Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Molecular characterization and potential function of Rxrγ in gonadal differentiation of Chinese soft-shelled turtle (Pelodiscus sinensis).

Retinoid X receptor (RXR) is a member of the ligand-dependent nuclear receptor family. Previous studies revealed that RXRs are involved in reproduction in vertebrates. However, information on the function of RXRs in turtles is scarce. In this study, the Rxrγ cDNA sequence of Pelodiscus sinensis was cloned and analyzed, and a polyclonal antibody was constructed. RXRγ protein showed a positive signal in both mature and differentiated gonads of the turtle. Subsequently, the function of the Rxrγ gene in gonadal differentiation was confirmed using short interfering RNA (RNAi). The full-length cDNA sequence of the Rxrγ gene in P. sinensis was 2152 bp, encoding 407 amino acids and containing typical nuclear receptor family domains, including the DNA-binding domain (DBD), ligand-binding domain (LBD), and activation function 1 (AF1). Moreover, gonadal Ps-Rxrγ showed sexual dimorphism expression patterns in differentiated gonads. Real-time quantitative PCR results revealed that the Rxrγ gene was highly expressed in the turtle ovary. RNAi treatment increased the number of Sertoli cells in ZZ embryonic gonads. Furthermore, RNA interference upregulated Dmrt1 and Sox9 in ZZ and ZW embryonic gonads. However, Foxl2, Cyp19a1, Stra8, and Cyp26b1 were downregulated in embryonic gonads. The results indicated that Rxrγ participated in gonadal differentiation and development in P. sinensis.

Oridonin employs interleukin 1 receptor type 2 to treat noise-induced hearing loss by blocking inner ear inflammation.

NOD-like receptor protein 3 (NLRP3) inflammasomes trigger the inflammatory cascades and participate in various inflammatory diseases, including noise-induced hearing loss (NIHL) caused by oxidative stress. Recently, the anti-inflammatory traditional medicine oridonin (Ori) has been reported to provide hearing protection in mice after noise exposure by blocking the NLRP3-never in mitosis gene A-related kinase 7 (NEK7)-inflammasome complex assembly. Using RNA sequencing analysis, we further elucidated that interleukin 1 receptor type 2 (IL1R2) may be another crucial factor regulated by Ori to protect NIHL. We observed that IL1R2 expression was localized in spiral ganglion neurons, inner and outer hair cells, in Ori-treated mouse cochleae. Additionally, we confirmed that ectopic overexpression of IL1R2 in the inner ears of healthy mice using an adeno-associated virus delivery system significantly reduced noise-induced ribbon synapse lesions and hearing loss by blocking the "cytokine storm" in the inner ear. This study provides a novel theoretical foundation for guiding the clinical treatment of NIHL.

Identification, characterization and differential expression analysis of a pteridine synthesis related gene, Ccptps, in koi carp (Cyprinus carpio L.).

6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second key enzyme of the pteridine biosynthetic pathway and it plays vital roles in fish body color formation. In this study, Ccptps of koi carp (Cyprinus carpio L.) was cloned, identified and characterized. The full-length cDNA of Ccptps was 1140 bp and encodes for 139 amino acids. Multiple alignments revealed that the amino acids sequence of CcPTPS shared the highest identity to that of C. carpio, and Ccptps was clustered with cyprinid fishes in phylogenetic tree. Liver tissues of koi carp exhibited the highest expression of Ccptps, followed by muscle and skin tissues. During early developmental stages, the expression of Ccptps declined from 2 dph to 4 dph, and increased from 4 dph to 12 dph. The expressions of Ccptps in three color-related tissues (skin, scale and caudal fin) of whole red (WR) koi carp were significantly higher than that of whole while (WW) koi carp. Immunohistochemistry results of skin tissues showed that CcPTPS was mainly located in epidermis, stratum compactum of dermis and muscle layer, with the signal intensities in stratum compactum and muscle layer were stronger in WR koi carp compared to WW koi carp. Co-expressions of CcPTPS, CcSPR and CcXDH were detected in skin tissues of WW and WR koi carps, with CcPTPS exhibited stronger signal intensity compared to CcSPR and CcXDH. These findings imply that Ccptps is potentially involved in koi carp body color formation through the pteridine synthesis pathway.

