/ml) to the HAPI culture medium for 24 h. To study the mechanism of the Sphk1-mediated inflammatory response in HAPI cells, we pretreated the cells with different doses of PF543 for 12 h before treating them with LPS (1 μg/ml) alone or LPS with S1P (1 μM). We also pretreated the cells with 5 μM BAY 11-7085 (an inhibitor of NF-κB activation), 10 μM CAY10444 (a selective antagonist of S1P binding to S1PR3), and 10 μM SB203580 (an inhibitor of p38 MAPK activation) for 2 h before treating them with different stimuli. All experiments were performed in triplicate.5 HAPI cells/insert and 5 × 105 PC12 cells/well) transwell plates for the coculture experiment. The HAPI cells were pretreated with or without 100 nM PF543 for 12 h before stimulation with 1 μg/ml LPS for another 24 h. After stimulation, the HAPI cell inserts were rinsed with PBS to eliminate the effects of any remaining LPS and PF543 before being placed into the PC12 wells. After 24 h of coculture, we measured neuronal apoptosis by TUNEL assay and Western blot.To mimic the environment and investigate the relationship between activated microglia and neuronal survival, we utilized a transwell coculture system  . HAPI an2 for 24 h. Then, different doses of PF543  were added. After treatment, the cells were washed with phosphate-buffered saline (PBS), 100 μl of DMEM containing 10 μl of CCK-8 solution was added to each well, and the plate was incubated for an additional 2 h. The absorbance of the wells was measured at 450 nm using a microplate reader .Cell viability was assessed with a cell counting kit-8 assay according to the manufacturer’s protocol. Briefly, HAPI cells were seeded in 96-well plates (5000 cells/well) and cultured in DMEM with 10% FBS at 37 °C and 5% COThe inflammatory cytokines tumor necrosis factor-α (TNF-α) in the cell culture supernatants were measured using commercial ELISA Kits following the manufacturer’s instructions.For immunofluorescence analysis, the samples were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% (v/v) Triton X-100 for 10 min, and then blocked with 1% (w/v) goat serum albumin for 1 h at 37 °C. The slides were incubated at 4 °C overnight with primary antibodies against Sphk1 (1:100), COX-2 (1:400), TNF-α (1:400), NF-κB p65 (1:200), and phospho-p38 (1:1600). The following day, the slices were washed three times with PBS and then incubated with AlexaFluor 568 and AlexaFluor 488 donkey anti-rabbit/mouse secondary antibodies for 1 h at 37 °C before being labeled with DAPI for 1 min at room temperature. Finally, three fields of view per slide were randomly selected for observation under a fluorescence microscope, and staining intensities were measured by observers who were blinded to the experimental groups using ImageJ software  .The TUNEL method is useful for measuring apoptotic DNA fragmentation. PC12 cells were seeded on slides in a 24-well plate and allowed to attach for 24 h. Then, HAPI cells were pretreated with or without 0.1 μM PF543 for 12 h before being treated with LPS and placed in the wells containing the PC12 cells for 24 h. Next, the PC12 cells were washed gently with PBS twice and fixed with freshly prepared 4% paraformaldehyde for 30 min before being incubated with 0.1% Triton X-100 for 5 min. The cells were washed with PBS three times after each step. The cells were then stained using the in situ cell death detection kit, fluorescein , and DAPI, according to the manufacturer’s instructions. Apoptosis was measured using a fluorescence microscope .For in vivo protein analysis, a spinal cord segment at the lesion epicenter was dissected at 3 days and immediately stored at − 80 °C. Proteins isolated from animal tissue or PC12 cells were first quantified with BCA reagents. Then, cellular samples containing 30 μg of protein and tissue samples containing 60 μg of protein were separated by SDS-PAGE before being transferred to PVDF membranes. Western blot was performed using standard protocols as we previously described . MembranP < 