Alkaloids from Corydalis saxicola and their antiproliferative activity against cancer cells.

Eight undescribed alkaloids named corydalisine D-K (1-7), including one isoquinoline benzopyranone alkaloid (1), one benzocyclopentanone alkaloid (2), four benzofuranone alkaloids (3, 4, and 5a/5b) and two protoberberine alkaloids (6 and 7), along with fourteen known ones, were isolated from the Corydalis saxicola. Their structures, including absolute configurations, were unambiguously identified using spectroscopic techniques, single-crystal X-ray diffraction and electron circular dichroism calculation. Compounds 2, 14 and 21 exhibit antiproliferative activity against five cancer cell lines. The aporphine alkaloid demethylsonodione (compound 14), which exhibited the best activity (IC50 = 3.68 ± 0.25 µM), was subjected to further investigation to determine its mechanism of action against the T24 cell line. The molecular mechanism was related to the arrest of cell cycle S-phase, inhibition of CDK2 expression, accumulation of reactive oxygen species (ROS), induction of cell apoptosis, inhibition of cell migration, and activation of p38 MAPK signaling pathway. The results indicated that 14 could be used as a potential candidate agent for further development of anti-bladder transitional cell carcinoma.

Network Pharmacology and Molecular Docking Reveal the Mechanism of Corydalis saxicola Bunting Total Alkaloids in Treating Radiation-Induced Oral Mucositis.

The study aims to explore the effect and mechanism of total alkaloids of Corydalis saxicola Bunting (CSBTA) in the treatment of radiation induced oral mucositis (RIOM) through network pharmacology and molecular docking. The components and corresponding targets of Corydalis saxicola Bunting were screened by literature review. RIOM related targets were obtained in GeneCards. Cytoscape software was used to construct the component-target-pathway network. Protein-Protein Interaction (PPI) networks was constructed by String database. GO and KEGG enrichment analyses were performed by Metascape. AutoDock Vina 4.2 software was used for molecular docking. There were 26 components of CSBTA targeting 61 genes related to RIOM. Through Cytoscape and PPI analysis, 15 core target genes of CSBTA for treating RIOM were identified. GO functional analysis indicated that CSBTA might play a role through kinase binding and protein kinase activation. KEGG pathway analysis showed that the core targets of CSBTA were mainly focused on cancer and reactive oxygen species (ROS) pathway. The results of molecular docking showed that CSBTA had strong binding energy with target protein including SRC, AKT and EGFR. The study demonstrates that CSBTA may treat RIOM by affecting SRC, AKT and EGFR through ROS pathway.

Integrating metabonomics and metagenomics sequencing to study the anti-liver fibrosis effects of palmatine in Corydalis saxicola Bunting.

ETHNOPHARMACOLOGICAL RELEVANCE: Corydalis saxicola Bunting (CS), a traditional Chinese folk medicine, has been effectively used for treating liver disease in Zhuang nationality in South China. However, the main anti-liver fibrosis ingredients in CS are incompletely understood. AIM OF THE STUDY: To elucidate the main anti-liver fibrosis ingredients in CS and its underlying mechanism. MATERIAL AND METHODS: Firstly, spectrum-effect relationship (SER) strategy was applied to identify the major ingredients against liver fibrosis in CS. Subsequently, 1H NMR metabonomics and metagenomics sequencing techniques were used to clarify the intervention of palmatine (PAL) on liver fibrosis. Furthermore, the expression of tight junction proteins and the levels of liver inflammation factors were examined, the effect of PAL on microbiota was verified by FMT. RESULTS: The SER model revealed that PAL was the most important active ingredient in CS. 1H NMR fecal metabonomics showed that PAL could reserve the abnormal levels of gut microbial-mediated metabolites of liver fibrosis, such as isoleucine, taurine, butyrate, propionate, lactate, glucose, which mainly involved in amino acid metabolism, intestinal flora metabolism and energy metabolism. Metagenomics sequencing found that PAL could callback the abundance of s__Lactobacillus_murinus, s__Lactobacillus_reuteri, s__Lactobacillus_johnsonii, s__Lactobacillus_acidophilus and s__Faecalibaculum_rodentium to varying degree. Furthermore, the intestinal barrier function and the levels of hepatic inflammation factors were significantly ameliorated by PAL. FMT demonstrated that the therapeutic efficiency of PAL was closely associated with gut microbiota. CONCLUSION: The effects of CS on liver fibrosis were attributed in part to PAL by alleviating metabolic disorders and rebalancing gut microbiota. The SER strategy may be a useful method for the discovery of active constituents in natural plants.