Integrating serum pharmacochemistry and network pharmacology to identify chemical markers for quality control of Apocyni Veneti Folium.

INTRODUCTION: Apocyni Veneti Folium (AVF) is a commonly used traditional Chinese medicinal herb for the treatment of hypertension. Chemical markers are crucial for the quality control of herbal medicines; however, the therapeutic components of AVF remain to be well elucidated. OBJECTIVES: This study was intended to integrate serum pharmacochemistry and network pharmacology to identify chemical markers of AVF and establish an efficacy-related quality control method of AVF. MATERIAL AND METHODS: Ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) was applied to identify the absorbed AVF constituents in rat serum. Network pharmacology was further used to identify anti-hypertension-related chemical markers. Subsequently, a quantitative method was established using UPLC with diode array detection (DAD) and applied for quality evaluation of commercial AVF samples. RESULTS: Thirteen prototype constituents were unequivocally or tentatively characterized in serum samples, among which quercetin, kaempferol, hyperoside, isoquercitrin, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid were identified as dominant chemicals related to anti-hypertensive efficacy. The quantitative data showed that the total contents of seven marker components even showed 2-fold variation among 14 batches of commercial AVF samples with RSD values ranging from 12.15% to 75.61%. Hierarchical cluster analysis and heatmap analysis showed that 14 batches of commercial AVF samples could be divided into three main groups. CONCLUSION: The chemical markers obtained from this study could be applicable for efficacy-related quality control of AVF.

Water Intake and Hydration Status among Pregnant Women in the Second Trimester in China: A Three-Day Metabolic Trial.

Adequate water intake and optimal hydration status during pregnancy are crucial for maternal and infant health. However, research on water intake by pregnant women in China is very limited. This study mainly aimed to observe the daily total water intake (TWI) of pregnant women and its different sources and to investigate the relationship between their water intake and hydration biomarkers. From October to November 2020, a convenience sample of pregnant women in the second trimester (n = 21) was recruited. Under conditions close to daily life, they undertook a 3-day metabolic trial. Each participant was provided with sufficient bottled water, and the weight of what they drank each time was measured. The intake of other beverages and foods was measured using a combination of weighing and duplicate portion method. Fasting venous blood and 24 h urine samples were collected and analyzed for the hydration biomarkers, including the serum/urine osmolality, urine pH, urine specific gravity, and the concentrations of major electrolytes in urine and serum. The results showed that the mean daily TWI was 3151 mL, of which water from beverages and foods accounted for 60.1% and 39.9%, respectively. The mean total fluid intake (TFI) was 1970 mL, with plain water being the primary contributor (68.7%, r = 0.896). Among the participants, 66.7% (n = 14, Group 1) met the TWI recommendation set by the Chinese Nutrition Society. Further analysis revealed that the TFI, water from beverages and foods, plain water, and milk and milk derivatives (MMDs) were significantly higher in Group 1 than those who did not reach the adequate intake value (Group 2) (p < 0.05). The results of hydration biomarkers showed that the mean 24 h urine volume in Group 1 was significantly higher than that in Group 2 (p < 0.05), while the 24 h urine osmolality, sodium, magnesium, phosphorus, chloride, and creatinine concentrations in Group 1 were significantly lower than those in Group 2 (p < 0.05). However, no significant differences were observed in serum biomarkers. Partial correlation analysis showed that TWI was moderately positively correlated with 24 h urine volume (r = 0.675) and negatively correlated with urine osmolality, sodium, potassium, magnesium, calcium, phosphorus, and chloride concentrations (r = from-0.505 to -0.769), but it was not significantly correlated with serum biomarkers. Therefore, under free-living conditions, increasing the daily intake of plain water and MMDs is beneficial for pregnant women to maintain optimal hydration. The hydration biomarkers in urine are more accurate indicators of water intake and exhibit greater sensitivity compared to serum biomarkers. These findings provide a scientific basis for establishing appropriate water intake and hydration status for pregnant women in China.