0.05 and **P < 0.01 versus the indicated group.Numerical data from at least three individual experiments are presented as mean ± SEM. One-way analysis of variance (one-way ANOVA) followed by Tukey’s post hoc analysis or Kruskal–Wallis analysis (non-parametric ANOVA) plus Dunn’s multiple comparisons (when the data failed the assumptions of one-way ANOVA) were used to test differences between groups at specific times. Between-group differences in BBB scores and inclined plane test results were analyzed using repeated measurement two-way mixed ANOVA, followed by Bonferroni’s test. Statistical significance was established as *P = 0.002, SCI-12 h group: 1.51 ± 0.09 vs. SCI-24 h group: 2.38 ± 0.21, P = 0.030, SCI-24 h group: 2.38 ± 0.21 vs. SCI-48 h group: 3.89 ± 0.24, P = 0.001). To investigate the role of Sphk1 following SCI, we examined the effects of PF543, a highly selective and efficient inhibitor of Sphk1 leading to reduced intracellular S1P [P = 0.001 for iNOS, 1.00 ± 0.11 vs. 2.14 ± 0.10, P = 0.002 for COX-2, 1.00 ± 0.10 vs. 3.16 ± 0.28, P < 0.001 for TNF-α, 1.00 ± 0.12 vs. 2.02 ± 0.16, P = 0.004 for IL-6). Elevated expression of these markers was suppressed by PF543 administration .To verify post-SCI inflammation and the induction of Sphk1 in a rat model of SCI, spinal cord tissue was harvested after SCI for double-immunofluorescence assays. The results showed enhanced staining of Sphk1 over time in activated microglia . Moreover, analysis of expression of proteins by Western blot, demonstrated that the pro-apoptotic proteins Bax and cleaved caspase-3 significantly increased and pro-survival protein Bcl-2 decreased in SCI rats . Consistent with the post-SCI inflammatory changes, these findings were reversed and neural apoptosis was ameliorated in the PF543 treatment group .We performed double-immunofluorescence staining for cleaved caspase 3 and NeuN (a neuron marker), and the results showed that the staining intensity of cleaved caspase 3 in neurons increased in the injured spinal cord segment and that PF543 treatment weakened this effect , indicating that inhibition of Sphk1 improves motor functional recovery after SCI.To evaluate the extent of post-SCI locomotion recovery, we observed behavioral changes after injury using blinded BBB scores and the inclined plane test. Rats showed no observable movement of their hindlimbs immediately following SCI, indicating that a severe SCI model was established. Throughout the course of post-SCI recovery, BBB scores and angle of incline estimates showed that the PF543 treatment group experienced greater functional recovery at 14 days post-SCI , indicating the protective effects of PF543 in SCI. Moreover, we counted the number of ventral neurons at high magnification. As shown in Fig. P < 0.001 for number of rostral neurons, 33.60 ± 1.47 vs. 15.60 ± 2.18, P < 0.001 for number of caudal neurons; SCI group vs. PF543 group: 12.60 ± 2.18 vs. 22.20 ± 1.28, P = 0.008 for number of rostral neurons; 15.60 ± 2.18 vs. 26.20 ± 2.29, P = 0.008 for number of caudal neurons), SCI induced significant neuronal loss, and PF543 treatment attenuated this phenomenon, demonstrating that PF543 can reduce neuronal loss and improve pathological injury. These data indicate that inhibition of Sphk1 attenuates microglial M1 polarization and prevents neuronal apoptosis after SCI, which are, in turn, related to improved functional recovery.In addition, we evaluated the histological morphology changes of injured spinal cord tissue in each group 7 days after surgery using H&E and Nissl staining. The results showed that rats in the SCI group exhibited severely damaged central gray matter and dorsal white matter compared with the sham group. Compared with damage in the SCI group, the PF543 treatment group exhibited attenuated damage in similar locations . Accordingly, the expression of Sphk1 showed the same trend .In our in vitro experiment, we performed LPS stimulation to mimic the post-SCI inflammatory response in the HAPI