Structure of GPR101-Gs enables identification of ligands with rejuvenating potential.

GPR101 is an orphan G protein-coupled receptor actively participating in energy homeostasis. Here we report the cryo-electron microscopy structure of GPR101 constitutively coupled to Gs heterotrimer, which reveals unique features of GPR101, including the interaction of extracellular loop 2 within the 7TM bundle, a hydrophobic chain packing-mediated activation mechanism and the structural basis of disease-related mutants. Importantly, a side pocket is identified in GPR101 that facilitates in silico screening to identify four small-molecule agonists, including AA-14. The structure of AA-14-GPR101-Gs provides direct evidence of the AA-14 binding at the side pocket. Functionally, AA-14 partially restores the functions of GH/IGF-1 axis and exhibits several rejuvenating effects in wild-type mice, which are abrogated in Gpr101-deficient mice. In summary, we provide a structural basis for the constitutive activity of GPR101. The structure-facilitated identification of GPR101 agonists and functional analysis suggest that targeting this orphan receptor has rejuvenating potential.

Research on Classification Algorithm of Silicon Single-Crystal Growth Temperature Gradient Trend Based on Multi-Level Feature Fusion.

In the process of silicon single-crystal preparation, the timely identification and adjustment of abnormal conditions are crucial. Failure to promptly detect and resolve issues may result in a substandard silicon crystal product quality or even crystal pulling failure. Therefore, the early identification of abnormal furnace conditions is essential for ensuring the preparation of perfect silicon single crystals. Additionally, since the thermal field is the fundamental driving force for stable crystal growth and the primary assurance of crystal quality, this paper proposes a silicon single-crystal growth temperature gradient trend classification algorithm based on multi-level feature fusion. The aim is to accurately identify temperature gradient changes during silicon crystal growth, in order to promptly react to early growth failures and ensure the stable growth of high-quality silicon single crystals to meet industrial production requirements. The algorithm first divides the temperature gradient trend into reasonable categories based on expert knowledge and qualitative analysis methods. Then, it fuses the original features of actual production data, shallow features extracted based on statistical information, and deep features extracted through deep learning. During the fusion process, the algorithm considers the impact of different features on the target variable and calculates mutual information based on the difference between information entropy and conditional entropy, ultimately using mutual information for feature weighting. Subsequently, the fused multi-level feature vectors and their corresponding trend labels are input into a Deep Belief Network (DBN) model to capture process dynamics and classify trend changes. Finally, the experimental results demonstrate that the proposed algorithm can effectively predict the changing trend of thermal field temperature gradients. The introduction of this algorithm will help improve the accuracy of fault trend prediction in silicon single-crystal preparation, thereby minimizing product quality issues and production interruptions caused by abnormal conditions.

Crystal structure and catalytic activity of the PPM1K N94K mutant.

Protein Phosphatase Mg2+ /Mn2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg2+ in the active site. Specifically, three Mg2+ were located in the active site of the PPM1K N94K instead of two Mg2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.

The role of AMPKα in high-glucose-induced dysfunction of cultured rat mesangial cells.

AIM: Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is known as a mammalian cell energy sensor, which could regulate cellular energy metabolism via sensing the alterations of energy balance, such as oversupply or lack of glucose and fatty acid. Recent studies have suggested that AMPK could also regulate many other biological processes, including cell cycling, inflammation, protein synthesis, and so on. In this study, AMPK signaling in high-glucose-induced dysfunction of mesangial cells (MCs) was investigated. METHODS: Established rat glomerular MCs were treated under normal glucose (5.6 mM glucose) or high-glucose conditions (30 mM glucose). mRNA levels of AMPK subunits were detected by reverse transcriptase-polymerase chain reaction. Expressions of AMPKα, phosphorylated AMPKα (p-AMPKα), phosphorylated acetyl-CoA carboxylase (p-ACC), and collagen IV were measured by Western blot. RESULTS: Under high-glucose conditions, AMPKα protein expression and mRNA levels were significantly decreased. High-glucose treatment also induced a notable decrease in p-AMPKα and p-ACC expression. AMPKα activation by 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) effectively ameliorated high-glucose-induced dysfunction of MCs, including cell proliferation, cell-cycle progression, and collagen IV production. CONCLUSION: High glucose impaired AMPKα in its expression and activity; AICAR significantly ameliorated high-glucose-induced proliferation of MCs and collagen IV production, indicating a role of AMPKα in high-glucose-induced dysfunction of MCs.