Traditional Chinese Medicine in Treatment of COVID-19 and Viral Disease: Efficacies and Clinical Evidence.

Coronavirus disease 2019 (COVID-19) remains an uncontained, worldwide pandemic. While battling the disease in China, the Chinese government has actively promoted the use of traditional Chinese medicine, and many studies have been conducted to determine the efficacy of traditional Chinese medicine for treating COVID-19. The present review discusses the effectiveness and safety of traditional Chinese medicine in curing COVID-19 and provides clinical evidence from all confirmed cases in China. Applications of traditional Chinese medicine and specific recipes for treating other viral infections, such as those caused by severe acute respiratory syndrome coronavirus and influenza A viruses (including H1N1), are also discussed. Studies have reported that traditional Chinese medicine treatment plays a significant role in improving clinical symptoms. Therefore, further investigation may be of high translational value in revealing novel targeted therapies for COVID-19.

ROS-Responsive Nanoparticle as a Berberine Carrier for OHC-Targeted Therapy of Noise-Induced Hearing Loss.

Overproduction of reactive oxygen species (ROS) and inflammation are two key pathogeneses of noise-induced hearing loss (NIHL), which leads to outer hair cell (OHC) damage and hearing loss. In this work, we successfully developed ROS-responsive nanoparticles as berberine (BBR) carriers (PL-PPS/BBR) for OHC-targeted therapy of NIHL: Prestin-targeting peptide 2 (PrTP2)-modified nanoparticles (PL-PPS/BBR), which effectively accumulated in OHC areas, and poly(propylene sulfide)120 (PPS120), which scavenged ROS and converted to poly(propylene sulfoxide)120 in a ROS environment to disintegrate and provoke the rapid release of BBR with anti-inflammatory and antioxidant effects. In this study, satisfactory anti-inflammatory and antioxidant effects of PL-PPS/BBR were confirmed. Immunofluorescence and scanning electron microscopy (SEM) images showed that PL-PPS/BBR effectively accumulated in OHCs and protected the morphological integrity of OHCs. The auditory brainstem response (ABR) results demonstrated that PL-PPS/BBR significantly improved hearing in NIHL guinea pigs after noise exposure. This work suggested that PL-PPS/BBR may be a new potential treatment for noise-associated injury with clinical application.

A novel sample-preparation method for the generic and rapid determination of pesticides and mycotoxins in tea by ultra-performance liquid chromatography-tandem mass spectrometry.

A rapid, simple, and generic analytical method for the simultaneous determination of 140 undesirable low-weight pesticides and mycotoxins from different chemical classes in black tea was developed. The method involved swelling the sample in ammonium acetate buffer, extraction with acetonitrile-dimethyl sulfoxide, cleanup by dual dispersive solid-phase extraction (D-SPE) with the assistance of low-temperature centrifugation, and analysis by ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry using multiple reaction monitoring mode. The interferences in the extract were eliminated by the combination of dual d-SPE using only C18 sorbent and anhydrous magnesium sulfate, which maintained the chromatographic column under the ideal condition for a long time and enabled satisfactory recoveries of hydrophobic and hydrophilic analytes simultaneously. Matrix-matched calibration curves were obtained for most target compounds with linear regression coefficients above 0.9900. The limits of quantification (LOQs) ranged within 0.5-10.0 µg/kg, which were usually sufficient to verify the compliance of products with legal tolerances. Satisfactory recoveries of 64.5%-138.1% were obtained in black ta samples with the relative standard deviation (RSD) values between 1.8 and 25.9%. The inter-day precision ranged within 2.2%-24.9%. For over 90% of the analytes, the recoveries were between 70% and 120%, with RSD values below 15.0%. The application of this method in routine monitoring programs can drastically reduce effort and time.