microglial cell line. To verify the results, we analyzed the expression of specific M1 phenotypic proteins iNOS and COX-2 by Western blot. Both markers increased in a time-dependent manner after LPS stimulation in the corresponding group . These findings suggest that LPS treatment can induce microglial activation and M1 polarization, which is linked to proinflammatory cytokine production and induces nerve cell apoptosis. In addition, the inflammatory amplification cascade may be partly regulated by the induction of Sphk1 in activated microglia.Immunofluorescence of Sphk1 in LPS-stimulated microglia displayed staining intensity, which increased in a time-dependent manner . In the three groups for LPS and PF543 10 nM, PF543 100 nM groups, respectively, the iNOS rankings were 2.54 ± 0.11 > 2.01 ± 0.14 > 1.39 ± 0.06, LPS group vs. PF543 10 nM group, P = 0.028, PF543 10 nM group vs. PF543 100 nM group, P = 0.012; the COX-2 rankings were 3.14 ± 0.19 > 2.10 ± 0.19 > 1.36 ± 0.12, LPS group vs. PF543 10 nM group, P = 0.006, PF543 10 nM group vs. PF543 100 nM group, P = 0.036; the TNF-α rankings were 2.07 ± 0.05 > 1.67 ± 0.13 > 1.20 ± 0.10, PF543 10 nM group vs. PF543 100 nM group, P = 0.041, LPS group vs. PF543 100 nM group, P = 0.001; the IL-6 rankings were 1.93 ± 0.15 > 1.60 ± 0.12 > 1.13 ± 0.07, LPS group vs. PF543 100 nM group, P = 0.005).To study the role of Sphk1 in neuroinflammation in vitro, we used PF543 to suppress Sphk1 activity. PF543 is a validated inhibitor of Sphk1 . WesternP < 0.01. LPS group: 3.80 ± 0.29 vs. PF543 10 nM group: 2.69 ± 0.16, P = 0.003, PF543 10 nM group: 2.69 ± 0.16 vs. PF543 100 nM group: 2.01 ± 0.33, P = 0.048). To investigate potential PF543 cytotoxicity, we treated microglia with or without PF543  for 48 h and evaluated the outcome of treatment by CCK-8 assay. We confirmed that PF543 did not cause significant cytotoxicity at concentrations below 1 μM . These data indicate that blocking the activity of Sphk1 in microglia may attenuate M1 phenotype polarization and may safely be achieved with PF543.These results were consistent with our immunofluorescence assay, which showed gradually declining staining for COX-2 in treated microglia .To further investigate whether neuronal apoptosis is related to Sphk1-mediated neuroinflammation, we established a microglia/neuron coculture system as previously mentioned with specific treatments. TUNEL assay was then performed to detect neuronal apoptosis. Results showed that PC12 cells cocultured with LPS-activated microglia underwent apoptosis at a significantly increased rate compared with cells in the control group. Moreover, cells that were cocultured with PF543-pretreated microglia underwent apoptosis at a significantly decreased rate compared with cells cocultured with M1 microglia with no pretreatment . These results indicate that inhibition of Sphk1 in activated microglia can protect neurons against inflammation-facilitated apoptosis.In addition, Western blot analysis showed the reduced expression of the apoptotic protein Bax and increased expression of the protective protein Bcl-2 in neurons cocultured with PF543-treated microglia. Expression of the apoptotic protein cleaved caspase-3 was non-significantly reduced in the PF543 pretreated coculture .Previous studies have demonstrated that NF-κB p65 activation is a required component of the inflammatory response and is, in part, regulated by intracellular and extracellular lipid molecules , 32. To P < 0.01. LPS group vs. PF543 group: 4.36 ± 0.23 vs 2.37 ± 0.36 P = 0.021. PF543 group vs. S1P group: 2.37 ± 0.36 vs 4.51 ± 0.52 P = 0.014). These data suggest that Sphk1-mediated neuroinflammation is dependent on the production of S1P, which then activates NF-κB p65 nuclear translocation.Through confocal observation of immunofluorescence staining, we found that NF-κB p65 significantly translocated from the cytoplasm to the nucleus after LPS treatment for 6 h and that this translocation was blocked by PF-543 pretreatment. Conversely, addition