Lignin bioconversion based on genome mining for ligninolytic genes in Erwinia billingiae QL-Z3.

BACKGROUND: Bioconversion of plant biomass into biofuels and bio-products produces large amounts of lignin. The aromatic biopolymers need to be degraded before being converted into value-added bio-products. Microbes can be environment-friendly and efficiently degrade lignin. Compared to fungi, bacteria have some advantages in lignin degradation, including broad tolerance to pH, temperature, and oxygen and the toolkit for genetic manipulation. RESULTS: Our previous study isolated a novel ligninolytic bacterial strain Erwinia billingiae QL-Z3. Under optimized conditions, its rate of lignin degradation was 25.24% at 1.5 g/L lignin as the sole carbon source. Whole genome sequencing revealed 4556 genes in the genome of QL-Z3. Among 4428 protein-coding genes are 139 CAZyme genes, including 54 glycoside hydrolase (GH) and 16 auxiliary activity (AA) genes. In addition, 74 genes encoding extracellular enzymes are potentially involved in lignin degradation. Real-time PCR quantification demonstrated that the expression of potential ligninolytic genes were significantly induced by lignin. 8 knock-out mutants and complementary strains were constructed. Disruption of the gene for ELAC_205 (laccase) as well as EDYP_48 (Dyp-type peroxidase), ESOD_1236 (superoxide dismutase), EDIO_858 (dioxygenase), EMON_3330 (monooxygenase), or EMCAT_3587 (manganese catalase) significantly reduced the lignin-degrading activity of QL-Z3 by 47-69%. Heterologously expressed and purified enzymes further confirmed their role in lignin degradation. Fourier transform infrared spectroscopy (FTIR) results indicated that the lignin structure was damaged, the benzene ring structure and groups of macromolecules were opened, and the chemical bond was broken under the action of six enzymes encoded by genes. The abundant enzymatic metabolic products by EDYP_48, ELAC_205 and ESOD_1236 were systematically analyzed via liquid chromatography-mass spectrometry (LC-MS) analysis, and then provide a speculative pathway for lignin biodegradation. Finally, The activities of ligninolytic enzymes from fermentation supernatant, namely, LiP, MnP and Lac were 367.50 U/L, 839.50 U/L, and 219.00 U/L by orthogonal optimization. CONCLUSIONS: Our findings provide that QL-Z3 and its enzymes have the potential for industrial application and hold great promise for the bioconversion of lignin into bioproducts in lignin valorization.

The association of preconceptional folic acid supplementation with gestational age and preterm birth risk in twin pregnancies: a prospective cohort study in China.

PURPOSE: This study aims to explore the association of maternal preconceptional folic acid (FA) supplementation with gestational age and preterm birth in twin pregnancies, and whether the association varies by chorionicity or conception mode. METHODS: From November 2018 to December 2021, the information of FA supplementation and pregnancy outcomes were collected in twin pregnant women. The linear regression models and the logistic regression were used to test the association of preconceptional FA supplementation with gestational age at delivery and preterm birth and premature rupture of membranes (PROM). RESULTS: A total of 416 twin pregnancies were included. Compared with no use in twins, maternal preconceptional FA use was associated with a 0.385-week longer gestational age (95% CI 0.019-0.751) and lower risk of preterm birth < 36 weeks (adjusted OR 0.519; 95% CI 0.301-0.895) and PROM (adjusted OR 0.426; 95% CI 0.215-0.845). The protective effect on preterm birth < 36 weeks and PROM is similar whether taking FA supplements alone or multivitamins. However, the associations varied by chorionicity and conception mode of twins or compliance with supplementation. The positive associations between preconceptional FA use and gestational age only remained significant among twins via assisted reproductive technology or dichorionic diamniotic twins. Significant protective effects on preterm birth < 36 weeks and PROM were only found among women who took FA at least 4 times a week before conception. CONCLUSION: Maternal preconceptional FA supplementation was associated with longer gestation duration and lower risk of preterm birth < 36 weeks and PROM in twin pregnancies. To improve the success of their pregnancies, reproductive women should start taking FA supplements well before conception and with good compliance.

Whole-Genome Resequencing Reveals the Diversity of Patchouli Germplasm.

