Construction of prognostic risk model of bladder cancer based on cuproptosis-related long non-coding RNAs. / åŸºäºŽé“œæ­»äº¡ç›¸å ³é•¿é“¾éžç¼–ç RNAæž„å»ºè†€èƒ±ç™Œæ‚£è€ é¢„åŽé£Žé™©è¯„ä¼°æ¨¡åž‹.

OBJECTIVES: To construct a prognosis risk model based on long noncoding RNAs (lncRNAs) related to cuproptosis and to evaluate its application in assessing prognosis risk of bladder cancer patients. METHODS: RNA sequence data and clinical data of bladder cancer patients were downloaded from the Cancer Genome Atlas database. The correlation between lncRNAs related to cuproptosis and bladder cancer prognosis was analyzed with Pearson correlation analysis, univariate Cox regression, Lasso regression, and multivariate Cox regression. Then a cuproptosis-related lncRNA prognostic risk scoring equation was constructed. Patients were divided into high-risk and low-risk groups based on the median risk score, and the immune cell abundance between the two groups were compared. The accuracy of the risk scoring equation was evaluated using Kaplan-Meier survival curves, and the application of the risk scoring equation in predicting 1, 3 and 5-year survival rates was evaluated using receiver operating characteristic (ROC) curves. Univariate and multivariate Cox regression were used to screen for prognostic factors related to bladder cancer patients, and a prognostic risk assessment nomogram was constructed, the accuracy of which was evaluated with calibration curves. RESULTS: A prognostic risk scoring equation for bladder cancer patients was constructed based on nine cuproptosis-related lncRNAs. Immune infiltration analysis showed that the abundances of M0 macrophages, M1 macrophages, M2 macrophages, resting mast cells and neutrophils in the high-risk group were significantly higher than those in the low-risk group, while the abundances of CD8+ T cells, helper T cells, regulatory T cells and plasma cells in the low-risk group were significantly higher than those in the high-risk group (all P<0.05). Kaplan-Meier survival curve analysis showed that the total survival and progression-free survival of the low-risk group were longer than those of the high-risk group (both P<0.01). Univariate and multivariate Cox analysis showed that the risk score, age and tumor stage were independent factors for patient prognosis. The ROC curve analysis showed that the area under the curve (AUC) of the risk score in predicting 1, 3 and 5-year survival was 0.716, 0.697 and 0.717, respectively. When combined with age and tumor stage, the AUC for predicting 1-year prognosis increased to 0.725. The prognostic risk assessment nomogram for bladder cancer patients constructed based on patient age, tumor stage, and risk score had a prediction value that was consistent with the actual value. CONCLUSIONS: A bladder cancer patient prognosis risk assessment model based on cuproptosis-related lncRNA has been successfully constructed in this study. The model can predict the prognosis of bladder cancer patients and their immune infiltration status, which may also provide a reference for tumor immunotherapy.

Transcriptome analysis reveals the potential mechanism of dietary carotenoids improving antioxidative capability and immunity of juvenile Chinese mitten crabs Eriocheir sinensis.

Carotenoids are known to be involved in the regulation of the antioxidative capability, immune response and stress resistance in crustacean species; however, very limited information is available on their underlying molecular mechanisms. This study performed transcriptome sequencing of hemolymph and hepatopancreas of juvenile Chinese mitten crabs (Eriocheir sinensis) that fed with three diets, i.e. diet A containing 90 mg kg-1 dry weight of astaxanthin, diet B containing 200 mg kg-1 dry weight of ß-carotene and control diet without supplementation of dietary carotenoids. The results showed that there were 2955 and 497 differentially expressed genes (DEGs) in the hemolymph between the astaxanthin treatment and control groups, and between the ß-carotene treatment and control groups, respectively. Moreover, compared with the control group, 833 and 1886 DEGs were obtained in the hepatopancreas of the astaxanthin treatment and the ß-carotene treatment groups, respectively. The DEGs in the three groups were enriched in 255 specific KEGG metabolic pathways according to KEGG enrichment analysis. Through this study, a series of key genes involved in Nrf2 signalling, ROS production, intracellular antioxidant enzymes and chaperones were significantly affected by dietary carotenoids. Dietary carotenoids also significantly altered the expression levels of immune-related molecules associated with signal transduction, prophenoloxidase cascade, apoptosis, pattern recognition proteins/receptors and antimicrobial peptides. In conclusion, this transcriptomic study provides valuable information for understanding the molecular mechanism and potential pathway of dietary carotenoids improved the antioxidative capability and immunity of juvenile E. sinensis.