of exogenous S1P enhanced NF-κB p65 translocation .To determine the relationship between S1P-induced NF-κB p65 activation and the inflammatory response, we used BAY11-7085, an inhibitor of NF-κB activation. Western blot assay for expression of phosphorylated-p65 (p-p65), p65, and IκB indicated that LPS enhanced p65 phosphorylation and IκB degradation. Adding exogenous S1P amplified NF-κB p65 activation, the effect of which was reversed by BAY11-7085 . ELISA assay also indicated that the secretion of TNF-α was significantly induced 24 h after LPS stimulation, and S1P treatment increased LPS-induced secretion of TNF-α, while BAY11-7085 antagonized its effects . These findings indicate that S1P-induced NF-κB p65 activation, which may result from extracellular regulation, may play a crucial role in the inflammatory amplification cascade.Using immunofluorescence analysis for TNF-α, we found that S1P facilitated the release of this proinflammatory mediator, and BAY11-7085 countered this effect . Double-immunofluorescence staining for S1PR3 and Iba-1 showed increased S1PR3 expression in the SCI group compared with the sham group, and staining intensity decreased in the PF543-treatment group .To elucidate the signaling pathways associated with Sphk1 in more detail, we hypothesized that both the activation of Sphk1 and subsequent S1P binding on surface receptors are required for the effects of downstream inflammatory molecules. In our in vivo study, Western blot analysis demonstrated that levels of S1PR3 expression and p38 MAPK phosphorylation were significantly increased in the SCI group compared with that of the sham group and were decreased in the PF543-treated group compared with that of the SCI group . The results indicated that the activation of p38 MAPK and NF-κB p65 was enhanced after LPS stimulation, PF543 pretreatment reduced phosphorylation of p38 MAPK and NF-κB p65 and reduced degradation of IκB when compared to no pretreatment. Moreover, the inhibition of p38 MAPK and NF-κB p65 were strengthened in the group treated with CAY10444 2 h prior to LPS. We also performed immunofluorescence analysis for p-p38 in microglia, and consistent results were observed .Next, our group explored whether NF-κB activation is associated with the S1P/S1PR3/p38 MAPK pathway in vitro. Microglia were pretreated with CAY10444, a selective antagonist of S1P binding to S1PR3, in addition to PF543 pretreatment and LPS treatment. Expression levels of p38, phospho-p38, p65, phospho-p65, and IκB were analyzed by Western blot as shown in Fig. P < 0.05; LPS group vs. S1P group: 1.67 ± 0.08 vs. 2.08 ± 0.13, P > 0.05 for p-p65/p65 ratio, 1.72 ± 0.15 vs. 2.27 ± 0.13, P = 0.030 for p-p38/p38 ratio, 0.70 ± 0.07 vs. 0.45 ± 0.03, P = 0.042 for IκB; S1P group vs. SB group: 2.08 ± 0.13 vs. 1.25 ± 0.11, P = 0.002 for p-p65/p65 ratio, 2.27 ± 0.13 vs. 1.42 ± 0.08, P = 0.003 for p-p38/p38 ratio, 0.45 ± 0.03 vs. 0.81 ± 0.06, P = 0.006 for IκB). Collectively, these findings indicate that S1P acting on S1PR3 enables NF-κB p65 transcription through the p38 MAPK pathway, which participates in Sphk1-mediated neuroinflammation in SCI.We next detected NF-κB p65 activity via another inhibitor, SB203580, which selectively acts on p38 MAPK. Western blot analysis showed that activation of p38 MAPK and NF-κB p65 induced by LPS was amplified by S1P, and the amplification effect was inhibited by SB203580  . S1P/S1PThe present in vivo study discovered the neuroinflammatory signaling pathway of Sphk1/S1P/S1PR3 in SCI Fig. b. We demThese findings unravel the important role of the S1P/S1PR3/p38 MAPK pathway in the secondary neuroinflammatory response of acute spinal cord injury and the role of dysregulated lipid metabolism in this pathology. Contributing a novel understanding of the molecular mechanisms of the inflammatory cascade in this specific pathology now provides a new translational implication for targeted drug therapies in SCI. We have demonstrated the feasibility of