A Single Vaccination of Chimeric Bivalent Virus-Like Particle Vaccine Confers Protection Against H9N2 and H3N2 Avian Influenza in Commercial Broilers and Allows a Strategy of Differentiating Infected from Vaccinated Animals.

H9N2 and H3N2 are the two most important subtypes of low pathogenic avian influenza viruses (LPAIV) because of their ongoing threat to the global poultry industry and public health. Although commercially available inactivated H9N2 vaccines are widely used in the affected countries, endemic H9N2 avian influenza remains uncontrolled. In addition, there is no available avian H3N2 vaccine. Influenza virus-like particles (VLPs) are one of the most promising vaccine alternatives to traditional egg-based vaccines. In this study, to increase the immunogenic content of VLPs to reduce production costs, we developed chimeric bivalent VLPs (cbVLPs) co-displaying hemagglutinin (HA) and neuraminidase (NA) of H9N2 and H3N2 viruses with the Gag protein of bovine immunodeficiency virus (BIV) as the inner core using the Bac-to-Bac baculovirus expression system. The results showed that a single immunization of chickens with 40µg/0.3mL cbVLPs elicited an effective immune response and provided complete protection against H9N2 and H3N2 viruses. More importantly, cbVLPs with accompanying serological assays can successfully accomplish the strategy of differentiating infected animals from vaccinated animals (DIVA), making virus surveillance easier. Therefore, this cbVLP vaccine candidate would be a promising alternative to conventional vaccines, showing great potential for commercial development.

Ligand recognition, unconventional activation, and G protein coupling of the prostaglandin E2 receptor EP2 subtype.

Selective modulation of the heterotrimeric G protein α S subunit-coupled prostaglandin E2 (PGE2) receptor EP2 subtype is a promising therapeutic strategy for osteoporosis, ocular hypertension, neurodegenerative diseases, and cardiovascular disorders. Here, we report the cryo-electron microscopy structure of the EP2-Gs complex with its endogenous agonist PGE2 and two synthesized agonists, taprenepag and evatanepag (CP-533536). These structures revealed distinct features of EP2 within the EP receptor family in terms of its unconventional receptor activation and G protein coupling mechanisms, including activation in the absence of a typical W6.48 "toggle switch" and coupling to Gs via helix 8. Moreover, inspection of the agonist-bound EP2 structures uncovered key motifs governing ligand selectivity. Our study provides important knowledge for agonist recognition and activation mechanisms of EP2 and will facilitate the rational design of drugs targeting the PGE2 signaling system.

Identification of Sp1-elements in the promoter region of human homeobox gene NKX3.1.

NKX3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. However, little is known about the mechanism for regulation of NKX3.1 in prostate cancer. With RT-PCR and western blot, we found that NKX3.1 expression was enhanced by over-expression of Sp1 at both the mRNA and protein levels in prostate cancer LNCaP cells. To identify the Sp1-elements in the promoter region of NKX3.1, a 521 bp-promoter of human NKX3.1 gene containing three possible Sp1-elements was cloned into the upstream of the luciferase reporter gene in pGL(3)-basic plasmid. With deletion mutation analysis, plasmid construction, EMSA and oligonucleotide decoy technique, two Sp1-elements which located between ?29 to ?43 and -60 to -46 of NKX3.1 gene were identified and proven to be functional elements. It will be important to further study on the functions and the regulatory mechanisms of Sp1 element in NKX3.1 gene expression.

[Distribution, Sources, and Ecological Risk Assessment of Polycyclic Aromatic Hydrocarbons in the Surface Waters of the Yinchuan Wetlands].