Penctrimertone, a bioactive citrinin dimer from the endophytic fungus Penicillium sp. T2-11.

Penctrimertone (1), a novel citrinin dimer bearing a 6/6/6/6 tetracyclic ring scaffold, along with two known compounds xerucitrinic acid A (2) and citrinin (3) were isolated from the endophytic fungus Penicillium sp. T2-11. Their structures were unequivocally established by a comprehensive interpretation of the spectroscopic data, with the stereochemistry for 1 was defined by a combination of TDDFT-ECD calculations and the DP4+ probability analysis based on NMR chemical shift calculations. Bioassays revealed that compound 1 exhibited noticeable antimicrobial activities and moderate cytotoxicity. A plausible biosynthetic pathway of 1 was also proposed.

Generic and rapid determination of low molecular weight organic chemical contaminants in protein powder by using ultra-performance liquid chromatography-tandem mass spectrometry.

A rapid, simple, and generic analytical method that could simultaneously determine 291 undesirable low molecular weight chemical contaminants from different drug families in protein powder, such as veterinary drugs and pesticides, etc, had been developed. This method comprised the extraction with acetonitrile-dimethyl sulfoxide (DMSO), clean-up through dispersive solid phase extraction (D-SPE) and low temperature filtration, and analysis by ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry at multiple-reaction monitoring mode. Acetonitrile-DMSO was more generic than acetonitrile or methanol for the extraction of large-scale organic chemical contaminants with different polarities in protein powder. Most interferences in the extract were eliminated by the combination of D-SPE and low temperature filtration, which simultaneously provided satisfactory recoveries of both hydrophobic and hydrophilic analytes. In particular, besides the purification function, the sorbent of D-SPE also played an important role in grinding samples to improve extraction efficiency during homogenization. This streamlined approach allowed the processes of extraction and the main purification were carried out in one-step, and dramatically reduced sample preparation turnaround times and solvent consumption. For quantification, matrix-fortified calibration curves showed competent linearity for most of the target compounds with linear regression coefficients (r) higher than 0.9900, except for two analytes. The limits of quantification ranged from 0.1 µg/kg to 50 µg/kg, which was usually sufficient to verify the compliance of products with legal tolerances. The average recoveries for spiked protein powder ranged from 65.6% to 142.2% with associated RSD values between 0.5% and 28.5%. For over 90% of the analytes, the recoveries were between 70% and 120% with RSD values in the range of 1%-15%. Applying this method in routine monitoring programs would drastically reduce both effort and time.

Isoliquiritigenin inhibits the proliferation, migration and metastasis of Hep3B cells via suppressing cyclin D1 and PI3K/AKT pathway.

The overall survival rate of patients with hepatocellular carcinoma (HCC) has remained unchanged over the last several decades. Therefore, novel drugs and therapies are required for HCC treatment. Isoliquiritigenin (ISL), a natural flavonoid predominantly isolated from the traditional Chinese medicine Glycyrrhizae Radix (Licorice), has a high anticancer potential and broad application value in various cancers. Here, we aimed to investigate the anticancer role of ISL in the HCC cell line Hep3B. Functional analysis revealed that ISL inhibited the proliferation of Hep3B cells by causing G1/S cell cycle arrest in vitro. Meanwhile, the inhibitory effect of ISL on proliferation was also observed in vivo. Further analysis revealed that ISL could suppress the migration and metastasis of Hep3B cells in vitro and in vivo. Mechanistic analysis revealed that ISL inhibited cyclin D1 and up-regulated the proteins P21, P27 that negatively regulate the cell cycle. Furthermore, ISL induced apoptosis while inhibiting cell cycle transition. In addition, phosphatidylinositol 3'-kinase/protein kinase B (PI3K/AKT) signal pathway was suppressed by ISL treatment, and the epithelial marker E-cadherin was up-regulated when the mesenchymal markers Vimentin and N-cadherin were down-regulated. In brief, our findings suggest that ISL could be a promising agent for preventing HCC tumorigenesis and metastasis.

