Isopropyl 3-(3,4-dihydroxyphenyl) 2-hydroxypropanoate protects septic myocardial injury via regulating GAS6/Axl-AMPK signaling pathway.

In a previous study, we used metabolomic techniques to identify a new metabolite of Danshen Dripping Pills called isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), which has potential as a drug candidate for cardiovascular diseases. This study aimed to explore the protective effects of IDHP against septic myocardial injury, as well as its molecular mechanism. Wild type or GAS6 knockout mice injured by cecal ligation and puncture (CLP) were used to observe the effect of IDHP. Here, we found that a specific concentration of IDHP (60 mg/kg) significantly increased the survival rate of septic mice to about 75 % at 72 h post CLP, and showed improvements in sepsis score, blood biochemistry parameters, cardiac function, and myocardial tissue damage. Furthermore, IDHP inhibited myocardial oxidative stress, inflammatory response, apoptosis, and mitochondrial dysfunction. Molecularly, we discovered that IDHP treatment reversed the CLP-induced downregulation of GAS6, Axl, and p-AMPK/AMPK expression. In addition, GAS6 knockout reversed the positive effect of IDHP in septic mice, indicated by more severe myocardial tissue damage, oxidative stress, inflammatory response, and mitochondrial dysfunction. GAS6 knockout also resulted in decreased levels of GAS6, Axl, and p-AMPK/AMPK. Taken together, our study provides evidence that IDHP has significant cardioprotective effects against sepsis by regulating the GAS6/Axl-AMPK signaling pathway. This finding has important therapeutic potential for treating sepsis.

Production of hydroxytyrosol through whole-cell bioconversion from L-DOPA using engineered Escherichia coli.

Hydroxytyrosol (HT), a polyphenolic molecule of high value, is used in the nutraceutical, cosmetic, food, and livestock nutrition industries. As a natural product, HT is chemically manufactured or extracted from olives; nevertheless, the increasing demand mandates the exploration and development of alternative sources, such as heterologous production by recombinant bacteria. In order to achieve this purpose, we have molecularly modified Escherichia coli to carry two plasmids. For conversion of L-DOPA (Levodopa) into HT efficiently, it is necessary to enhance the expression of DODC (DOPA decarboxylase), ADH (alcohol dehydrogenases), MAO (Monoamine oxidase) and GDH (glucose dehydrogenases). The step that significantly affects the rate of ht biosynthesis is likely to be associated with the reaction facilitated by DODC enzymatic activity, as suggested by the result of in vitro catalytic experiment and HPLC. Then Pseudomonas putida, Sus scrofa, Homo sapiens and Levilactobacillus brevis DODC were taken into comparsion. The DODC from H. sapiens is superior to that of P. putida, S. scrofa or L. brevis for HT production. Seven promoters were introduced to increase the expression levels of catalase (CAT) to remove the byproduct H2O2 and optimized coexpression strains were obtained after screening. After the 10-hour operation, the optimized whole-cell biocatalyst produced HT at a maximum titer of 4.84 g/L with over 77.5% molar substrate conversion rate.

Design and synthesis of ligustrazine derivatives as potential anti-Alzheimer's agents.

A novel series of ligustrazine derivatives was designed, synthesized, and evaluated as acetylcholinesterase (AChE) and butylcholinesterase (BuChE) inhibitors for the treatment of Alzheimer's disease (AD). In vitro studies displayed that some of the synthesized compounds revealed promising AChE and BuChE inhibitory effects. Particularly, compounds E12 and E27, indicated highly AChE inhibitory activity with IC50 values of 1.85 µM and 0.98 µM, respectively and showed noteworthy protective effects against on glutamate-induced SH-SY5Y cells damage at 1 µM and 10 µM concentrations. Furthermore, molecular simulation docking elucidates compounds E12 and E27 interacting with residues in the binding site of AChE (PDB code: 4EY7) and BuChE (PDB code: 1P0I), emphasizing the protein residues that participate in the main interactions with the two targets. Taken together, these results revealed that compounds E12 and E27 might be potential lead compounds for further structure optimization in the drug-discovery process against AD.