As an important medicinal and aromatic plant, patchouli is distributed throughout most of Asia. However, current research on patchouli's genetic diversity is limited and lacks genome-wide studies. Here, we have collected seven representative patchouli accessions from different localities and performed whole-genome resequencing on them. In total, 402,650 single nucleotide polymorphisms (SNPs) and 153,233 insertions/deletions (INDELs) were detected. Based on these abundant genetic variants, patchouli accessions were primarily classified into the Chinese group and the Southeast Asian group. However, the accession SP (Shipai) collected from China formed a distinct subgroup within the Southeast Asian group. As SP has been used as a genuine herb in traditional Chinese medicine, its unique molecular markers have been subsequently screened and verified. For 26,144 specific SNPs and 16,289 specific INDELs in SP, 10 of them were validated using Polymerase Chain Reaction (PCR) following three different approaches. Further, we analyzed the effects of total genetic variants on genes involved in the sesquiterpene synthesis pathway, which produce the primary phytochemical compounds found in patchouli. Eight genes were ultimately investigated and a gene encoding nerolidol synthetase (PatNES) was chosen and confirmed through biochemical assay. In accession YN, genetic variants in PatNES led to a loss of synthetase activity. Our results provide valuable information for understanding the diversity of patchouli germplasm resources.

The underlying mechanisms of cisplatin-induced nephrotoxicity and its therapeutic intervention using natural compounds.

Cisplatin is an effective chemotherapeutic drug widely used for the treatment of various solid tumors; however, its clinical use and efficacy are limited by its inherent nephrotoxicity. The pathogenesis of cisplatin-induced nephrotoxicity is complex and has not been fully elucidated. Cellular uptake and transport, DNA damage, apoptosis, oxidative stress, inflammatory response, and autophagy are involved in the development of cisplatin-induced nephrotoxicity. Currently, despite some deficiencies, hydration regimens remain the major protective measures against cisplatin-induced nephrotoxicity. Therefore, effective drugs must be explored and developed to prevent and treat cisplatin-induced kidney injury. In recent years, many natural compounds with high efficiency and low toxicity have been identified for the treatment of cisplatin-induced nephrotoxicity, including quercetin, saikosaponin D, berberine, resveratrol, and curcumin. These natural agents have multiple targets, multiple effects, and low drug resistance; therefore, they can be safely used as a supplementary regimen or combination therapy for cisplatin-induced nephrotoxicity. This review aimed to comprehensively describe the molecular mechanisms underlying cisplatin-induced nephrotoxicity and summarize natural kidney-protecting compounds to provide new ideas for the development of better therapeutic agents.

Identification and functional characterization of three cytochrome P450 genes for the abietane diterpenoid biosynthesis in Isodon lophanthoides.

MAIN CONCLUSION: We identify two ferruginol synthases and a 11-hydroxyferruginol synthase from a traditional Chinese medicinal herb Isodon lophanthoides and propose their involvement in two independent abietane diterpenoids biosynthetic pathways. Isodon lophanthoides is a traditional Chinese medicinal herb rich in highly oxidized abietane-type diterpenoids. These compounds exhibit a wide range of pharmaceutical activities, yet the biosynthesis is barely known. Here, we describe the screening and functional characterization of P450s that oxidize the abietane skeleton abietatriene. We mainly focused on CYP76 family and identified 12 CYP76AHs by mining the RNA-seq data of I. lophanthoides. Among the 12 CYP76AHs, 6 exhibited similar transcriptional expression features as upstream diterpene synthases, including root or leaf-preferential expression pattern and highly MeJA inducibility. These six P450s were considered as first-tier candidates and functionally characterized in yeast and plant cells. In yeast assays showed that both CYP76AH42 and CYP76AH43 were ferruginol synthases hydroxylating the C12 position of abietatriene, whereas CYP76AH46 was characterized as a 11-hydroxyferruginol synthase which catalyzes two successive oxidations at C12 and C11 of abietatriene. Heterologous expression of three CYP76AHs in Nicotiana benthamiana resulted in the formation of ferruginol. qPCR analysis showed CYP76AH42 and CYP76AH43 were mainly expressed in the root, which was consistent with the distribution of ferruginol in the root periderms. CYP76AH46 was primarily expressed in the leaves where barely ferruginol or 11-hydroxyferruginol was detected. In addition to distinct organ-specific expression pattern, three CYP76AHs exhibited different genomic structures (w or w/o introns), low protein sequence identities (51-63%) and were placed in separate subclades in the phylogenetic tree. These results suggest that the identified CYP76AHs may be involved in at least two independent abietane biosynthetic pathways in the aerial and underground parts of I. lophanthoides.