Hypothalamic molecular correlates of photoperiod-induced spring migration in intact and castrated male redheaded buntings.

This study investigated the molecular changes associated with neural plasticity in photoperiodic induction of spring migration in intact and castrated redheaded bunting, Emberiza bruniceps. We measured the hypothalamic mRNA expression of genes in birds that were photostimulated into winter non-migratory and spring (vernal) migratory phenotypes under short and long photoperiods, respectively. These included genes associated with the appetitive phase of reproduction (spring migration drive, th and ddc genes encoding for tyrosine hydroxylase and dopamine decarboxylase enzymes, respectively), sleep/awake state (pmch gene encoding for pro-melanin concentrating hormone; hcrt and hcrtr2 encoding for the hypocretin/orexin and its receptor, respectively) and neurogenesis (dcx and neuN coding for doublecortin and neuronal nuclear proteins, respectively). Higher th mRNA levels suggested an upregulated dopamine synthesis in the hypothalamus of spring migrants. Similarly, elevated hcrt and hcrtr2 mRNA levels suggested an increased wakefulness, and those of dcx and neuN genes suggested an enhanced neurogenesis during the spring migration state. Further, compared to intact birds, the lower th and pmch, and higher hcrtr2 and neuN mRNA levels in castrates suggested a role of testicular steroids in modulation of the appetitive phase of reproduction, sleep and awake states, and neurogenesis during spring migration period. These results provide insights into molecular changes linked with important hypothalamic molecular pathways and steroidal influence in the photoperiodic induction of spring migration in obligate migratory songbirds.

Corydalis saxicola Bunting Total Alkaloids ameliorate diet-induced non-alcoholic steatohepatitis by regulating hepatic PI3K/Akt and TLR4/NF-κB pathways in mice.

Corydalis saxicola Bunting (Yanhuanglian), distributed in Southwest China, is mainly used for treatment of hepatitis, oral mucosal erosion, conjunctivitis, dysentery, acute abdominal pain and hemorrhoids in the folk. Corydalis saxicola Bunting Total Alkaloids (CSBTA) are the active ingredients extracted from the root of C. saxicola bunting. Non-alcoholic steatohepatitis (NASH) is the hinge between steatosis and cirrhosis in the spectrum of Non-alcoholic fatty liver disease (NAFLD), which has become one of the most common chronic liver diseases in the world. CSBTA can reduce tumors and brain diseases through anti-inflammatory and antioxidant pathways. Our study was designed to clarify the effects of CSBTA on the HFHC (High fat and high carbohydrate drinking) diet induced mice. In our research, A HFHC diet induced NASH mice model was applied to investigate the effects of CSBTA in vivo and obeticholic acid (OA) was set as positive control. Moreover, the underlying mechanisms were explored by palmitic acid (PA) and lipopolysaccharide (LPS) stimulated HepG2 cells in vitro. The in vivo study illustrated that CSBTA could alleviate mice away from the onset of NASH, and reduce intrahepatocellular lipid accumulation and hepatocyte inflammation under high fat condition. Further in vitro analysis confirmed that CSBTA attenuated inflammation and hepatic lipid accumulation by improving hepatic PI3K/Akt and suppressing hepatic TLR4/NF-κB pathways. In summary, this study demonstrated that CSBTA might be a promising compound for the treatment of NAFLD.

The Traditional Uses, Phytochemistry, Pharmacokinetics, Pharmacology, Toxicity, and Applications of Corydalis saxicola Bunting: A Review.