In order to explore the composition, sources and ecological risks of polycyclic aromatic hydrocarbons (PAHs) in water from Yinchuan wetlands, water samples were collected in the dry season and plentiful season from 15 wetlands. Sixteen species of PAHs were analyzed by gas-mass spectrometry, and source identification of PAHs was investigated by PCA and EPA positive matrix factorization 5.0. Ecological risk was assessed using the risk entropy method based on the neglected concentrations (NCs) and the maximum permissible concentrations (MPCs). The results showed that:① in the dry season, eight kinds of PAHs were detected, the concentrations of which ranged from 1455.38 ng·L-1to 2538.84 ng·L-1. In the plentiful season, 12 kinds of PAHs were detected, the concentrations of which ranged from 818.69 ng·L-1 to 1582.14 ng·L-1. The concentrations of PAHs in the dry season in Yinchuan were higher than that during the plentiful season. Compared with other domestic and overseas surface waters, PAH pollution was high; ② in the dry season, PAHs were mainly composed of 3-5 rings, and 2-3 and 4-6rings accounted for 35.6%-59.2% and 40.8%-59.7%, respectively. In the plentiful season, PAHs were mainly composed of 4-5 rings, and 2-3 and 4-6rings accounted for 10.2%-45.07% and 54.92%-89.76%, respectively; ③ the source analysis showed that in both the dry season and in the plentiful season, the main source were combustion and automobile emissions; ④ the ecological risk assessment indicated that the RQMPCs of BaA, BbF, InP, DBA, and BghiP during both the dry and plentiful seasons, and RQMPCs of Phe during the dry season, were higher than 1.0, indicating that attention needs to be paid to pollution levels. The RQNCs of Nap, Ace, Fla, Pyr, and BaP during the plentiful season and the RQNCs of Nap during the dry season were higher than 1.0, indicating the pollution risk was moderate and control and prevision of pollution from PAHs are required in the region.

An effective method for isolation of DNA from pig faeces and comparison of five different methods.

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.

Emodin induces apoptosis in human prostate cancer cell LNCaP.

AIM: To elucidate effects and mechanisms of emodin in prostate cancer cells. METHODS: Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor (AR), prostate-specific antigen (PSA), p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. RESULTS: In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax /Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. CONCLUSION: In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.

Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli.

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using TA cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M.bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

Effects of 9-cis retinoic acid on human homeobox gene NKX3.1 expression in prostate cancer cell line LNCaP.

AIM: To study the regulatory effects of 9-cis retinoic acid (RA) on the expression of human homeobox gene NKX3.1 in prostate cancer cell line LNCaP. METHODS: Flow cytometry, reverse transcriptase polymerase chain reaction and Western blotting were performed to evaluate the effects of 9-cis RA on NKX3.1 expression and cell cycle of LNCaP cells. To identify a regulatory region within the NKX3.1 promoter contributing to the regulation induced by 9-cis RA, we have constructed an NKX3.1 promoter-reporter plasmid, pGL3-1040bp, and its 5'-deletion mutants, which were transfected into LNCaP cells with treatment of 9-cis RA in indicated concentrations. RESULTS: With the treatment of 9-cis RA, the NKX3.1 promoter activity was increased in reporter gene assay and NKX3.1 expression was enhanced at both mRNA and protein levels in LNCaP cells. We found that the region between -936 and -921 in the upstream of NKX3.1 gene involved the inducible regulation by 9-cis RA treatment. In flow cytometry, 9-cis RA treatment caused accumulation of cells in the G(1) phase of the cell cycle and a fewer cells pass through to G(2)/M. CONCLUSION: Our results demonstrated that 9-cis RA as a differentiating agent can arrest prostate cancer cells in G(1) phase and reduce cell mitosis, and upregulate the expression of human homeobox gene NKX3.1, which is thought to play an important role in prostate differentiation and to act as a tumor suppressor gene in the prostate.

[Up-regulates the expression of maspin gene in prostate cancer cell line LNCaP].