Mood, hormone levels, metabolic and sleep across the menopausal transition in VCD-induced ICR mice.

AIMS: Menopausal transition is the transitional period before menopause in women, often accompanied by abnormal fluctuations in hormone levels that increase the risk of aging-related diseases. 4-vinylcyclohexene dioxide (VCD) is a chemical agent that induces gradual depletion of ovarian follicles, which can mimic the natural human process of transition from menopausal transition to post-menopause. Previous studies have shown that the onset of menopausal transition or menopause in VCD-injected mice is associated with a specific strain, even in inbred animals. Institute of Cancer Research (ICR) mice constitute general purpose outbred population, which has not been well-characterized in the VCD-induced model. Thus, the current study aimed to explore the characteristic features, including sleep, mood, and metabolism, of the model by examining the effect of timing of VCD injection in ICR mice to extend the applications of this model. MATERIALS AND METHODS: ICR mice were randomly divided into six groups: 20d VCD and 20d Control, 35d VCD and 35d Control, 52d VCD and 52d Control. VCD mice were intraperitoneally injected with VCD (160 mg/kg), while Control mice were injected intraperitoneally with sesame oil for 4 consecutive weeks, five times a week daily. A vaginal smear was used to observe the estrous cycle of the mice. On the 20th, 35th, and 52nd day after VCD or sesame oil injection, the ovarian morphology, the number of atretic cells, hormone levels, anxiety, depression-like behaviors, sleep phase, and energy metabolism were observed. KEY FINDINGS: The menopausal transition model was successfully replicated by injecting VCD into ICR mice. On the specific days after VCD treatment, the number of atretic follicles increased, the level of E2 decreased and FSH increased, the depressive- and anxiety-like behavior increased, the time of REM and NREM sleep time decreased, and energy metabolism was reduced. SIGNIFICANCE: These results suggested that the ICR mice model has human-like characteristics during the menopause transition. Moreover, the ICR model has a long menopausal transition duration.

Exploring the mechanism of Cassiae semen in regulating lipid metabolism through network pharmacology and experimental validation.

BACKGROUND: Multiple studies have assessed the role of Cassiae semen (CS) in regulating lipid metabolism. However, the mechanism of action of CS on non-alcoholic fatty liver disease (NAFLD) has seen rare scrutiny. OBJECTIVE: The objective of this study was to explore the regulatory mechanism of CS on lipid metabolism in NAFLD. METHODS: Components of CS ethanol extract (CSEE) were analyzed and identified using UPLC-Q-Orbirap HRMS. The candidate compounds of CS and its relative targets were extracted from the Traditional Chinese Medicine Systems Pharmacology, Swiss-Target-Prediction, and TargetNet web server. The Therapeutic Target Database, Genecards, Online Mendelian Inheritance in Man, and DisGeNET were searched for NAFLD targets. Binding affinity between potential core components and key targets was established employing molecular docking simulations. After that, free fatty acid (FFA)-induced HepG2 cells were used to further validate part of the network pharmacology results. RESULTS: Six genes, including Caspase 3 (CASP3), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA), epidermal growth factor receptor (EGFR), and amyloid ß (A4) precursor protein (APP) were identified as key targets. The mitogen-activated protein kinase (MAPK) signaling pathway was found to associate closely with CS's effect on NAFLD. Per molecular docking findings, toralactone and quinizarin formed the most stable combinations with hub genes. About 0.1 (vs. FFA, P<0.01) and 0.2 (vs. FFA, P<0.05) mg/ml CSEE decreased lipid accumulation in vitro by reversing the up-regulation of CASP3, EGFR, and APP and the down-regulation of PIK3CA. CONCLUSION: CSEE can significantly reduce intracellular lipid accumulation by modulating the MAPK signaling pathway to decrease CASP3 and EGFR expression.