Alpha-asaronol promoted oligodendrocyte precursor cell differentiation and improved myelination as an activator PPARγ.

Preterm white matter injury (PWMI), characterized by oligodendrocyte precursor cell (OPC) differentiation disorder and dysmyelination, is a prevalent demyelinating disease of the central nervous system in premature infants, necessitating the development of mitigating strategies. Convincing evidence suggests that peroxisome proliferator-activated receptor γ (PPARγ) activation is a stimulative factor against the hindered process of oligodendrocyte (OL) differentiation. However, much remains unknown about its promotive mechanism. Our previous study indicated that alpha-asaronol (α-asaronol) could alleviate myelination disorder in a neonatal PWMI rat model, but the mechanism remained unclear. In this study, we demonstrated that α-asaronol attenuated cognitive deficits, repaired myelin damage, and stimulated OL differentiation in the corpus callosum of PWMI rats. Co-immunoprecipitation analysis confirmed that α-asaronol induced the binding of PPARγ with its coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which in turn activated oligodendroglial PPARγ. This activation subsequently upregulated the expression of phosphatase and tensin homolog (PTEN) and pro-differentiation-associated genes of Cnp1 and Klk6 and downregulated the expression of Clk1. However, the benefits of α-asaronol were blocked by GW9662, an antagonist of PPARγ. Moreover, α-asaronol also promoted OPC differentiation under oxygen-glucose deprivation conditions. In conclusion, α-asaronol can promote OL differentiation and myelination and alleviate cognitive deficits in neonatal PWMI rats by activating PPARγ and modulating OL differentiation-associated gene expression. This study suggests that α-asaronol may be a potential therapeutic drug for myelination failure in PWMI.

221S-1a inhibits endothelial proliferation in pathological angiogenesis through ERK/c-Myc signaling.

Pathological angiogenesis plays a major role in many disease processes, including cancer and diabetic retinopathy. Antiangiogenic therapy is a potential management for pathologic angiogenesis. The novel synthetic compound 221S-1a, derived from captopril, tanshinol and borneol, may have antiangiogenic properties. On the basis of MS, NMR and HPLC analysis, the structure of 221S-1a was identified. The cellular uptake and metabolism of this compound was also observed. Next, the antiangiogenic properties of 221S-1a were evaluated in tumor-xenograft and OIR models in vivo. The inhibitory properties of 221S-1a on endothelial cell proliferation, migration, tube formation and sprouting were detected in vitro. Furthermore, 221S-1a induced G1/S phase arrest was detected by PI staining flow cytometry analysis and Cyclin D, Cyclin E expression. 221S-1a inhibited ERK1/2 activation and nuclear translocation, in addition to downregulation of c-Myc, a transcription factor that regulates cell cycle progression. Molecular docking indicated the interaction of 221S-1a with the ATP-binding site of ERK2, leading to the inhibition of ERK2 phosphorylation and a concomitant inhibition of ERK1 phosphorylation. In conclusion, 221S-1a inhibited the G1/S phase transition by blocking the ERK1/2/c-Myc pathway to reduce tumor and OIR retinal angiogenesis. These novel findings suggest that 221S-1a is a potential pharmacologic candidate for treating pathological angiogenesis.

Microspore-expressed SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen development in wheat.