Gene expression and hormone secretion profile of urotensin I associated with osmotic challenge in caudal neurosecretory system of the euryhaline flounder, Platichthys flesus.

The caudal neurosecretory system (CNSS) is a part of stress response system, a neuroendocrine structure unique to fish. To gain a better understanding of the physiological roles of CNSS in fluid homeostasis, we characterized the tissue distribution of urotensin I (UI) expression in European flounder (Platichthys flesus), analyzed the effect chronic exposure to seawater (SW) or freshwater (FW), transfer from SW to FW, and reverse transfer on mRNA levels of UI, L-type Ca2+ channels and Ca-activated K+ channels transcripts in CNSS. The tissue distribution demonstrated that the CNSS is dominant sites of UI expression, and UI mRNA level in fore brain appeared greater than other non-CNSS tissues. There were no consistent differences in CNSS UI expression or urophysis UI content between SW- and FW-adapted fish in July and September. After transfer from SW to FW, at 8 h CNSS UI expression was significantly increased, but urophysis UI content was no significantly changes. At 24 h transfer from SW to FW, expression of CNSS UI was no apparent change and urophysis UI content was reduced. At 8 h and 24 h after transfer from FW to SW UI expression and urophysis UI content was no significantly effect. The expression of bursting dependent L-type Ca2+ channels and Ca-activated K+ channels in SW-adapted fish significantly decreased compared to those in FW-adapted. However, there were no differences in transfer from SW to FW or from FW to SW at 8 h and 24 h. Thus, these results suggest CNSS UI acts as a modulator in response to osmotic stress and plays important roles in the body fluid homeostasis.

Quantitative proteomic reveals the dynamic of protein profile during final oocyte maturation in zebrafish.

The molecular mechanisms underlying final oocyte maturation in zebrafish (Danio rerio) remain poorly understood. The present study aimed to employ iTRAQ approach for a comprehensive characterization of during zebrafish oocyte maturation proteome and for comparison between fully-grow immature and mature oocytes prior to ovulation. A total of 1568 proteins were identified, which was representing the largest zebrafish isolated oocytes proteome dataset to date. Differential expression analysis revealed 190 proteins significantly changes between immature and mature oocytes, which 136 proteins were up-regulated and 54 proteins were down-regulated in mature oocytes comparison with immature oocytes. Functional analysis revealed that these differential proteins were mostly involved in cellular response to estrogen stimulus, cellular components, extracellular region, and enzyme regulator activity, etc. The revealed differentially changes in protein expression patterns associated with oocyte maturation suggest that several of the examined proteins, such as vitellogenin(Vtg3), protein S100(S100A10), 17-beta hydroxysteroid dehydrogenase(HSD17B1), pentaxin, zona pellucida (ZP3.2), elongation factor1-alpha, caluemnin B, and 14-3-3 protein may play a specific role during zebrafish final oocyte maturation. These data will provide powerful information for understanding the molecular mechanism underlying zebrafish oocyte maturation, and these proteins may potentially act as markers to predict control oocyte maturation of zebrafish oocytes.

Dynamic expression pattern of corticotropin-releasing hormone, urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish.

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIß genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIß mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIß mRNA from levels in control fish except at 6dpf when uIIα and uIIß were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIß apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIß genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIß in zebrafish.

Dynamic changes of urotensin II and its receptor during ovarian development of olive flounder Paralichthys olivaceus.