Acidity-Biodegradable Iridium-Coordinated Nanosheets for Amplified Ferroptotic Cell Death Through Multiple Regulatory Pathways.

Ferroptosis-based treatment strategies display the potential to suppress some malignant tumors with intrinsic apoptosis resistance. However, current related cancer treatments are still hampered by insufficient intracellular reactive oxygen species (ROS) levels and Fe2+ contents, posing considerable challenges for their clinical translation. Herein, an intracellular acid-biodegradable iridium-coordinated nanosheets (Ir-Hemin) with sonodynamic therapy (SDT) properties to effectively induce ferroptosis in tumor cells through multiple regulatory pathways are proposed. Under ultrasound (US) irradiation, Ir-Hemin nanosheets act as nanosonosensitizers to effectively generate ROS, subsequently causing the accumulation of lipid peroxides (LPO) and inducing ferroptotic cell death. Furthermore, these Ir-Hemin nanosheets decompose quickly to release hemin and Ir(IV), which deplete intracellular glutathione (GSH) to deactivate the enzyme glutathione peroxidase 4 (GPX4) and initiate the ferroptosis pathway. Specifically, the released hemin enables heme oxygenase 1 (HO-1) upregulation for endogenous ferrous ion supplementation, which compensates for the toxicity concerns brought about by the large uptake of exogenous iron. Surprisingly, Ir-Hemin nanosheets exhibit high tumor accumulation and trigger effective ferroptosis for tumor therapy. These Ir-Hemin nanosheets display pronounced synergistic anticancer efficacy under US stimulation both in vitro and in vivo, providing a strong rationale for the application of ferroptosis in cancer treatment.

Two new cytochalasins from the endophytic fungus Xylaria sp. GDGJ-77B.

Two new open-chain cytochalasins, xylarchalasins A and B (1 and 2), together with six known analogues (3-8), were isolated from the endophytic fungus Xylaria sp. GDGJ-77B from the Chinese medicinal plant Sophora tonkinensis. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. Compound 2 displayed moderate antibacterial activities against Bacillus subtilis and Escherichia coli with MIC values of 25 and 12.5 µg/mL, respectively.

In Vitro Biotransformation of Total Glycosides in Qiwei Baizhu Powder by the Gut Microbiota of Normal and Diarrheal Mice: Novel Insight Into the Biotransformation of Multi-Glycosides by the Gut Microbiota.

The gut microbiota (GM) is involved in the metabolism of glycosides and is beneficial for enhancing their bioactivity. However, the metabolism of multi-glycosides by the GM under normal and pathological conditions is unclear. In this study, the total glycosides (TG) of the traditional Chinese medicine (TCM) formula Qiwei Baizhu Powder (QWBZP) were extracted to represent a multi-glycoside system. Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to rapidly identify the components and in vitro metabolites of QWBZP-TG. The metabolic profiles of QWBZP-TG in the GM of normal and diarrheal mice were also compared. A total of 68 compounds and seven metabolites were identified in the QWBZP-TG and metabolic samples, respectively. Deglycosylation was the main metabolic pathway of in vitro multi-glycoside metabolism. Liquiritin apioside, isoliquiritin apioside, liquiritin, protopanaxadiol (PPD)-type, and oleanane (OLE)-type ginsenosides were relatively easy to metabolize by the GM. At first, the deglycosylation capability of the GM of normal mice was superior to that of diarrheal mice, but the deglycosylation capability of diarrheal mice gradually recovered and produced abundant deglycosylation metabolites. In conclusion, deglycosylation metabolites may be the bioactive components of QWBZP. Glycoside-bacteria interaction may be a key mechanism for QWBZP to therapy diarrhea.

Hypothalamic mechanisms of obesity-associated disturbance of hypothalamic-pituitary-ovarian axis.