Sporopollenin is one of the most structurally sophisticated and chemically recalcitrant biopolymers. In higher plants, sporopollenin is the dominant component of exine, the outer wall of pollen grains, and contains covalently linked phenolics that protect the male gametes from harsh environments. Although much has been learned about the biosynthesis of sporopollenin precursors in the tapetum, the nutritive cell layer surrounding developing microspores, little is known about how the biopolymer is assembled on the microspore surface. We identified SCULP1 (SKS clade universal in pollen) as a seed plant conserved clade of the multicopper oxidase family. We showed that SCULP1 in common wheat (Triticum aestivum) is specifically expressed in the microspore when sporopollenin assembly takes place, localized to the developing exine, and binds p-coumaric acid in vitro. Through genetic, biochemical, and 3D reconstruction analyses, we demonstrated that SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen viability. Moreover, we found that SCULP1 accumulation is compromised in thermosensitive genic male sterile wheat lines and its expression partially restored exine integrity and male fertility. These findings identified a key microspore protein in autonomous sporopollenin polymer assembly, thereby laying the foundation for elucidating and engineering sporopollenin biosynthesis.

Discovery and therapeutic implications of bioactive dihydroxylated phenolic acids in patients with severe heart disease and conditions associated with inflammation and hypoxia.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled; their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. In DHPLA-positive patients' fecal samples, Proteus vulgaris and P. mirabilis were more abundant than healthy volunteers. In culture, these gut bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest bioactive DPAs are generated de novo as part of a human's defense mechanism against disease. Because DHPLA isolated from Radix Salvia miltiorrhizae has a multitude of pharmacological activities, these data underpin the scientific basis of this medicinal plant's ethnopharmacological applications as well as highlighting the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans.

Highly efficient biosynthesis of spermidine from L-homoserine and putrescine using an engineered Escherichia coli with NADPH self-sufficient system.

Spermidine is an important polyamine that can be used for the synthesis of various bioactive compounds in the food and pharmaceutical fields. In this study, a novel efficient whole-cell biocatalytic method with an NADPH self-sufficient cycle for spermidine biosynthesis was designed and constructed by co-expressing homoserine dehydrogenase (HSD), carboxyspermidine dehydrogenase (CASDH), and carboxyspermidine decarboxylase (CASDC). First, the enzyme-substrate coupled cofactor regeneration system from co-expression of NADP+-dependent ScHSD and NADPH-dependent AfCASDH exactly provides an efficient method for cofactor cycling. Second, we identified and characterized a putative CASDC with high decarboxylase activity from Butyrivibrio crossotus DSM 2876; it showed an optimum temperature of 35 °C and an optimum pH of 7.0, which make it better suited for the designed synthetic route. Subsequently, the protein expression level of each enzyme was optimized through the variation of the gene copy number, and a whole-cell catalyst with high catalytic efficiency was constructed successfully. Finally, a yield of 28.6 mM of spermidine was produced in a 1-L scale of E. coli whole-cell catalytic system with a 95.3% molar conversion rate after optimization of temperature, the ratio of catalyst-to-substrate, and the amount of NADP+, and a productivity of 0.17 g·L-1·h-1 was achieved. In summary, this novel pathway of constructing a whole-cell catalytic system from L-homoserine and putrescine could provide a green alternative method for the efficient synthesis of spermidine. KEY POINTS: • A novel pathway for spermidine biosynthesis was developed in Escherichia coli. • The enzyme-substrate coupled system provides an NADPH self-sufficient cycle. • Spermidine with 28.6 mM was obtained using an optimized whole-cell system.

Simultaneous and rapid analysis of chiral Danshensu and its ester derivatives by supercritical fluid chromatography.

The analysis and separation of chiral compounds with wide polar range by supercritical fluid chromatography is of major importance in the process of drug development and quality control. In this work, a fast and reliable enantioselective method for the simultaneous quantitative determination of 8 DBZ-related enantiomers has been successfully developed by supercritical fluid chromatography using an amylose-based reversed-chiral stationary phase. Within less than seven minutes all target compounds could be baseline resolved, using a mobile phase comprising supercritical carbon dioxide and methanol with 0.05 % H3PO4. The optimum chiral stationary phase showed to be a CHIRALPAK® AD-RH column, operated at flow rate of 3.0 mL/min, back pressure of 150 bar and temperature of 40 °C. Method validation confirmed that the developed procedure was selective, linear (r2 > 0.998), accurate (recovery rates: 98.02-100.02 %), and precise (intra-day: 0.05-1.98 %, inter-day: 0.08-1.98 %); the limit of detection and limit of quantification were 0.13-0.55 µg/mL and 0.37-1.68 µg/mL on column, respectively. After initial evaluation of stability according to the ICH Q1A (R2) guideline, R-DBZ showed good stability. Thus, this developed method can be used for assessing the stability of bulk DBZ samples, dosage forms of DBZ and also for monitoring the synthetic procedures of DBZ.