Urotensin II (UII) is a kind of fish somatostatins cyclic peptide, which was originally extracted from the caudal neurosecretory system (CNSS). The system of UII and UII receptor (UIIR) has been reported to have multiple physiological regulatory functions, such as cardiovascular control, osmoregulation, and lipid metabolism. However, the effect of UII and UIIR on the ovarian development has not been covered. This study investigated the expression pattern of UII and UIIR in the ovarian follicles and explored their impact on ovarian development in olive flounder Paralichthys olivaceus. The results showed that the highest UII and UIIR mRNA levels were observed at stage II and stage III follicles during ovarian development, respectively. In situ hybridization revealed that a strong signal of UII was expressed in the oocyte nuclei of stage II follicles, however, UIIR was found in the follicle cells and oocyte cytoplasm of stage II and stage III follicles. Similarly, immunohistochemistry found positive signal of UII was detected in the oocyte nuclei of stage II follicles. The results from in vitro culture of olive flounder follicles suggested the expression of UII and UIIR mRNA levels significantly increased by 10 IU/ml human chorionic gonadotropin (hCG) for 9 h. Furthermore, the transcriptional expression of UII and UIIR was not statistically significantly changed by 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP). These results firstly suggested that UII and UII receptor may play vital roles in regulating ovarian growth in olive flounder.

Molecular characterization of growth differentiation factor 9 and its spatio-temporal expression pattern in gibel carp (Carassius auratus gibelio).

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor ß (TGF-ß) superfamily with a key role in regulating follicle development. In this study, the GDF9 full-length genomic DNA and cDNA were isolated and characterized from the gibel carp ovary using rapid-amplification of cDNA ends (RACE) and LD-PCR. The full-length genomic DNA and cDNA sequences of GDF9 are 3979 and 2044 bp which code 428 amino acid residues with a specific RKKR protease cleavage site of TGF-ß superfamily. Sequence analysis showed that gibel carp was similar to zebrafish and other fish species. Spatio-temporal expression analysis using real-time quantitative PCR revealed that GDF9 mRNA was largely expressed in ovary and testis. GDF9 is mainly present at stage I follicles indicating its important role in early follicles development. The same result was obtained in immunohistochemistry localization of GDF9 protein. Within the follicle, the follicle layer cells were barely expressed whereas GDF9 mRNA was mostly expressed in the oocytes. Supplemented with human chorionic gonadotropin (hCG) in isolated follicles, the expression of GDF9 mRNA was increased firstly and then decreased. The results of this study indicated that GDF9 gene played a role in fish during development of follicles, especially in the early stage follicles.

Label-free quantification proteomics analysis reveals acute hyper-osmotic responsive proteins in the gills of Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) is a typical euryhaline crustacean to study osmotic regulation of crustaceans. Osmotic-regulation of Chinese mitten crab is a complex process. In order to study the osmotic-regulation related proteins of Chinese mitten crab, we domesticated Chinese mitten crab for 144 h with 25 salinity sea water (SW) and 0 salinity fresh water (FW) respectively, and then analyzed the proteome of its posterior gills. A total of 1453 proteins were identified by label free proteomics. Under the threshold of 2 fold change (FC), 242 differentially expressed proteins (DEPs) were screened, including 122 up-regulated DEPs and 120 down-regulated DEPs. GO database and KEGG database were used to annotate and enrich DEPs. It was found that DEPs were significantly enriched in energy metabolism, signal transduction, ion transport, actin cytoskeleton, immunity, lipid metabolism, amino acid metabolism and other biological functions. After 144 h of high salinity stress, the energy metabolism of Chinese mitten crab decreased and the expression of actin and cytoskeleton protein increased. In order to cope with oxidative damage caused by high salinity, Chinese mitten crab improved its immunity and antioxidant capacity.

Moderate acidification mitigates the toxic effects of phenanthrene on the mitten crab Eriocheir sinensis.