[The Research Advancement and Conception of the Deep-underground Medicine].

The 21th century is the century of exploring and utilizing the underground space. In the future, more and more people will spend more and more time living or/and working in the underground space. However,we know little about the effect on the health of human caused by the underground environment. Herein,we systematically put forward the strategic conception of the deep-underground medicine,in order to reveal relative effects and mechanism of the potential factors in the deep underground space on human's physiological and psychological healthy,and to work out the corresponding countermeasures. The original deep-underground medicine includes the following items. ①To model different depth of underground environment according to various parameters (such as temperature,radiation,air pressure, rock,microorganism), and to explore their quantitative character and effects on human health and mechanism. ② To study the psychological change, maintenance of homeostasis and biothythm of organism in the deep underground space. ③ To learn the association between psychological healthy of human and the depth, structure, physical environment and working time of underground space. ④ To investigate the effect of different terrane and lithology on healthy of human and to deliberate their contribution on organism growth. ⑤ To research the character and their mechanism of growth,metabolism,exchange of energy,response of growth, aging and adaptation of cells living in deep underground space. ⑥ To explore the physiological feature,growth of microbiome and it's interaction with host in the deep underground space. ⑦ To develop deep-underground simulation space, the biologically medical technology and equipments. As a research basis,a deep-underground medical lab under a rock thickness of about 1 470 m has been built,which aims to operate the research of the effect on living organism caused by different depth of underground environment.

Neo-clerodane and abietane diterpenoids with neurotrophic activities from the aerial parts of Salvia leucantha Cav.

Four new neoclerodane diterpenoids, leucansalvialins FI (1-4), and one rare 18(4 → 3)-abeo-abietane diterpenoid, leucansalvialin J (5), were isolated from the aerial part of Salvia leucantha Cav., along with 14 known analogues. Leucansalvialin F (1) represents the first rearranged salvigenane type clerodane-17,12:18,6-diolide. Their structures were elucidated by 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of 1, 2, 3, and 5 were determinded by X-ray diffraction crystal analysis and the ECD technique. All of the isolated components were evaluated for their neurotrophic activities on PC12 cells and all new compounds were evaluated for their cytotoxicity against five human cancer cell lines (HL-60, A-549, SMMC-7721, MCF-7, and SW480). Compounds 2 and 5 showed moderate neuroprotective effects in an in vitro assay.

Anisucoumaramide, a Bioactive Coumarin from Clausena anisum-olens.

A new coumarin, anisucoumaramide (1), and a new δ-truxinate derivative, anisumic acid (2), were isolated from Clausena anisum-olens. Their structures were elucidated from extensive NMR and MS data. The absolute configurations of the coumarins were assigned using the experimental and calculated electronic circular dichroism data. Anisucoumaramide (1) represents the first example of a naturally occurring coumarin of which the terpenoidal side chain does not comply with the biosynthesis isoprene rule due to the presence of an unprecedented acetamido motif directly connected with the terpenoidal side chain. The δ-truxinate derivative was isolated from Clausena species for the first time. Compound 1 showed high selectivity for the MAO-B isoenzyme and inhibitory activity in the nanomolar range. Putative biosynthesis pathways toward 1 and 2 are proposed.

Cloning and expression of two hepcidin genes in the mudskipper (Boleophthalmus pectinirostris) provides insights into their roles in male reproductive immunity.