Synthesis, crystal structure and bioactivities of α-asaronol.

α-Asaronol [or (E)-3'-hydroxyasarone; systematic name: (E)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-ol; C12H16O4] was synthesized towards the development of a potential antiepileptic drug. Following purification by recrystallization, single crystals of α-asaronol were obtained by a liquid interface diffusion method at room temperature. The product was characterized by 1H and 13C NMR, and FT-IR spectroscopic analysis. X-ray crystallography revealed the title crystal to belong to the orthorhombic space group P212121. Preliminary bioassays with mouse neuroblastoma N2a cells demonstrated the neuroprotective activities of the synthesized α-asaronol.

Alpha-Asaronol Alleviates Dysmyelination by Enhancing Glutamate Transport Through the Activation of PPARγ-GLT-1 Signaling in Hypoxia-Ischemia Neonatal Rats.

Preterm white matter injury (PWMI) is the most common form of brain damage in premature infants caused by hypoxia-ischemia (HI), inflammation, or excitotoxicity. It is characterized by oligodendrocyte precursor cell (OPC) differentiation disorder and dysmyelination. Our previous study confirmed that alpha-asarone (α-asaronol), a major compound isolated from the Chinese medicinal herb Acorus gramineus by our lab, could alleviate neuronal overexcitation and improve the cognitive function of aged rats. In the present study, we investigated the effect and mechanism of α-asaronol on myelination in a rat model of PWMI induced by HI. Notably, α-asaronol promoted OPC differentiation and myelination in the corpus callosum of PWMI rats. Meanwhile, the concentration of glutamate was significantly decreased, and the levels of PPARγ and glutamate transporter 1 (GLT-1) were increased by α-asaronol treatment. In vitro, it was also confirmed that α-asaronol increased GLT-1 expression and recruitment of the PPARγ coactivator PCG-1a in astrocytes under oxygen and glucose deprivation (OGD) conditions. The PPARγ inhibitor GW9662 significantly reversed the effect of α-asaronol on GLT-1 expression and PCG-1a recruitment. Interestingly, the conditioned medium from α-asaronol-treated astrocytes decreased the number of OPCs and increased the number of mature oligodendrocytes. These results suggest that α-asaronol can promote OPC differentiation and relieve dysmyelination by regulating glutamate levels via astrocyte PPARγ-GLT-1 signaling. Although whether α-asaronol binds to PPARγ directly or indirectly is not investigated here, this study still indicates that α-asaronol may be a promising small molecular drug for the treatment of myelin-related diseases.

A Novel Compound, Tanshinol Borneol Ester, Ameliorates Pressure Overload-Induced Cardiac Hypertrophy by Inhibiting Oxidative Stress via the mTOR/ß-TrCP/NRF2 Pathway.

Tanshinol borneol ester (DBZ) exerts anti-atherosclerotic and anti-inflammatory effects. However, its effects on cardiac hypertrophy are not well understood. In this work, we investigated the treatment effects and potential mechanisms of DBZ on the hypertrophic heart under oxidative stress and endoplasmic reticulum (ER) stress. A hypertrophic model was established in rats using transverse-aortic constriction (TAC) surgery and in neonatal rat cardiomyocytes (NRCMs) using angiotensin II (Ang II). Our results revealed that DBZ remarkably inhibited oxidative stress and ER stress, blocked autophagy flow, and decreased apoptosis in vivo and in vitro through nuclear NRF2 accumulation, and enhanced NRF2 stability via regulating the mTOR/ß-TrcP/NRF2 signal pathway. Thus, DBZ may serve as a promising therapeutic for stress-induced cardiac hypertrophy.