Background: Corydalis saxicola Bunting (CSB) is a perennial herb belonging to genus Corydalis (Papaveraceae), called "Yan-huang-lian" in the Chinese folk. Traditionally, it is used to treat acute conjunctivitis, corneal pannus, acute abdominal pain, hemorrhoidal bleeding, haematochezia, swelling, hepatitis, cirrhosis and liver cancer based on traditional Chinese medicine (TCM) concepts. Purpose: This review aims to summarize and analyze the pharmacokinetics, pharmacological and toxicological properties of CSB and its extracts; to highlight the relevance of modern pharmacology to traditional pharmacology; also to assess its therapeutic potential. Methods: CSB related literatures were searched and screened from databases including PubMed, Web of Science and CNKI. The selected literatures provided reliable source identification evidences. Results: In traditional medicine concepts, CSB has the effects of clearing away heat and detoxification, eliminating dampness, relieving pain, and stopping bleeding. Its modern pharmacology includes hepatoprotective, anticancer, anti-inflammatory, analgesic, antibacterial, anti-oxidative effects. Further, some pharmacological effects support its traditional uses. The CSB total alkaloids (CSBTA) are the main constituents isolated from this plant, and they exert the major of the pharmacological effects. Toxicological studies have shown that the toxicity of CSBTA is mild and reversible in rodents and beagle dogs. Conclusion: Although the present study summarizes the botany, phytochemistry, pharmacokinetics, pharmacology, toxicity, and applications of this plant, it is still necessary to systemically evaluate the chemistry, safety and parameters related to drug metabolism of the extracts or compounds from this plant before or in clinical trials in the future. Meanwhile, cancers and inflammatory-related diseases may be new research directions of this ethnomedicine.

Investigation of the Therapeutic Effect of Total Alkaloids of Corydalis saxicola Bunting on CCl4-Induced Liver Fibrosis in Rats by LC/MS-Based Metabolomics Analysis and Network Pharmacology.

Liver fibrosis is a pathological result of liver injury that usually leads to a pathophysiological wound healing response. The total alkaloids of Corydalis saxicola Bunting (TACS) have been used for hepatoprotective effects on the liver. However, its exact therapeutic mechanisms of liver fibrosis are not yet well understood. To explore the potential anti-fibrosis mechanism of TACS, metabolomics coupled with network pharmacology were applied to reveal the underlying mechanisms. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) combined with multivariate statistical analyses were performed to estimate changes in metabolic profiles. As a result, a total of 23 metabolites in rats with liver fibrosis were altered; of these, 11 had been downregulated and 12 had been upregulated compared with the control group. After TACS treatment, the levels of 13 metabolites were significantly restored compared with the CCl4-treated group, of which 4 metabolites were up-regulated and 9 metabolites were down-regulated. Many of these metabolites are involved in the bile acid metabolism, glutathione metabolism, tryptophan metabolism and purine metabolism. Then, three key targets, including cytochrome P450 family1 subfamily A member 1 (CYP1A1), ornithine decarboxylase 1 (OCD1) and monoamine oxidase Type B (MAOB) were predicted as potential therapeutic targets of TACS against liver fibrosis through network pharmacology analysis. Finally, palmatine, tetrahydropalmatine and dehydrocavidine were screened as potential active compounds responsible for the anti-fibrosis effect of TACS by molecular docking analysis. This study reveals that TACS exerted anti-fibrosis effects by regulating the liver metabolic pathway with multiple components and multiple targets, which is helpful to further clarify the hepatoprotective mechanisms of natural plant extracts.

Investigation of the hepatoprotective effect of Corydalis saxicola Bunting on carbon tetrachloride-induced liver fibrosis in rats by 1H-NMR-based metabonomics and network pharmacology approaches.

Liver fibrosis is a common consequence of chronic liver diseases resulting from multiple etiologies. Furthermore, prolonged unresolved liver fibrosis may gradually progress to cirrhosis, and eventually evolve into hepatocellular carcinoma (HCC). Corydalis saxicola Bunting (CS), a type of traditional Chinese folk medicine, has been reported to have hepatoprotective effects on the liver. However, the exact mechanism of how it cures liver fibrosis requires further elucidation. In this work, an integrated approach combining proton nuclear magnetic resonance (1H-NMR)-based metabonomics and network pharmacology was adopted to elucidate the anti-fibrosis mechanism of CS. Metabonomic study of serum biochemical changes by carbon tetrachloride (CCl4)-induced liver fibrosis in rats after CS treatment were performed using 1H-NMR analysis. Metabolic profiling by means of partial least squares-discriminate analysis (PLS-DA) indicated that the metabolic perturbation caused by CCl4 was reduced after CS treatment. As a result, lipids, leucine, alanine, acetate, O-acetyl-glycoprotein and creatine were significantly restored after CS treatment, which regulated valine, leucine and isoleucine metabolism; arginine and proline metabolism; lipid metabolism and pyruvate metabolism. Additionally, 157 potential targets of CS and 265 targets of liver fibrosis were identified by means of network pharmacology. Subsequently, 5 target proteins, which are the intersection of potential CS targets and liver fibrosis targets, indicated that CS has potential anti-fibrosis effects through regulating alanine aminotransferase (ALT) activity, the farnesoid X receptor (FXR), cyclooxygenase-2 (COX-2), matrix metalloproteinase-1 (MMP-1) and angiotensinogen. Chelerythrine and sanguinarine were the potential active compounds in CS for treating liver fibrosis through regulating ALT activity. This study is the first report to study the anti-fibrosis effects of CS on the basis of combining a metabonomics and network pharmacology approaches, and it may be a potentially powerful tool to study the efficacy and mechanisms of traditional Chinese folk medicines.