Ovulatory disorders are the most common clinical feature exhibited among obese women. Initiation of ovulation physiologically requires a surge of gonadotropin-releasing hormone (GnRH) released from GnRH neurons located in the hypothalamus. These GnRH neurons receive metabolic signals from circulation and vicinal neurons to regulate GnRH release. Leptin acts indirectly on GnRH via adjacent leptin receptor (LEPR)-expressing neurons such as proopiomelanocortin (POMC), neuropeptide Y (NPY)/agouti-related peptide (AgRP), and neuronal nitric oxide (NO) synthase (nNOS) neurons to affect GnRH neuronal activities. Additionally, hypothalamic inflammation also affects ovulation independent of obesity. Therefore, this review focuses on hypothalamic mechanisms that underlie the disturbance of hypothalamic-pituitary-ovarian (HPO) axis during obesity with an attempt to promote future studies and/or novel therapeutic strategies for ovulatory disorders in obesity.

Action Mechanism Underlying Improvement Effect of Fuzi Lizhong Decoction on Nonalcoholic Fatty Liver Disease: A Study Based on Network Pharmacology and Molecular Docking.

OBJECTIVE: This study aimed to decipher the bioactive compounds and potential mechanism of traditional Chinese medicine (TCM) formula Fuzi Lizhong Decoction (FLD) for nonalcoholic fatty liver disease (NAFLD) treatment via an integrative network pharmacology approach. METHODS: The candidate compounds of FLD and its relative targets were obtained from the TCMSP and PharmMapper web server, and the intersection genes for NAFLD were discerned using OMIM, GeneCards, and DisGeNET. Then, the PPI and component-target-pathway networks were constructed. Moreover, GO enrichment and KEGG pathway analysis were performed to investigate the potential signaling pathways associated with FLD's effect on NAFLD. Eventually, molecular docking simulation was carried out to validate the binding affinity between potential core components and key targets. RESULTS: A total of 143 candidate active compounds and 129 relative drug targets were obtained, in which 61 targets were overlapped with NAFLD. The PPI network analysis identified ALB, MAPK1, CASP3, MARK8, and AR as key targets, mainly focusing on cellular response to organic cyclic compound, steroid metabolic process, and response to steroid hormone in the biological processes. The KEGG pathway analysis demonstrated that 16 signaling pathways were closely correlated with FLD's effect on NALFD with cancer pathways, Th17 cell differentiation, and IL-17 signaling pathways as the most significant ones. In addition, the molecular docking analysis revealed that the core active compounds of FLD, such as 3'-methoxyglabridin, chrysanthemaxanthin, and Gancaonin H, had a high binding activity with such key targets as ALB, MAPK1, and CASP3. CONCLUSIONS: This study suggested that FLD exerted its effect on NAFLD via modulating multitargets with multicompounds through multipathways. It also demonstrated that the network pharmacology-based approach might provide insights for understanding the interrelationship between complex diseases and interventions of the TCM formula.

Pien-Tze-Huang attenuates neuroinflammation in cerebral ischaemia-reperfusion injury in rats through the TLR4/NF-κB/MAPK pathway.

CONTEXT: Pien-Tze-Huang (PTH) is traditionally applied to treat various inflammation-related diseases including stroke. However, literature regarding the anti-inflammatory effects and possible mechanisms of PTH in ischaemic stroke is unavailable. OBJECTIVE: This study investigates the anti-inflammatory effects and its underlying mechanism of PTH on ischaemic stroke. MATERIALS AND METHODS: Cerebral ischaemia-reperfusion injury was induced through 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion in male Sprague-Dawley (SD) rats receiving oral pre-treatment with PTH (180 mg/kg) for 4 days. TLR4 antagonist TAK-242 (3 mg/kg) was injected intraperitoneally at 1.5 h after MCAO. MRI, HE staining, qRT-PCR, western blot, and immunofluorescence methods were employed. RESULTS: PTH treatment markedly reduced cerebral infarct volume (by 51%), improved neurological function (by 33%), and ameliorated brain histopathological damage in MCAO rats. It also reduced the levels of four inflammatory mediators including IL-1ß (by 70%), IL-6 (by 78%), TNF-α (by 60%) and MCP-1 (by 58%); inhibited microglia and astrocyte activation; and decreased protein expression of iNOS and COX-2 in injured brains. Moreover, PTH down-regulated the protein expressions of TLR4, MyD88, and TRAF6; reduced the expression and nuclear translocation of NF-κB; and lowered the protein expressions of p-ERK1/2, p-JNK, and p-p38. Similar effects were observed in MCAO rats with TAK-242 treatment. However, combined administration of PTH and TAK-242 did not significantly reinforce the anti-inflammatory effects of PTH. DISCUSSION AND CONCLUSION: PTH improved cerebral ischaemia-reperfusion injury by inhibiting neuroinflammation partly via the TLR4/NF-κB/MAPK signalling pathway, which will help guide its clinical application.