Revealing the Antiepileptic Effect of α-Asaronol on Pentylenetetrazole-Induced Seizure Rats Using NMR-Based Metabolomics.

α-Asaronol from Acorus tatarinowii (known as "Shichangpu" in Traditional Chinese medicine) has been proved to possess more efficient antiepileptic activity and lower toxicity than α-asarone (namely "Xixinnaojiaonang" as an antiepileptic drug in China) in our previous study. However, the molecular mechanism of α-asaronol against epilepsy needs to be known if to become a novel antiepileptic medicine. Nuclear magnetic resonance (NMR)-based metabolomics was applied to investigate the metabolic patterns of plasma and the brain tissue extract from pentylenetetrazole (PTZ)-induced seizure rats when treated with α-asaronol or α-asarone. The results showed that α-asaronol can regulate the metabolomic level of epileptic rats to normal to some extent, and four metabolic pathways were associated with the antiepileptic effect of α-asaronol, including alanine, aspartate, and glutamate metabolism; synthesis and degradation of ketone bodies; glutamine and glutamate metabolism; and glycine, serine, and threonine metabolism. It was concluded that α-asaronol plays a vital role in enhancing energy metabolism, regulating the balance of excitatory and inhibitory neurotransmitters, and inhibiting cell membrane damage to prevent the occurrence of epilepsy. These findings are of great significance in developing α-asaronol into a promising antiepileptic drug derived from Traditional Chinese medicine.

Biosynthesis of phenylpyruvic acid from l-phenylalanine using chromosomally engineered Escherichia coli.

The efficiency of whole-cell biotransformation is often affected by the genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for the synthesis of phenylpyruvic acid (PPA) from l-phenylalanine. First, l-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into Escherichia coli BL21 (DE3) chromosome and the copy numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). Fifty-nine copies of pmLAAD were obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of isopropyl-ß-d-thiogalactopyranoside, the resulting strain, E. coli BL8△recA△lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37% increase in PPA production compared to E. coli BL8△recA. Finally, the engineered E. coli BL8△recA△lacI strain achieved 19.14 g/L PPA at 24 h in a 5-L bioreactor.

Converting the 3-quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4-chloroacetoacetate reductase by site-directed mutagenesis.

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat /Km ) for COBE was 51.36 s-1 ·mM-1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.

Characterization of a putative tropinone reductase from Tarenaya hassleriana with a broad substrate specificity.

A novel short-chain alcohol dehydrogenase from Tarenaya hassleriana labeled as putative tropinone reductase was heterologously expressed in Escherichia coli. Purified recombinant protein had molecular weight of approximately 30 kDa on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T. hassleriana tropinone reductase-like enzyme (ThTRL) had not detected oxidative activity. The optimum pH for enzyme activity of ThTRL was weakly acidic (pH 5.0). 50°C was the optimum temperature for ThTRL. The highest catalytic efficiency and substrate affinity for recombinant ThTRL were observed with (+)-camphorquinone (kcat /Km  = 814.3 s-1  mM-1 , Km  = 44.25 µM). ThTRL exhibited a broad substrate specificity and reduced various carbonyl compounds, including small lipophilic aldehydes and ketones, terpene ketones, and their structural analogs.

Overexpression and biochemical characterization of a carboxyspermidine dehydrogenase from Agrobacterium fabrum str. C58 and its application to carboxyspermidine production.