The mudskipper Boleophthalmus pectinirostris is a burrow-dwelling fish inhabiting intertidal mudflats. During the spawning season, in a spawning chamber located at the center of their burrow, a pair of male and female fish mate and fertilized eggs adheres onto the inner walls and ceiling with filamentous attachments. During 5 days of incubation, the fertilized eggs are kept clean and hatch with a very high hatching rate under the natural conditions filled with microorganisms. This suggests that the male and/or female reproductive tract may synthesize antimicrobial substances to offer protection against microorganisms that may be deleterious to fertility. To study the antimicrobial strategy of this fish in the spawning season, we first cloned the two hepcidin isoforms from B. pectinirostris, and designated them as Hepcidin-1 and Hepcidin-2 based on phylogenetic analyses. Both of these hepcidin isoforms were highly expressed in the liver, but only Hepcidin-1 showed significant change in response to iron overload. Interestingly, these two hepcidin isoforms were expressed in male reproductive tracts, i.e. the testes and seminal vesicles. The monthly expression pattern indicated that Hepcidin-1 transcript levels showed a peak point only in March (before spawning) in the seminal vesicle, while Hepcidin-2 transcript levels were correlated with male reproductive status and reached their highest level in May (the peak spawning period). Under experimental conditions, the expression of these two hepcidin isoforms showed no response to iron overload in the male gonad. However, after lipopolysaccharide injection, the Hepcidin-1 transcript level was significantly up-regulated in the testes and seminal vesicle 6 h post injection, while Hepcidin-2 transcript levels exhibited a clear time-course dependent upregulation pattern and reached the highest levels 24 h post injection. More interestingly, after injection with LHRH-A3, the expression of Hepcidin-2 was significantly up-regulated in both testes and seminal vesicle. Results from in situ hybridization showed that Hepcidin-2 was expressed in the Leydig cells of the testes and in the epithelium of the seminal vesicle. Taken together, the results from our study indicated that these two hepcidin isoforms in the mudskipper may have different functions: Hepcidin-1 may play a dual role in both iron metabolism regulation in the liver and a short antimicrobial response in male reproductive tracts, while Hepcidin-2 is more specialized in reproductive immunity in male reproductive tracts.

Effects and mechanisms of waterborne copper exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco.

The present study was conducted to determine the effects and mechanism of waterborne copper (Cu) exposure influencing ovary development and related hormones secretion in yellow catfish Pelteobagrus fulvidraco. To this end, two experiments were conducted. In Exp. 1, the partial cDNA sequences of three steroidogenesis-related genes (androgen receptor (ar), steroidogenic factor 1 (sf-1) and steroidogenic acute regulatory protein (star)) were firstly characterized from P. fulvidraco. The predicted amino acid sequences for the P. fulvidraco ar, sf-1 and star contained the main structural features characteristic in other species. In Exp. 2, P. fulvidraco were exposed to three waterborne Cu concentrations (control, 30µg/l and 60µg/l, respectively) for 56days. Sampling occurred on day 28 and day 56, respectively. On day 28, the levels of serum sex-steroid hormones (FSH and LH) and the mRNA levels of steroidogenesis-related genes (3ß-hsd, cyp11a1, cyp17, cyp19a, sf-1 and star) were significantly increased in ovary of P. fulvidraco exposed to 30µg Cu/l. The immunohistochemical analysis showed the positive reaction of ER, VTG and aromatase in low dose exposure group. These indicated that in low dose and relative short-term exposure, Cu was beneficial. In contrast, 60µg Cu/l exposure significantly reduced the levels of serum FSH, LH, E2 and P, and the mRNA levels of ovarian 20ß-hsd, cyp19a and erα in P. fulvidraco. On day 56, waterborne Cu concentration exposure reduced the levels of serum gonadotropins and sex hormones, and down-regulated the mRNA levels of steroidogenesis-related genes, indicating long-term Cu exposure had toxic effect on the secretion of sex-steroid hormone in P. fulvidraco. For the first time, our study cloned cDNA sequences of ar, sf-1 and star in P. fulvidraco, and demonstrated the effects and mechanism of waterborne Cu exposure influencing hormones secretion and synthesis in dose- and time-dependent manner in P. fulvidraco, which will help to understand the Cu-induced reproductive toxicity at both protein and transcriptional levels in fish.