Freshwater acidification and phenanthrene may result in complex adverse effects on aquatic animals. Juvenile Chinese mitten crabs (Eriocheir sinensis) were exposed to different pH levels (7.8, 6.5, and 5.5) under phenanthrene (PHE) (0 (control) and 50 µg/L) conditions for 14 days. Antioxidant and transcriptomic responses were determined under stress conditions to evaluate the physiological adaptation of crabs. Under the control pH 7.8, PHE led to significantly reduced activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione S-transferase (GST), but increased glutathione peroxidase (GSH-Px), 7-ethoxyresorufin-o-deethylase (EROD) activities, and malondialdehyde (MDA) levels. However, moderate acidification (pH 6.5) changed PHE effects by increasing antioxidant enzymes. Acidification generally reduced SOD, GPx, GST and EROD activities, but increased CAT, GR, MDA. Compared with pH7.8 group, pH7.8 × PHE and pH6.5 × PHE groups had 1148 and 1498 differentially expressed genes, respectively, with "Biological process" being the main category in the two experimental groups. pH7.8 × PHE treatment caused significant enrichment of disease and immune-related pathways, while under pH6.5 × PHE, more pathways related to metabolism, detoxification, environmental information processing, and energy supply were significantly enriched. Thus, PHE had a significant inhibitory effect on antioxidant performance in crabs, while moderate acidification (pH6.5) mitigated the toxic effects of PHE. Overall, moderate acidification has a positive effect on the defense against the negative effects of PHE in Chinese mitten crabs, and this study provides insights into the defense mechanism of crustaceans in response to combined stress of acidification and PHE.

Polystyrene microplastics increase Pb bioaccumulation and health damage in the Chinese mitten crab Eriocheir sinensis.

Microplastics may be potential vectors for environmental contaminants such as heavy metals in the aquatic ecosystem due to their highly hydrophobic surfaces and fugacity property. To investigate the combined effects of microplastics with Pb, we exposed juvenile Chinese mitten crabs Eriocheir sinensis to different Pb concentrations (0, 5 and 50 µg/L) combined with microplastics (0 and 400 µg/L) for 21 days to determine the Pb bioaccumulation, oxidative stress, lipid anabolism, and histopathology of hepatopancreas. In general, the results showed that compared to single Pb exposure, the combination of MPs and Pb significantly increased the bioaccumulation of Pb, activities/content of antioxidant biomarkers and lipid metabolism enzymes, and liver injury parameters in crabs, indicating MPs are potential vector of heavy metals and co-exposure exerts more severe effects on crabs. This study provides the insights into the oxidative defense and preliminary lipid anabolism of economic crustaceans in response to combined stress of Pb and MPs.

Effects of copper exposure on lipid metabolism and SREBP pathway in the Chinese mitten crab Eriocheir sinensis.

Copper (Cu) is not only a common metal pollutant in the aquatic environment but also an essential trace element for aquatic organisms such as the Chinese mitten crab (Eriocheir sinensis). Cu is known to regulate lipid metabolism yet exert toxic effects if ingested in excess. However, the molecular regulatory roles of Cu in the lipid metabolism of crabs remains unclear. Thus, this study investigated the potential regulatory mechanism of Cu onto lipid metabolism of E. sinensis following acute Cu exposure. Crabs were exposed to environmental concentration of Cu (50 µg/L) for 96 h, and the expression of sterol regulatory element binding protein (SREBP) was knocked down by RNA interference (RNAi) to test its effect on Cu exposure. The results showed that RNAi significantly attenuated the Cu exposure-induced increase in lipid synthesis and triglycerides (TG) hydrolysis, while significantly inhibited the Cu exposure-induced decrease in fatty acid ß-oxidation, suggesting that SREBP is involved in Cu-induced lipid metabolism. Subsequent analyses of the transcriptome results further revealed potential responsive genes of SREBP that were linked to lipid metabolism and immune regulation. Moreover, Cu may affect lipid metabolism through the TOR-SREBP pathway in E. sinensis. This work provides a reference for exploring the effects of Cu on lipid metabolism disorders in crustaceans.

Corticotropin-releasing hormone reduces basal estradiol production in zebrafish follicular cells.