manipulating this pathway via specific Sphk1 inhibitors to successfully alter the inflammatory cascade and improve functional outcomes. Future investigation should focus on the ideal limb of this cascade for inhibition and intervention and translational routes of administration. Therapies that target Sphk1/S1P/S1PR are complex and multifaceted and may in the future replace the current inefficient conservative therapies such as NSAIDs and corticosteroids while avoiding the invasiveness of surgical intervention, with the hope of providing the first targeted biologic therapy for the treatment of acute SCI.Sphingomyelin is abundant in the nervous system, and S1P receptors are widely expressed in various cells, with many cell responses depending on the Sphk1/S1P/S1PRs axis. For instance, one study demonstrated that localized delivery of FTY720 can reduce pathological astrogliosis by targeting the S1P1 receptor of astrocytes and provide a new therapeutic strategy for SCI treatment . Thus, oIn conclusion, our study has uncovered the role of abnormal lipid metabolism via the Sphk1 cascade as an etiology of secondary inflammatory injury after traumatic SCI. SCI enhances Sphk1 expression and activity in injured spinal cord tissue. Sphk1 modulates microglial M1 polarization and proinflammatory cytokine production via intracellular S1P signaling, as well as extracellular S1P binding to S1PR3, with ultimate phosphorylation of p38 MAPK and nuclear translocation and activation of NF-κB p65. This S1PR3/p38 MAPK pathway results not only in the release of proinflammatory molecules, but also results in loss of neuronal cells via apoptosis. Inhibition along the Sphk1/S1P/S1PR3 axis is feasible, reduces neuronal apoptosis, improves neurological functional recovery, and is presented as a translational therapeutic target for mitigating the inflammatory cascade amplification that exacerbates secondary injury in SCI.


A series of novel-sulfonated furanic polyesteramides have been synthesized by polycondensation in the melt-phase. The copolymers were obtained with reasonable molecular weights and high inherent viscosities between 0.2 and 0.4 dL/g. The hydrolytic degradability of polyesteramides was enhanced by the increasing of amide units in the copolymers backbone. The thermal properties indicate that the ensuing oligoesters are amorphous or semicrystalline that essentially depend on the nature of monomers [Polymeric materials synthesized from renewable monomers have already gained an established position among synthetic materials both in academia and in industry. Predominantly due to two principal major reasons (i) firstly environmental concerns and (ii) secondly the realization that our petroleum resources are finite. In this context, several biosourced chemicals such as furanic, isosorbide, succinic acid and itaconic acid … monomers are considered as alternative, from the moment they make possible to obtain environmentally friendly polymers and with properties similar to their fossil counterparts . In thismonomers . On the monomers , 23.1 H-NMR spectroscopy, they detected minor side reactions [Few studies concerning the preparation of biobased poly-esteramides have been carried out, for example, from saccharide by the polycondensation of bis(p-nitrophenyl) aliphatic dicarboxylates with p-toluene sulfonate salts of isosorbide or isomannide diesters of natural amino acids . Recentleactions . These seactions , 29.As a continuation of our studies on this family of polymer, a series of furanic poly(ester-amide)s bearing sulfonated groups in the main chain were synthesized from 5.5ʹ-Isopropylidene-bis(ethyl-2-furoate), dimethyl 5-sodiosulfoisophthalate, ethylene glycol and hexamethylene diamine by melt polycondensation using zinc acetate as a catalyst. These copolymers have potential interesting attributes: their biosourced origin, their relatively high thermal stability, the strong intermolecular hydrogen bonding interactions between amide functions and water molecules