Design, green synthesis, antioxidant activity screening, and evaluation of protective effect on cerebral ischemia reperfusion injury of novel monoenone monocarbonyl curcumin analogs.

Antioxidants with high efficacy and low toxicity have the potential to treat cerebral ischemia reperfusion injury (CIRI). Dienone monocarbonyl curcumin analogs (DMCA) capable of overcoming the instability and pharmacokinetic defects of curcumin possess notable antioxidant activity but are found to be significantly toxic. In this study, a novel skeleton of the monoenone monocarbonyl curcumin analogue sAc possessing reduced toxicity and improved stability was designed on the basis of the DMCA skeleton. Moreover, 32 sAc analogs were obtained by applying a green, simple, and economical synthetic method. Multiple sAc analogs with an antioxidant protective effect in PC12 cells were screened using an H2O2-induced oxidative stress damage model, and quantitative evaluation of structure-activity relationship (QSAR) model with regression coefficient of R2 = 0.918921 was built through random forest algorithm (RF). Among these compounds, the optimally active compound sAc15 elicited a potent protective effect on cell growth of PC12 cells by effectively eliminating ROS generation in response to oxidative stress injury by activating the Nrf2/HO-1 antioxidant signaling pathway. In addition, sAc15 exhibited good protection against CIRI in the mice middle cerebral artery occlusion (MCAO) model. In this paper, we provide a novel class of antioxidants and a potential compound for stroke treatment.

Pien-Tze-Huang, a Chinese patent formula, attenuates NLRP3 inflammasome-related neuroinflammation by enhancing autophagy via the AMPK/mTOR/ULK1 signaling pathway.

NLRP3 inflammasome is a key mediator in ischemic stroke-induced neuroinflammation and subsequent brain injury. Our previous study demonstrated the potent activity of Pien-Tze-Huang (PTH), a well-known Chinese patent formula, in reducing mitochondria-mediated neuronal apoptosis in cerebral ischemia/reperfusion impaired rats. This study aims to elucidate the mechanistic action of PTH related to neuroinflammation in LPS-induced BV2 microglial cells and cerebral ischemia/reperfusion impaired rats. BV2 cells were stimulated with LPS for 12 h and treated with PTH with various concentrations. Modulation by PTH of relevant genes (IL-6, IL-1ß, IL-18, TNF-α, COX-2 and iNOS mRNA) and proteins (NLRP3 inflammasome, autophagy and AMPK/mTOR/ULK signaling) was analyzed by real-time PCR and western blot, respectively. Similar analyses were conducted in middle cerebral artery occlusion rat model including neurological deficit, infarct volume, microglial activation, and key genes and proteins in modulating autophagy and NLRP3. Our results showed that PTH significantly inhibited the production of key proinflammatory mediators and protein expressions of NLRP3 and caspase-1 p20 in LPS induced BV2 cells. It also enhanced the autophagy response by modulating the key autophagy proteins via AMPK/mTOR/ULK related pathway. The reduced inflammatory responses and NLRP3 expressions by PTH were partially blocked by the autophagy inhibitor (3-MA) and AMPK blocker (compound C). In rats, PTH significantly reduced infarct size, suppressed microglial activation, and improved neuron deficit. It also promoted autophagy and reduced NLRP3 activity. Our study demonstrated that PTH inhibited NLRP3 inflammasome-mediated neuroinflammation, which was associated with enhanced autophagy via AMPK/mTOR/ULK1 pathway in vitro and in vivo.

Discovery and Functional Characterization of a Diverse Diterpene Synthase Family in the Medicinal Herb Isodon lophanthoides Var. gerardiana.