Corticotropin-releasing hormone (CRH) plays a key regulatory role in coordinating the regulation of endocrine, autonomic nervous, immune, and reproductive systems. Two CRH (CRHα and CRHß) and their receptors (CRHR1 and CRHR2) had been identified in zebrafish. However, their functions remained uncovered in the ovary of zebrafish. Therefore, this study aimed to determine whether CRH acts directly on the ovary to regulate steroidogenesis in cultured zebrafish follicular cells. Firstly, CRH and its receptors are expressed in the zebrafish ovary. The expression profile of CRHß fluctuated during ovarian development in zebrafish, and the highest CRHα mRNA levels were observed at the mature follicle. The highest CRHR1 and CRHR2 mRNA levels existed in mid-vitellogenic (MV) and early vitellogenic (EV) stages, respectively. In primary cultured zebrafish follicular cells, both of the CRHα and CRHß inhibited expression of hsd17b3 mRNA levels and decreased content of estradiol (E2) in the medium. Furthermore, CRH activated p38 MAPK and p38 MAPK inhibitor SB203580 attenuated the phosphorylation of p38 MAPK induced by CRHα. Simultaneously, SB203580 changed the effect of CRH on cyp19a1a expression but not hsd17b1 and hsd17b3. SB203580 alone or combined with CRH inhibited the E2 content. Finally, the CRHR inhibitor α-helical 9-41 also blocked the phosphorylation of p38 MAPK induced by CRHα but did not change the inhibitory effect of CRH on the mRNA expression of the steroidogenic gene and the content of E2 in the culture medium. Taken together, our findings suggest that the anti-steroidogenic effects of CRH may be mediated partly through activation of the p38 MAPK signaling pathway.

Antiproliferation and apoptosis induced by evodiamine in human colorectal carcinoma cells (COLO-205).

Evodiamine (1), a biologically active alkaloid isolated from Evodia rutaecarpa (known in Chinese as Wu-Chu-Yu), has antioxidant, anti-inflammatory, and anticancer activities. It has recently been demonstrated that the cytotoxic activities of 1 might be due to its ability to inhibit cell growth and induce apoptosis. In this study, we investigated the effects of 1 on growth and apoptosis in COLO-205 cells by MTT assay, fluorescence microscopy, transmission electron microscopy, DNA fragmentation assay, flow cytometry, immunohistochemical analysis, Western blotting, and caspase-3 activity assay. Our data revealed that 1 could significantly inhibit COLO-205 cell proliferation in a dose-dependent manner, and 1-treated COLO-205 cells displayed typical morphological apoptotic characteristics and formation of DNA ladders in agarose gel electrophoresis. The COLO-205 cell cycle was arrested in G(2)/M phase by 1. Meanwhile, 1 increased the expression of Bax and p53, decreased the expression of Bcl-2, lowered the mitochondrial transmembrane potential, and induced the activation of caspase-3. These activities may contribute to the anticarcinogenic action of 1.

Dynamic Expression and Regulation of Urotensin I and Corticotropin-Releasing Hormone Receptors in Ovary of Olive Flounder Paralichthys olivaceus.

Urotensin I (UI), a fish corticotropin-releasing hormone (CRH) like peptide, has been found throughout vertebrate species that has great effects on adaptive physiology comprising stress-related responses, and osmotic regulation by binding with CRH type I receptor (CRHR1) and CRH type II receptor (CRHR2) in fish. Dynamic expression and regulation of UI and CRH receptors in the olive flounder ovarian follicle were studied so as to make further efforts to understand the role of UI in the development of teleost ovary. The results showed that stage-specific change in UI mRNA levels in ovarian follicles of olive flounder. UI and CRHR1 mRNA levels were higher in stage III follicles (300∼500 µm diameter) compared to stage II (90∼300 µm diameter) and IV (500∼800 µm diameter) follicles, however, the levels of CRHR2 mRNA were decreased in line with the ovarian development from stage II to stage IV. A strong signal of UI protein was observed in the follicular cells and oocyte in stage III and IV follicles by immunohistochemistry. In vitro treatment of olive flounder ovarian follicles with human chorionic gonadotropin (hCG) showed that the mRNA expression of UI increased significantly at low concentration and decreased at high concentration at 6 h, but the CRHR1 and CRHR2 mRNA did not change obviously. In addition, the results of incubation with 17α, 20ß-dihydroxy-4-oregnen-3-one (DHP) show that UI and CRHR1 mRNA expression were elevated with increasing concentrations at 9 h. All above results indicated that UI and CRH receptors may have a vital effect on olive flounder ovarian development.

Effects of dietary lipids on the hepatopancreas transcriptome of Chinese mitten crab (Eriocheir sinensis).