increases the hydrophilicity of the macromolecular chains and consequently their hydrolytic degradation. The hydrolytic degradation in acidic aqueous conditions (pH = 4.35) at 37°C over the period of 4 weeks show that the mechanism of the hydrolysis of poly(ester-amide)s was elucidated in relation with the microstructure.2.2.1.4 (97.00%), zinc acetate (Zn(OAc)2) (99.99%), and sodium acetate (NaOAc) were purchased from Sigma-Aldrich.Dimethyl 5-sodiosulfoisophthalate (Na-DMSI) (98.00%), 2-ethyl furoate (99.00%), hexane-1.6- diamine , ethylene glycol (EG) (99.80%), tetrabutoxytitanium Ti(OBu)All chemicals were used as received, without further purification.2.1.12SO4, 30 ml), at 60°C for 8 h. The ensuing product was isolated in 95% yield by extraction, double distillation and recrystallisation from hexane.The synthesis of 5.5ʹ-Isopropylidene-bis(ethyl 2-furoate) (DEBF) followed a previously reported procedure . Typical3.3.1.The polyesters were prepared by polycondensation in the melt from a mixture of variable amount of DEBF (4.68 mmol), Na-DMSI (1.17 mmol) with excess quantity of EG (17.5 mmol).4(0.1 wt%) as a catalyst. In the second step, the reaction was performed under reduced pressure for 6 h at 240°C. The resulting polymers are mentioned to as PEBF-Co-PESI.In the first step, the transesterification was carried out, under nitrogen atmosphere for 6 h at 200°C using Ti(OBu)3.2.2(0.1 wt%) was added to the mixture to act as a transesterification catalyst. The reaction was carried out at 200°C under a nitrogen flow. Methanol and ethanol formed in the transesterification reaction was continuously removed by distillation for a period of 6 h. The excess of HMD was removed by high vacuum distillation at temperature ranged from 70°C to 200°C. The pressure was slowly reduced to 0.01 mbar and reaction continued for 4 h at 220°C. PASI was analyzed without further purification.DEBF (4.7 mmol) and Na-DMSI (2 mmol) and excess of HMD (20.1 mmol) were charged to a 50-mL, threenecked, round-bottom flask equipped with a mechanical stirrer, a nitrogen inlet, and a distillation column. Zn(OAc)3.3.1-3 were prepared according to the general procedure described below for PEASI 1: DEBF (6.2 mmol), ED (35 mmol), Na-DMSI (2.6 mmol) and HMD (1.76 mmol) were introduced in a 50-mL glass kettle equipped with a central mechanical stirrer, a nitrogen inlet, a distillation head connected to a condenser and a receiver flask. The kettle was placed in a salt bath at 200°C. After 5 min of heating, the reaction medium was homogeneous and10−3 of total mass of zinc acetate Zn(OAc)2 was introduced in the mixture. Bath temperature was raised gradually to 220 C and ethanol distilled off. After 6 h heating, pressure was slowly reduced to 0.07 mbar and reaction continued for 4 h. PEASI1 was analyzed after cooling, without further purification.Polyesteramides PEASI4.1H, 13C NMR spectra were recorded in DMSO-d6 using a Bruker Avance III 400 spectrometer at 90°C. All chemical shifts (δ) downfield from the tetramethylsilane TMS .−1 were collected and signal-averaged.Infrared spectra were recorded on a PerkinElmer 2000 Fourier transform infrared (FTIR) spectrophotometer at room temperature from powder pressed pellet samples in KBr. For each sample, 64 scans at a resolution of 1 cmThe inherent viscosities of the polymers were measured from DMSO solutions with an Ubbelohde viscometer maintained at 25.0 ± 0.1°C.The hydrolytic degradation essays were performed with films prepared via solvent casting process.After immersion in water (pH = 4.35) at 37°C for a fixed period of time, the samples were rinsed thoroughly in water and dried to constant weight. Sample weighting was used to follow the evolution of the hydrodegradation.1-3 and film PEBF-Co-PESI were prepared by casting a sample of PEASI1-3 polyesteramides and PEBF-Co-PESI polyesters at room temperature from a 10% (W/V) solution in CHCl3/MeOH (8/1 V/V) on silanized Petri dish. The films were cut and dried in vacuum at 50°C to constant