BACKGROUND: Carboxyspermidine (C-Spd) is a potentially valuable polyamine carboxylate compound and an excellent building block for spermidine synthesis, which is a critical polyamine with significant implications for human health and longevity. C-Spd can also be used to prepare multivalent cationic lipids and modify nucleoside probes. Because of these positive effects on human health, C-Spd is of considerable interest as a food additive and pharmaceutical target. RESULTS: A putative gene afcasdh from Agrobacterium fabrum str. C58, encoding carboxyspermidine dehydrogenase with C-Spd biosynthesis activity, was synthesized and transformed into Escherichia coli BL21 (DE3) for overexpression. The recombinant AfCASDH was purified and fully characterized. The optimum temperature and pH for the recombinant enzyme were 30 °C and 7.5, respectively. The coupled catalytic strategy of AfCASDH and various NADPH regeneration systems were developed to enhance the efficient production of C-Spd compound. Finally, the maximum titer of C-Spd production successfully achieved 1.82 mmol L-1 with a yield of 91% by optimizing the catalytic conditions. CONCLUSION: A novel AfCASDH from A. fabrum str. C58 was characterized that could catalyze the formation of C-Spd from putrescine and l-aspartate-ß-semialdehyde (L-Asa). A whole-cell catalytic strategy coupled with NADPH regeneration was established successfully for C-Spd biosynthesis for the first time. The coupled system indicated that AfCASDH might provide a feasible method for the industrial production of C-Spd. © 2021 Society of Chemical Industry.

[Applications of chromatography in giant complex drug-organism system].

Chromatography is an important branch of analytical chemistry that focuses on the separation and analysis of complex structures. Following more than 100 years of development and improvement, chromatography theory and technology have gradually become sophisticated. It has become a coalition of science, technology, and art. Recently, chromatography has been successfully used in combination with mass spectrometry, nuclear magnetic resonance spectroscopy, and atomic emission spectroscopy. Chromatography and the combination with other techniques has significantly improved the analysis of complex systems, such as the environment, food, petrochemicals, biological specimens, and medicine. As one of the oldest healing systems, Traditional Chinese Medicine (TCM) has served to maintain the health of people in China and worldwide for thousands of years. Therefore, it has become a core representative of traditional Chinese culture. In the past two years, TCM has been widely used to treat COVID-19, especially in patients with mild symptoms. Recently, Chinese government emphasized the inheritance and innovation of TCM and stepped up efforts to promote its modernization. TCM includes herbal medicine, acupuncture, moxibustion, massage, food therapy, and physical exercise, such as Tai Chi. In most cases, the patients are administered a mixture of TCM formulas containing more than two herbal medicines, resulting in a highly complicated compound mixture. There is no doubt that long-term clinical practices have demonstrated the safety and therapeutic effect of TCM. However, the compound mixture must be simplified to identify the active compounds. This is mainly because of the existence of carcinogenic compounds, pesticides, and heavy metal residues introduced through plantation and production processes. Moreover, enzymes within the human system generate further new compounds in response to the entry of the TCM containing thousands of components. Consequently, the complex TCM and organism systems interact with each other, constituting a giant complex drug-organism system. The analysis of this giant complex system is acknowledged as a key aspect in the modernization process of TCM. In the last 20 years, many studies have been conducted to screen and identify effective compounds in TCM. These effective compounds can be either the original compounds or new metabolic components generated in vivo. All these efforts are aimed at simplifying the components of TCM and elucidating the therapeutic mechanism. It is well known that chromatography can provide technical support for complex systems owing to its unique advantage of outstanding separation and analysis capabilities. Therefore, chromatography and its combination with other technologies have become mainstream technologies for promoting the compilation of molecular structure, information, digitalization, and modernization of TCM. This paper reviews the research and application of chromatography and combination technologies in a giant complex TCM formula-organism system. Furthermore, the authors briefly introduce and summarize the understanding, research ideas, and activities of the authors' team on the modernization of TCM. "Liang Guanxi" and "He strategy" are proposed as novel strategies for studying the giant complex drug-organism system. A distinguished technology integrated with mathematical model of causal relation, combined receptor chromatography, identification of chemical molecular structure and evaluating of pharmacological activities was established. It was successfully employed to determine the core effector-response substances of "Liang Guanxi" herb pairs in a giant complex drug-organism system. Subsequently, utilizing the proposed technology of Combination of Traditional Chinese Medicine Molecular Chemistry, the author's team designed and developed four series of innovative drugs. Inspired by the hundred years of chromatography history and thousands of years of TCM culture, the future development of chromatographic technology is expected. Furthermore, the mechanisms of TCM in medical healthcare, prevention, and treatment of diseases are likely be explained through chromatography, leading to a new strategy to realize the molecularization and digitalization of TCM, which is beneficial to the development of original new drugs.