Inhibitory and inductive effects of Corydalis saxicola Bunting total alkaloids (CSBTA) on cytochrome P450s in rats.

Corydalis saxicola Bunting, a well-known traditional Chinese medicine in south China, has been widely used for the treatment of various hepatic diseases. Its active ingredients are Corydalis saxicola Bunting total alkaloids (CSBTA), which primarily include dehydrocavidine, palmatine, and berberine. These representative alkaloids could be metabolized by hepatic CYP450s. Hence, it is necessary to investigate the potential influences of CSBTA on CYP450s to explore the possibility of herb-drug interactions. In present study, in vitro inhibition and in vivo induction studies were performed to evaluate the potential effects of CSBTA extract on CYP450s in rats. Inhibition assay illustrated that CSBTA exerted inhibitory effects on CYP1A2 (IC50 , 38.08 µg/ml; Ki , 14.3 µg/ml), CYP2D1 (IC50 , 20.89 µg/ml; Ki , 9.34 µg/ml), CYP2C6/11 (IC50 for diclofenac and S-mephenytoin, 56.98 and 31.59 µg/ml; Ki, 39.0 and 23.8 µg/ml), and CYP2B1 (IC50 , 48.49 µg/ml; Ki , 36.3 µg/ml) in a noncompetitive manner. Induction study showed CSBTA had obvious inhibitory rather than inductive effects on CYP1A2 and CYP2C6/11. Interestingly, neither inhibition nor induction on CYP3A was observed for CSBTA. In conclusion, CSBTA-drug interactions might occur through CYP450s inhibition, particularly CYP1A and CYP2D. Further studies are still needed to elucidate the underlying mechanisms of inhibition.

Temporal Expression of c-fos and Genes Coding for Neuropeptides and Enzymes of Amino Acid and Amine Neurotransmitter Biosynthesis in Retina, Pineal and Hypothalamus of a Migratory Songbird: Evidence for Circadian Rhythm-Dependent Seasonal Responses.

This study investigated whether, in photoperiodic songbirds, the circadian pacemaker system (CPS) connects to the seasonal photoperiodic responses, by changes at transcriptional level in the level and 24-h rhythm of its constituent neurotransmitters. We used black-headed buntings (Emberiza melanocephala), which exhibit distinct seasonal states in captivity under appropriate photoperiods and hence served as a useful model system. Under short days, buntings remain in the photosensitive state (Pse) (winter phenotype: non-migratory, non-breeding). Under long days, however, buntings undergo through early-photostimulated (spring phenotype: pre-migratory, pre-breeding), late photostimulated (summer phenotype: migratory, breeding) and photorefractory (autumn phenotype: post-breeding) states. During all four seasonal states, we measured in the retina, pineal and hypothalamus, which together form avian CPS, 4-hourly mRNA expression of c-fos (a neuronal-activity marker) and of genes coding for neuropeptides (vasoactive intestinal peptide, vip; somatostatin, sst; neuropeptide Y, npy) and for intermediary enzymes of amino acid (glutamate: glutaminase, gls and glutamic-oxaloacetic transaminase 2, got2; GABA: glutamic acid decarboxylase, gad65) and amine (dopamine: tyrosine hydroxylase, th) neurotransmitters biosynthetic pathway. There was a significant alteration in level and 24-h pattern of mRNA expression, albeit with seasonal differences in presence, waveform parameters and phase relationship of 24-h rhythm, of different genes. Particularly, mRNA expression of all candidate genes (except hypothalamic vip, pineal gls and retinal th) was arrhythmic in late photostimulated state. These results underscore that circadian rhythm of peptide, amino acid and amine neurotransmitter biosynthesis in CPS plays a critical role in the photoperiodic regulation of seasonal states in birds.