Isodon lophanthoides var. gerardiana (Lamiaceae), also named xihuangcao, is a traditional Chinese medicinal herb that exhibits a broad range of pharmacological activities. Abietane-type diterpenoids are the characteristic constituents of I. lophanthoides, yet their biosynthesis has not been elucidated. Although the aerial parts are the most commonly used organs of I. lophanthoides, metabolite profiling by gas chromatography-mass spectrometry showed the underground parts also contain large amounts of labdane diterpenoids including abietatriene, miltiradiene and ferruginol, which are distinct from the 13-hydroxy-8(14)-abietene detected in the aerial parts. Comparative transcriptome analysis of root and leaf samples identified a diverse diterpene synthase family including 6 copalyl diphosphate synthase (IlCPS1-6) and 5 kaurene synthase-like (IlKSL1-5). Here we report the functional characterization of six of these enzymes using yeast heterologous expression system. Both IlCPS1 and IlCPS3 synthesized (+)-copalyl diphosphate (CPP), in combination with IlKSL1 resulted in miltiradiene, precursor of abietane-type diterpenoids, while coupling with IlKSL5 led to the formation of hydroxylated diterpene scaffold nezukol. Expression profiling and phylogenetic analysis further support the distinct evolutionary relationship and spatial distribution of IlCPS1 and IlCPS3. IlCPS2 converted GGPP into labda-7,13E-dien-15-ol diphosphate. IlCPS6 was identified as ent-CPS, indicating a role in gibberellin metabolism. We further identified a single residue that determined the water addition of nezukol synthase IlKSL5. Substitution of alanine 513 with isoleucine completely altered the product outcome from hydroxylated nezukol to isopimara-7,15-diene. Together, these findings elucidated the early steps of bioactive abietane-type diterpenoid biosynthesis in I. lophanthoides and the catalytic mechanism of nezukol synthase.

[Effect of fucoxanthin on insulin resistance in obese mice induced by high fat diet].

The aim of this paper was to study the effect and mechanism of fucoxanthin on insulin resistance of obese mice induced by high-fat diet. Fifty C57 BL/6 J male mice were randomly divided into control group and high-fat diet group. The insulin resistance model was induced with high-fat diet for 12 weeks, and model mice were randomly divided into model group, fucoxanthin-0.2% group, fucoxanthin-0.4% group and metformin group. After dietary treatment for 6 weeks, the body weight and epididymal fat weight in each group were measured. Fasting blood glucose(FBG), fasting insulin(FINS), total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL-C) and high-density lipoprotein(HDL-C) were measured, and insulin resistance index(HOMA-IR) was calcula-ted. The pathological morphology in liver was observed by hematoxylin eosin staining, and the expressions of some key proteins in insulin receptor substrate 1(IRS-1)/posphoinositide 3-kinase(PI3 K)/serine-threonine kinase(Akt) and peroxisome proliferators-activated receptor-γ(PPARγ)/sterol regulatory element binding protein-1(SREBP-1)/fatty acid synthetase(FAS) pathways in liver were detected by Western blot. According to the findings, compared with the model group, levels of body weight, epididymal fat weight, FBG, FINS, TC, TG, LDL-C and HOMA-IR, as well as protein expressions of PPARγ, SREBP-1 and FAS in liver were significantly reduced(P<0.05 or P<0.01), while level of HDL-C and protein expressions of p-IRS-1, IRS-1, PI3 K and p-Akt in liver were signi-ficantly increased after treatment with fucoxanthin(P<0.05 or P<0.01). And the pathological changes of liver tissue in fucoxanthin-treated mice were also improved obviously. The results showed that fucoxanthin could improve obesity, hyperglycemia and hyperlipidemia, and alleviate insulin resistance in obese mice, and its mechanism is possibly related to the regulation of IRS-1/PI3 K/Akt and PPARγ/SREBP-1/FAS pathways.

Effects and molecular mechanism of pachymic acid on ferroptosis in renal ischemia reperfusion injury.