Endoplasmic reticulum stress and dysregulation of calcium homeostasis mediate Cu-induced alteration in hepatic lipid metabolism of javelin goby Synechogobius hasta.

The present study was conducted to investigate the effect of Cu exposure on endoplasmic reticulum (ER) stress and Ca(2+) homeostasis, and also explore the underlying mechanism of the ER stress and Ca(2+) homeostasis in the Cu-induced change of hepatic lipid metabolism in javelin goby Synechogobius hasta. To this end, four experiments were conducted. In experiment 1, the full-length cDNA sequences of two ER molecular chaperones [glucose-regulated protein 78 (GRP78) and calreticulin (CRT)] and three ER stress sensors [PKR-like ER kinase (PERK), inositol requiring enzyme (IRE)-1α, and activating transcription factor (ATF)-6α] cDNAs were firstly characterized from S. hasta. The predicted amino acid sequences for the S. hasta GRP78, CRT, PERK, IRE-1α and ATF-6α revealed that the proteins contained all of the structural features characteristic in other species. mRNAs of the five genes were expressed in various tissues, but their mRNA levels varied among tissues. In experiment 2, S. hasta were exposed to four waterborne Cu concentrations (control, 19µg/l, 38µg/l, and 57µg/l, respectively) for 60days. Cu exposure evoked ER stress in liver of S. hasta in a time- and concentration-course change. In experiment 3, specific inhibitors, 2-aminoethyldiphenyl borate (2-APB) and dantrolene, were used to explore whether Ca(2+) release from ER was involved in the Cu-induced ER stress change. Dantrolene and 2-APB prevented Cu-induced intracellular Ca(2+) elevation, which demonstrated the release of Ca(2+) from the ER was mediated by both RyR and IP3R. In experiment 4, a chemical chaperone, 4-phenyl butyric acid (4-PBA), was used to demonstrate whether Cu-induced alteration in lipid metabolism was suppressed through the attenuation of ER stress. Cu exposure evoked ER stress and sterol-regulator element-binding protein-1c (SREBP-1c) activation in hepatocytes of S. hasta, resulting in dysregulation of hepatic lipid metabolism. 4-PBA attenuated the Cu-induced elevation of mRNA expression of ER stress-related genes. For the first time, our study cloned GRP78, CRT, PERK, IRE-1α and ATF-6α genes in S. hasta and demonstrated their differential expression among tissues. Moreover, the study demonstrated the molecular mechanism by which ER stress might underlie the change of lipid metabolism induced by Cu in S. hasta.

[Clinical implication of urinary protein markers in diabetic nephropathy and interventional effects of Chinese herbal medicine].

In clinic, some urinary protein makers can dynamically and noninvasively reflect the degree of renal tubular injury in patients with diabetic nephropathy (DN). These urinary biomarkers of tubular damage are broadly divided into two categories. One is newfound, including kidney injury molecule-1 (Kim-1), neutrophil getatinase-associated lipocalin (NGAL), liver-type fatty acid-binding protein (L-FABP) and cystatin C (CysC); the other one is classical, including beta2 microglobulin (beta2-MG), retinal binding protein (RBP) and N-acetyl-beta-D-glucosaminidase (NAG). It is reported that, the increases in urinary protein markers are not only closely related to the damage of tubular epithelial cells in DN patients, but also can be ameliorated by the treatment with Chinese herbal compound preparations or Chinese herbal medicine. Recently, although urinary proteomics are used in the protein separation and identification, the traditional associated detection of urinary protein markers is more practical in clinic. At present, it is possible that the associated detection of urinary biomarkers of glomerular and tubular damages may be a feasible measure to reveal the clinical significance of urinary protein markers in DN patients and the interventional effects of Chinese herbal medicine.