Urinary metabonomics study of the hepatoprotective effects of total alkaloids from Corydalis saxicola Bunting on carbon tetrachloride-induced chronic hepatotoxicity in rats using 1H NMR analysis.

Chronic liver injury has been shown to cause liver fibrosis due to the sustained pathophysiological wound healing response of the liver, and eventually progresses to cirrhosis. The total alkaloids of Corydalis saxicola Bunting (TACS), a collection of important bioactive ingredients derived from the traditional Chinese folk medicine Corydalis saxicola Bunting (CS), have been reported to have protective effects on the liver. However, the underlying molecular mechanisms need further elucidation. In this study, the urinary metabonomics and the biochemical changes in rats with carbon tetrachloride (CCl4)-induced chronic liver injury due to treatment TACS or administration of the positive control drug-bifendate were studied via proton nuclear magnetic resonance (1H NMR) analysis. Partial least squares-discriminate analysis (PLS-DA) suggested that metabolic perturbation caused by CCl4 damage was recovered with TACS and bifendate treatment. A total of seven metabolites including 2-oxoglutarate, citrate, dimethylamine, taurine, phenylacetylglycine, creatinine and hippurate were considered as potential biomarkers involved in the development of CCl4-induced chronic liver injury. According to pathway analysis using identified metabolites and correlation network construction, the tricarboxylic acid (TCA) cycle, gut microbiota metabolism and taurine and hypotaurine metabolism were recognized as the most affected metabolic pathways associated with CCl4 chronic hepatotoxicity. Notably, the changes in 2-oxoglutarate, citrate, taurine and hippurate during the process of CCl4-induced chronic liver injury were significantly restored by TACS treatment, which suggested that TACS synergistically mediated the regulation of multiple metabolic pathways including the TCA cycle, gut microbiota metabolism and taurine and hypotaurine metabolism. This study could bring valuable insight to evaluating the efficacy of TACS intervention therapy, help deepen the understanding of the hepatoprotective mechanisms of TACS and enable optimal diagnosis of chronic liver injury.

Extra-hypothalamic brain clocks in songbirds: Photoperiodic state dependent clock gene oscillations in night-migratory blackheaded buntings, Emberiza melanocephala.

The avian circadian pacemaker system is comprised of independent clocks in the retina, pineal and hypothalamus, as shown by daily and circadian oscillations of core clock genes (Per2, Cry1, Bmal1 and Clock) in several birds including migratory blackheaded buntings (Emberiza melanocephala). This study investigated the extra-hypothalamic brain circadian clocks in blackheaded buntings, and measured Per2, Cry1, Cry2, Bmal1 and Clock mRNA expressions at 4h intervals over 24h beginning 1h after light-on in the left and right telencephalon, optic tectum and cerebellum, the brain regions involved in several physiological and cognitive functions. Because of seasonal alterations in the circadian clock dependent brain functions, we measured daily clock gene oscillations in buntings photoperiod-induced with the non-migratory state under short days (SDnM), and the pre-migratory (LDpM), migratory (LDM) and post-migratory (refractory, LDR) states under long days. Daily Per2 oscillations were not altered with changes in the photoperiodic states, except for about 2-3h phase difference in the optic tectum between the SDnM and LDpM states. However, there were about 3-5h differences in the phase and 2 to 4 fold change in the amplitude of daily Bmal1 and Cry1 mRNA oscillations between the photoperiod-induced states. Further, Cry2 and Clock genes lacked a significant oscillation, except in Cb (Cry2) and TeO and Rt (Clock) under LDR state. Overall, these results show the presence of circadian clocks in extra-hypothalamic brain regions of blackheaded buntings, and suggest tissue-dependent alterations in the waveforms of mRNA oscillations with transitions in the photoperiod-induced seasonal states in a long-day species.

Seasonal alterations in the daily rhythms in hypothalamic expression of genes involved in the photoperiodic transduction and neurosteroid-dependent processes in migratory blackheaded buntings.