Fish oil supplies worldwide have declined sharply over the years. To reduce the use of fish oil in aquaculture, many studies have explored the effects of fish oil substitutions on aquatic animals. To illustrate the effects of dietary lipids on Chinese mitten crab and to improve the use of vegetable oils in the diet of the crabs, 60 male juvenile Chinese mitten crabs were fed one of five diets for 116 days: fish oil (FO), soybean oil (SO), linseed oil (LO), FO + SO (1:1, FSO), and FO + LO (1:1, FLO). Changes in the crab hepatopancreas transcriptome were analyzed using RNA sequencing. There were a total 55,167 unigenes obtained from the transcriptome, of which the expression of 3030 was significantly altered in the FLO vs. FO groups, but the expression of only 412 unigenes was altered in the FSO vs. FO groups. The diets significantly altered the expression of many enzymes involved in lipid metabolism, such as pancreatic lipase, long-chain acyl-CoA synthetases, carnitine palmitoyltransferase I, acetyl-CoA carboxylase, fatty acid synthase, and fatty acyl Δ9-desaturase. The dietary lipids also affected the Toll-like receptor and Janus activated kinase-signal transducers and activators of transcription signaling pathways. Our results indicate that substituting fish oil with vegetable oils in the diet of Chinese mitten crabs might decrease the digestion and absorption of dietary lipids, fatty acids biosynthesis, and immunologic viral defense, and increase ß-oxidation by altering the expression of the relevant genes. Our results lay the foundation for further understanding of lipid nutrition in Chinese mitten crab.

Characterization of bmp15 and its regulation by human chorionic gonadotropin in the follicle of gibel carp (Carassius auratus gibelio).

Bone morphogenetic protein (BMP15) is a member of the transforming growth factor ß (TGF-ß) superfamily with a key role in regulating follicle development in mammals and birds. However, potential ovarian roles of BMPs remain unexplored in teleosts. In this study, the full-length sequences of bmp15 were obtained using rapid-amplification of cDNA ends (RACE). The full-length cDNA sequence of bmp15 is 2217 bp which contained 214 bp 5'-UTR and 845 bp 3'-UTR. The open reading frame (ORF) sequence of bmp15 is 1158 bp, encoding a predicted protein of 385 amino acid residues. BMP15 has a specific RXXR protease cleavage site of TGF-ß superfamily (is RIRR) and six conserved cysteine residues. Using real-time quantitative PCR revealed that bmp15 mRNA was largely expressed in the ovary and testis and mostly in oocytes within the follicle, slightly expressed in muscle, liver and pituitary. BMP15 is mainly present at stage I follicles by real-time quantitative PCR and immunohistochemistry. Phylogenetic analysis showed that gibel carp bmp15 was similar to bmp15 of zebrafish and other fish species. Treatment with human chorionic gonadotropin (hCG) in isolated follicles of gibel carp in vitro showed altered bmp15 mRNA expression: when treated with 10 ng/mL hCG for 10h, the expression level of bmp15 was significantly increased. However, with proceeding cultivation, the expression level of BMP15 mRNA decreased. The results of this study indicate that bmp15 may play a key role during development of follicles in gibel carp, especially in early stage follicles.

Ginsenoside Rd elicits Th1 and Th2 immune responses to ovalbumin in mice.

Ginsenoside Rd (Rd), a saponin isolated from the roots of panax notoginseng, was evaluated for inducing Th1 or Th2 immune responses in mice against ovalbumin (OVA). ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing alum (200 microg), or Rd (10, 25 or 50 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation was determined using MTT assay, and OVA-specific antibody titers and levels of cytokines in serum were measured by ELISA and microparticle-based flow cytometric immunoassay, as well as peripheral blood T-lymphocyte subsets analyzed using flow cytometer. Rd significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice. OVA-specific IgG, IgG1, and IgG2b antibody titers in serum were significantly enhanced by Rd compared with OVA control group. Meanwhile, Rd also significantly promoted the production of the Th1 and Th2 cytokines in OVA-immunized mice. Further, the effects of Rd on expression of cytokine mRNA in Con A-stimulated mice splenocytes were evaluated by RT-PCR analysis. Rd significantly enhanced the interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4, and IL-10 mRNA expression in mice splenocyte induced by Con A. These results suggested that Rd had immunological adjuvant activity, and elicited a Th1 and Th2 immune response by regulating production and gene expression of Th1 cytokines and Th2 cytokines.