weight. The thickness of the obtained films was 210 ± 10 mm.Films referenced as film PEASI5.1H-NMR spectrum relating to the final PEASI copolymer, we first proceeded to prepare the homopolyester and the homopolyamide separately, assign the different signals on their spectra, and then the attribution of the signals of the PEASI spectrum.In view of the complexity of the 5.1.4. Finally, the reaction pressure was returned to normal atmospheric pressure by the introduction of N2 and the target product was obtained.The synthesis of poly PEBF-Co-PESI the two-step melt polycondensation method (transesterification and polycondensation)between 5.5ʹ-isopropylidene-bis(ethyl 2-furoate) (DEBF), dimethyl 5-sodiosulfoisophthalate (Na-DMSI) and 1.2-ethanediol (ED). The first step was carried out under a nitrogen atmosphere in the presence of zinc acetate as transesterification catalyst and the sodium acetate as inhibitor of etherification side reaction. The temperature was gradually raised from 180°C to 200°C during this step in order to avoid sublimation of reagent and side reactions. Until no more methanol and ethanol were collected, this step was considered to be over, typically within 3 h. The second step was completed at higher temperature (240°C) under vacuum in order to release the excess of diol and to shift the reaction equilibrium toward the formation of high molecular weight. This latter step, i.e. the polycondensation was catalysed by the addition of Ti(OBu)1H-NMR. The molar fraction of furoate (FF) and sodiosulfoisophthalate (FSI) units in copolymers were determined from the integration of isophthalate  and furoate  NMR peaks (2-CH2-O-CH2-CH2) units are present in the copolyester.The structural characterization of the PEBF-Co-PESI copolyester was performed by MR peaks . The pre5.2.2 as catalyst. The reaction was first carried out under atmospheric pressure, then under vacuum with (HMD) elimination to obtain the final high-molar-mass of sulfonated polyamide.The sulfonated polyamide PASI was prepared as shown in by copolycondensation of 5.5ʹ-isopropylidene-bis(ethyl 2-furoate) (DEBF), dimethyl 5-sodiosulfoisophthalate (Na-DMSI) and an excess of hexane-1.6-diamine (HMD), in the presence of Zn(OAc)1H-NMR spectrum shown in 1, H2 and H3 at 6.26, 6.98 and 1.66 ppm, respectively, and those of sodium sulfoisophthalate protons H7, H7ʹ at 8.45 ppm. The (1.27–1.47) ppm and (3.16–3.25) ppm region of the 1 H-NMR spectrum, the resonances of amide methylene are easily assigned.For PASI, the 13,12) are observed at 8.21 and 8.71 ppm, respectively.The amide proton (-NH) of sulfonated and furanic units 1, and then extended the study to other compositions to obtain a series of sulfonated PEASIs with various ester/amide ratios , leading to PEASe ratios . The evo715 cm−1 . A repre1H-NMR spectrum of PEASI1 were assigned by comparison with the spectra of pure polyester and pure polyamide  arising the stiffening of the polymer backbone  at 37°C over a period of 4 weeks. After this period, each sample was rinsed thoroughly in water and dried to constant weight. The remaining weight of PEASI1-3 showed an overall decreasing trend over the entire period of degradation at (pH 4.35)  .Figure 1-3 are minor than theirs homologous polyesters PEBF-Co-PESI. Obviously, the decrease of the remaining weight depend on the sulfonated unit but as can be seen in As expected the remaining weight of the degraded samples of PEASI6.We investigated the synthesis of sulfonated bio-based polyesteramides by step-growth polymerization in melt conditions. Experimental results indicated that the resulting polyesteramides exhibited a regular structure, high inherent viscosities and reasonable molecular weights. This study also clearly shows the relationship between the monomer structure and the properties underlining the major role of the amide content on the copolymer hydrolytic degradation behaviour. Work is in progress to assess the properties and possible applications of these materials.