The present study investigated seasonal alterations in the daily rhythms of hypothalamic expression of genes involved in the photoperiodic regulation of annual cycles in birds. We measured the 4-hourly mRNA expression of genes involved in the photoperiodic transduction (OPN5, EYA3, CGA, TSHß, DIO2, DIO3) and neurosteroid-dependent processes (AR, CYP19, ERα, ERß) in the hypothalamus of migratory blackheaded buntings photoinduced with photosensitive, photostimulated (early and late stimulated) and photorefractory seasonal states. There were significant differences in daily mRNA profiles between the photoperiodic states. Particularly, increased CGA, TSHß and DIO2 and decreased DIO3 mRNA levels in the early photostimulated state, compared to the photosensitive state, suggest that thyroid hormones have a role in photostimulation in buntings. Similar differences in the expression of genes coding for the aromatase enzyme (CYP19) and receptors for oestrogen (ERα, ERß) (but not androgen; AR) indicate that there is seasonal alteration in the neuro-oestrogen-mediated functions. Furthermore, peak expression times of CGA, TSHß and DIO2 genes at hours 14-15 of the day in the early stimulated state indicated molecular regulation of the daily rhythm of photoinducibility in buntings. Most significantly, however, we found an attenuated daily rhythm in thyroid hormone modulatory genes and a switch of peak expression time from day to night in CYP19 mRNA rhythm in the subsequent late photostimulated state, although testicular maturation still persisted. These alterations in daily rhythms may have signalled the initiation of processes underlying other seasonal phenologies in parallel with the gonadal response, such as a manifestation of the night-time flight in buntings. These results show alterations in daily rhythms underlying the transcriptional regulation of the photoperiod-induced seasonal states in migratory blackheaded buntings.

Concurrent hypothalamic gene expression under acute and chronic long days: Implications for initiation and maintenance of photoperiodic response in migratory songbirds.

Hypothalamic expression of the thyroid hormone (TH) responsive gonadostimulatory (eya3, cga, tshß, dio2, dio3, gnrh, gnih) and neurosteroid pathway genes (androgen receptor [ar], aromatase [cyp19], estrogen receptor [er] α and ß) was examined in photosensitive redheaded buntings exposed to 2 (acute, experiment 1) or 12 (chronic, experiment 2) long days (16L:8D). Experiment 2 also included a photorefractory group. Acute long days caused a significant increase in eya3, cga, tshß, dio2 and gnrh and decrease in dio3 mRNA levels. eya3, cga and tshß expressions were unchanged after the chronic long days. We also found increased cyp19, erα and erß mRNA levels after acute, and increased cyp19 and decreased erß levels after the chronic long-day exposure. Photorefractory buntings showed expression patterns similar to that in the photosensitive state, except for high gnrh and gnih and low dio3 mRNA levels. Consistent with gene expression patterns, there were changes in fat deposition, body mass, testis size, and plasma levels of testosterone, tri-iodothyronine and thyroxine. These results show concurrent photostimulation of the TH-signalling and neurosteroid pathways, and extend the idea, based on differences in gene expression, that transitions in seasonal photoperiodic states are accomplished at the transcriptional levels in absolute photorefractory species.

Serum metabonomics study of the hepatoprotective effect of Corydalis saxicola Bunting on carbon tetrachloride-induced acute hepatotoxicity in rats by (1)H NMR analysis.

Corydalis saxicola Bunting (CS), a traditional Chinese folk medicine, has been effectively used for treating liver disease in Zhuang nationality in South China. However, the exact hepatoprotective mechanism of CS was still looking forward to further elucidation by far. In present work, metabonomic study of biochemical changes in the serum of carbon tetrachloride (CCl4)-induced acute liver injury rats after CS treatment were performed using proton nuclear magnetic resonance ((1)H-NMR) analysis. Metabolic profiling by means of principal components analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) indicated that the metabolic perturbation caused by CCl4 was reduced by CS treatment. A total of 9 metabolites including isoleucine (1), lactate (2), alanine (3), glutamine (4), acetone (5), succinate (6), phosphocholine (7), d-glucose (8) and glycerol (9) were considered as potential biomarkers involved in the development of CCl4-induced acute liver injury. According to pathway analysis by metabolites identified and correlation network construction by Pearson's correlation coefficency matrix, alanine, aspartate and glutamate metabolism and glycerolipid metabolism were recognized as the most influenced metabolic pathways associated with CCl4 injury. As a result, notably, deviations of metabolites 1, 3, 4, 7 and 9 in the process of CCl4-induced acute liver injury were improved by CS treatment, which suggested that CS mediated synergistically abnormalities of the metabolic pathways, composed of alanine, aspartate and glutamate metabolism and glycerolipid metabolism. In this study, it was the first report to investigate the hepatoprotective effect of the CS based on metabonomics strategy, which may be a potentially powerful tool to interpret the action mechanism of traditional Chinese folk medicines.