A novel compound DBZ ameliorates neuroinflammation in LPS-stimulated microglia and ischemic stroke rats: Role of Akt(Ser473)/GSK3ß(Ser9)-mediated Nrf2 activation.

Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, yet lacks effective therapeutic agents. Previously, we discovered one novel synthetic compound, tanshinol borneol ester (DBZ), possesses anti-inflammatory and anti-atherosclerotic activities, whereas little is known about its effects in CNS. Therefore, the present study aims to explore the effects and potential mechanism of DBZ on neuroinflammation and microglial function. Our studies revealed that DBZ significantly inhibited NF-κB activity, suppressed the production of pro-inflammatory mediators meanwhile promoted M2 mediators expression in LPS-stimulated BV2 cells and mouse primary microglia cells. DBZ also exhibited antioxidant activity by enhancing Nrf2 nuclear accumulation and transcriptional activity, increasing HO-1 and NQO1 expression, and inhibiting LPS-induced ROS generation in BV2 cells. Importantly, the anti-neuroinflammatory and antioxidant effects of DBZ above were reversed by Nrf2 knockdown. Additionally, DBZ ameliorated sickness behaviors of neuroinflammatory mice induced by systemic LPS administration, and significantly reduced infract volume, improved sensorimotor and cognitive function in rats subjected to transient middle cerebral artery occlusion (tMCAO); besides, DBZ restored microglia morphological alterations and shifted the M1/M2 polarization in both murine models. Mechanistically, DBZ-induced Nrf2 nuclear accumulation and antioxidant enzymes expression were accompanied by increased level of p-Akt(Ser473) (activation) and p-GSK3ß(Ser9) (inactivation), and decreased nuclear level of Fyn both in vitro and in vivo. Pharmacologically inhibiting PI3K or activating GSK3ß markedly increased nuclear density of Fyn in microglia cells, which blocked the promoting effect of DBZ on Nrf2 nuclear accumulation and its antioxidant and anti-neuroinflammatory activities. Collectively, these results indicated the effects of DBZ on microglia-mediated neuroinflammation were strongly associated with the nuclear accumulation and stabilization of Nrf2 via the Akt(Ser473)/GSK3ß(Ser9)/Fyn pathway. With anti-neuroinflammatory and antioxidant properties, DBZ could be a promising new drug candidate for prevention and/or treatment of cerebral ischemia and other neuroinflammatory disorders.

Unveiling the Multipath Biosynthesis Mechanism of 2-Phenylethanol in Proteus mirabilis.

Proteus mirabilis could convert l-phenylalanine into 2-phenylethanol (2-PE) via the Ehrlich pathway, the amino acid deaminase pathway, and the aromatic amino acid decarboxylase pathway. The aromatic amino acid decarboxylase pathway was proved for the first time in P. mirabilis. In this pathway, l-aromatic amino acid transferase demonstrated a unique catalytic property, transforming 2-penylethylamine into phenylacetaldehyde. Eleven enzymes were supposed to involve in 2-phenylethanol synthesis. The mRNA expression levels of 11 genes were assessed over time by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in vivo. As a result, the expression of 11 genes was significantly increased, suggesting that P. mirabilis could transform l-phenylalanine into 2-phenylethanol via three pathways under aerobic conditions; nine genes were significantly overexpressed, suggesting that P. mirabilis could synthesize 2-phenylethanol via the Ehrlich pathway under anaerobic conditions. This study reveals the multipath synthetic metabolism for 2-phenylethanol in P. mirabilis and will enrich the new ideas for natural (2-PE) synthesis.