Acute kidney injury (AKI) is a common clinical disease. Ferropotosis, a new type of regulatory cell death, serves an important regulatory role in AKI. Pachymic acid (PA), a lanostane­type triterpenoid from Poria cocos, has been reported to be protective against AKI. However, the protective mechanism of PA in AKI is not yet fully understood. The present study aimed to investigate the effect and molecular mechanism of PA on ferroptosis in renal ischemia reperfusion injury in vivo. A total of 30 mice were intraperitoneally injected with 5, 10 and 20 mg/kg PA for 3 days. A bilateral renal pedicle clip was used for 40 min to induce renal ischemia­reperfusion injury and establish the model. The results demonstrated that treatment with PA decreased serum creatinine and blood urea nitrogen, and ameliorated renal pathological damage. Transmission electron microscopy revealed no characteristic changes in ferroptosis in the mitochondria of the renal tissue in the high­dose PA group, and only mild edema. Furthermore, treatment with PA increased glutathione expression, and decreased the expression levels of malondialdehyde and cyclooxygenase 2. Treatment with PA enhanced the protein and mRNA expression levels of the ferroptosis related proteins, glutathione peroxidase 4 (GPX4), solute carrier family 7 (cationic amino acid transporter, y+ system) member 11 (SLC7A11) and heme oxygenase 1 (HO­1) in the kidney, and increased the expression levels of nuclear factor erythroid derived 2 like 2 (NRF2) signaling pathway members. Taken together, the results of the present study suggest that PA has a protective effect on ischemia­reperfusion induced acute kidney injury in mice, which may be associated with the inhibition of ferroptosis in the kidneys through direct or indirect activation of NRF2, and upregulation of the expression of the downstream ferroptosis related proteins, GPX4, SLC7A11 and HO­1.

Pregnancy, but not dietary octanoic acid supplementation, stimulates the ghrelin-pituitary growth hormone axis in mice.

Circulating growth hormone (GH) concentrations increase during pregnancy in mice and remain pituitary-derived. Whether abundance or activation of the GH secretagogue ghrelin increase during pregnancy, or in response to dietary octanoic acid supplementation, is unclear. We therefore measured circulating GH profiles in late pregnant C57BL/6J mice and in aged-matched non-pregnant females fed with standard laboratory chow supplemented with 5% octanoic or palmitic (control) acid (n = 4-13/group). Serum total and acyl-ghrelin concentrations, stomach and placenta ghrelin mRNA and protein expression, Pcsk1 (encoding prohormone convertase 1/3) and Mboat4 (membrane bound O-acyl transferase 4) mRNA were determined at zeitgeber (ZT) 13 and ZT23. Total and basal GH secretion were higher in late pregnant than non-pregnant mice (P < 0.001), regardless of diet. At ZT13, serum concentrations of total ghrelin (P = 0.004), but not acyl-ghrelin, and the density of ghrelin-positive cells in the gastric antrum (P = 0.019) were higher, and gastric Mboat4 and Pcsk1 mRNA expression were lower in pregnant than non-pregnant mice at ZT23. In the placenta, ghrelin protein was localised mostly to labyrinthine trophoblast cells. Serum acyl-, but not total, ghrelin was lower at mid-pregnancy than in non-pregnant mice, but not different at early or late pregnancy. In conclusion, dietary supplementation with 5% octanoic acid did not increase activation of ghrelin in female mice. Our results further suggest that increases in maternal GH secretion throughout murine pregnancy are not due to circulating acyl-ghrelin acting at the pituitary. Nevertheless, time-dependent increased circulating total ghrelin could potentially increase ghrelin action in tissues that express the acylating enzyme and receptor.

Therapeutic Potential of Pien-Tze-Huang: A Review on Its Chemical Composition, Pharmacology, and Clinical Application.

Pien-Tze-Huang (PTH) is a famous and commonly used traditional Chinese medicine formula in China. It was first formulated by a royal physician of the Ming Dynasty (around 1555 AD). Recently, PTH has attracted attention worldwide due to its beneficial effects against various diseases, especially cancer. This paper systematically reviewed the up-to-date information on its chemical composition, pharmacology, and clinical application. A range of chemical compounds, mainly ginsenosides and bile acids, have been identified and quantified from PTH. Pharmacological studies indicated that PTH has beneficial effects against various cancers, hepatopathy, and ischemic stroke. Furthermore, PTH has been used clinically to treat various diseases in China, such as colorectal cancer, liver cancer, and hepatitis. In summary, PTH is a potential agent with extensive therapeutic effects for the treatment of various diseases. However, the lack of information on the side effects and toxicity of PTH is a non-negligible issue, which needs to be seriously studied in the future.