Changes in brain peptides associated with reproduction and energy homeostasis in photosensitive and photorefractory migratory redheaded buntings.

Present study examined the expression of brain peptides associated with the reproduction and energy homeostasis (GnRH/GnIH, NPY/VIP), and assessed their possible functional association in the photosensitive (non-breeding, pre-breeding), photostimulated (breeding) and photorefractory (post-breeding) migratory redheaded buntings (Emberiza bruniceps), using double-labeled immunohistochemistry. Particularly, we measured immunoreactive (-ir) cell numbers, per cent cell area and cell optical density (OD) in the preoptic area (GnRH-I), midbrain (GnRH-II), paraventricular nucleus (GnIH), dorsomedial hypothalamus, DMH and infundibular complex, INc (NPY and VIP), and lateral septal organ (VIP) of buntings kept under natural photoperiods at the wintering latitude (26°55'N). There was a significant seasonal difference in GnRH-I, not GnRH-II, with reduced -ir cells in the photosensitive and photorefractory buntings, and notably with increased cell OD between the refractory and non-breeding states with no increase in testis size. Also, increased cell OD of GnIH neurons in non-breeding state indicated its role in the maintenance of small testes during the post-refractory period. Overall, seasonal changes in GnRH-I and GnIH were found consistent with their suggested roles in reproductive regulation of absolute photorefractory birds. Further, there was a significant seasonal change in cell OD of NPY neurons in DMH, not the INc. In contrast, VIP immunoreactivity was seasonally altered, with a significantly higher VIP-ir cells in breeding than the pre-breeding state. Finally, close proximity between perikarya with fibres suggested functional interactions between the GnRH and GnIH, and NPY and VIP. Thus, seasonal plasticity of brain peptides is perhaps the part of neural regulation of seasonal reproduction and associated energy homeostasis in migratory songbirds.

Pinealectomy abolishes circadian behavior and interferes with circadian clock gene oscillations in brain and liver but not retina in a migratory songbird.

In songbirds, the pineal gland is part of the multi-oscillatory circadian timing system, with participating component oscillators in the eyes and hypothalamus. This study investigated the role of the pineal gland in development of the nighttime migratory restlessness (Zugunruhe) and generation of circadian gene oscillations in the retina, brain and liver tissues in migratory redheaded buntings (Emberiza bruniceps). Pinealectomized (pinx) and sham-operated buntings entrained to short days (8h light: 16h darkness, 8L:16D) were sequentially exposed for 10days each to stimulatory long days (13L: 11D) and constant dim light (LLdim; a condition that tested circadian rhythm persistence). Whereas activity-rest pattern was monitored continuously, the mRNA expressions of clock genes (bmal1, clock, npas2, per2, cry1, rorα, reverα) were measured in the retina, hypothalamus, telencephalon, optic tectum and liver tissues at circadian times, CT, 1, 6, 13, 17 and 21 (CT 0, activity onset) on day 11 of the LLdim. The absence of the pineal gland did not affect the development of long-day induced Zugunruhe but caused decay of the circadian rhythm in Zugunruhe as well as the clock gene oscillations in the hypothalamus, but not in the retina. Further, there were variable effects of pinealectomy in the peripheral brain and liver tissue circadian gene oscillations, notably the persistence of per 2 and cry1 (optic tectum), rorα (telencephalon) and npas2 (liver) mRNA oscillations in pinx birds. We suggest the pineal gland dependence of the generation of circadian gene oscillations in the hypothalamus, not retina, and peripheral brain and liver tissues in migratory redheaded buntings.

Circadian timing in central and peripheral tissues in a migratory songbird: dependence on annual life-history states.

Predictable seasonal change in photoperiod triggers a sequential change in the daily activity-rest pattern, adaptive for migration in several bird species. The night-migratory black-headed bunting (Emberiza melanocephala) is day active under short photoperiods (8 h light:16 h dark, short day sensitive). Under long photoperiods (16 h light:8 h dark), the buntings are initially day active (long day premigratory) but subsequently become intensely night active (long day migratory) and after few weeks again return to a day active pattern (long day refractory). However, it is unclear how the daily expression of circadian genes changes during photoperiod-induced seasonal life-history states (LHSs). We measured period 2 (Per2), cryptochrome 1 (Cry1), brain and muscle arnt-like protein 1 (Bmal1), and circadian locomotor output cycles kaput (Clock) mRNA expressions in various neural and peripheral tissues of buntings in different LHSs and discovered differences of ∼2 to 6 h in the phase and 2- to 4-fold in amplitude of circadian oscillations of Per2, Cry1, and Bmal1 between photoperiod-induced LHSs. Phase relationship in mRNA oscillations was altered between oscillator components in the circadian pacemaker system (retina, pineal, hypothalamus) as well as in the peripheral (liver, muscle) tissues. These results show for the first time altered waveforms of clock gene expressions in all tissues in parallel with behavioral shifts and suggest the involvement of circadian system in photoperiod induction of seasonal LHSs in a migratory species.

Circadian synchronization determines critical day length for seasonal responses.

A photoperiodic species initiates fat deposition (in migrants) and gonadal recrudescence in response to a specific duration of natural daylight, called critical day length (CD), when light extends in the inductive phase of the endogenous circadian rhythm of photoinducibility (CRP). The molecular basis of species-specificCD, determined by the entrainment of the CRP, has been poorly understood. To investigate this, we measured expression levels of genes implicated in the photoperiod-induced changes in reproduction (EYA3, TSH beta, DIO2, DIO3, GNRH and GNIH) and metabolism (SIRT1, HMGCR, FASN and PPAR alpha) in photosensitive redheaded buntings subjected to light-dark cycles of varying period lengths (T-photocycles). Buntings were exposed to six T22, T24 or T26 photocycles, with 1h additional light at night falling at different phases of the entrained CRP (T2211L=6L:4D:1L:11D; T2411L=6L:4D:1L:13D,T2412L=6L:5D:1L:12D, T2413L=6L:6D:1L:11D; T2612L=6L:5D:1L:14D). Photoinduction at genetic and phenotypic levels in T2412L and T2413L, not T2411L, groups confirmed CD being close to 12h in buntings under T24. Compared to T24, exposure to T22 advanced CD by 1h, as evidenced by photoinduction in the T2211L, not T226L, group. Similarly, CD appeared to be delayed under T26, with no photoinduction in the T2612L group. Further, to show that induction of response under a T-photocycle was because of the interaction of inductive phase of the CRP with 1h during the dark period in each cycle, not with the 6h main light periods falling 2h earlier each successive 24hday in a T22 paradigm, a group of buntings was exposed to 6L:16D (T226L), to which they did not respond. The mRNA expression of genes, particularly TSH beta, DIO2, DIO3 and PPAR alpha, was significantly correlated with changes in reproductive and metabolic phenotypes. These results suggest CRP-entrainment based genetic regulation of the CD, and extend the idea that synchronization with environment is a critical measure in a seasonal species for its temporal adaptation in the wild.

Hypothalamic gene switches control transitions between seasonal life history states in a night-migratory photoperiodic songbird.

This study investigated photoperiodic plasticity in hypothalamic expression of genes implicated in the photoperiodic light perception (rhodopsin, melanopsin, neuropsin and peropsin), transduction (pax6, bmal1, clock, per2 and casr), induction (eya3, tshß, dio2 and dio3, gnrh and gnih) and metabolism (NPY, sirtuin1, foxO1, hmgcr, citrate synthase and dehydrogenases) in photosensitive and photorefractory redheaded buntings. There was a significant increase in eya3, tsh ß, dio2, pax6 and rhodopsin and decrease in dio3 mRNA expression at hour 15 and/or 19 on the day photosensitive buntings were subjected to a 13- or 16 h, but not to 8- and 11 h light exposure. Downstream reproductive and metabolic gene expression was not altered, except for an increase in those genes coding for succinate and malate dehydrogenase enzymes involved in lipogenesis. Photorefractory buntings had high dio3 mRNA expression which significantly declined after 1 short day exposure, suggesting possible involvement of dio3 in the maintenance of photorefractoriness. Positive correlation of rhodopsin on eya 3 and tshß indicates its role in photoperiodic timing, perhaps involving the peropsin and pax6 genes. These results suggest that rapid switching of hypothalamic gene expression underlies photoperiod-induced seasonal plasticity and regulates transitions from photosensitive to photostimulated and from photorefractory to photosensitive states in migratory songbirds.