AIM: To study the effect of curcumin on the apoptosis of prostate cancer cell line LNCaP and regulation of expression of maspin gene. METHODS: MTT and DNA electrophoresis were used to examine the cell growth and apoptosis of prostate cancer cell line LNCaP after treated with different doses of curcumin. The expression of maspin gene at transcription level and translation level was also detected by RT-PCR and Western blotting. pGL3-maspin luciferase expression vector, containing 847 bp (- 764 -/+ 83) DNA of maspin gene 5' promoter region, was transient transfected into LNCaP cell. Through detecting the activity of luciferase, the effect of curcumin on the promoter of maspin was studied. RESULTS: Curcumin inhibited cell growth, induced the apoptosis and enhanced the expression of maspin gene in LNCaP cells. CONCLUSION: Curcumin up-regulated expression of maspin gene in LNCaP cells through enhancing the transcription activity of promoter of maspin gene.

Identification of androgen-responsive element ARE and Sp1 element in the maspin promoter.

Maspin is a serine protease inhibitor (serpin) with tumor-suppressing function in mammary gland. It is down-regulated in primary prostate cancer cells and lost in metastatic cells. To better understand the transcriptional regulation of maspin gene, the 860bp (-765 approximately +95) of its promoter sequence was amplified by PCR from the human genomic DNA. Then this 860bp sequence and a series of deletions from 5' and 3' ends were inserted into the upstream of luciferase reporter gene respectively. Results from dual luciferase reporter assay and electrophoretic mobility shift assay indicated that there were a negative androgen-responsive element (ARE) in the region of -277 to -262 and a positive Sp1 element in the region of +14 to +35, respectively. In addition, androgen receptor (AR) can recognize and bind to the ARE element, and then inhibit the activity of maspin promoter.

[Inhibition of the expression of prostate specific antigen by curcumin].

AIM: To study the effect of curcumin on the expression of prostate specific antigen (PSA). METHODS: AXSYM system-chemical luciferase method was used to examine the content of PSA in prostate cancer cell lines, LNCap after treated with different doses of curcumin. pGL3-PSA luciferase expression vector, containing 640 bp DNA of PSA gene 5' promoter region was constructed and transfected into LNCap cell with lipofectin. Through detecting the activity of luciferase, the effect of curcumin on the promoter of PSA was studied. Western blotting was used to detect expression of androgen receptor (AR) in LNCap cell with different concentrations of curcumin. RESULTS: The expression of PSA was inhibited and activity of luciferase was reduced by curcumin. There was also significant difference in AR expression as shown by Western blotting experiment after treatment of different doses of curcumin. CONCLUSION: Through inhibiting AR expression, curcumin reduced the function of PSA promoter and inhibited PSA protein expression.

Panax notoginseng saponins inhibits atherosclerotic plaque angiogenesis by down-regulating vascular endothelial growth factor and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 expression.

OBJECTIVE: To investigate the mechanism of Panax notoginseng saponins (PNS), an effective component extracted from Panax notoginseng, on atherosclerotic plaque angiogenesis in atherosclerosis-prone apolipoprotein E-knockout (ApoE-KO) mice fed with high-fat, high-cholesterol diet. METHODS: Twenty ApoE-KO mice were divided into two groups, the model group and the PNS group. Ten normal C57BL/6J mice were used as a control group. PNS (60 mg/kg) was orally administered daily for 12 weeks in the PNS group. The ratio of plaque area to vessel area was examined by histological staining. The tissue sample of aortic root was used to detect the CD34 and vascular endothelial growth factor (VEGF) expression areas by immunohistochemistry. The expression of VEGF and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) were measured by reverse transcription polymerase chain reaction and Western blotting respectively. RESULTS: After treatment with PNS, the plaque areas were decreased (P<0.05). CD34 expressing areas and VEGF expression areas in plaques were significantly decreased (P<0.05). Meanwhile, VEGF and NOX4 mRNA expression were decreased after treatment with PNS. VEGF and NOX4 protein expression were also decreased by about 72% and 63%, respectively (P<0.01). CONCLUSION: PNS, which decreases VEGF and NOX4 expression, could alleviate plaque angiogenesis and attenuate atherosclerosis.

The miRNA let-7a1 inhibits the expression of insulin-like growth factor 1 receptor (IGF1R) in prostate cancer PC-3 cells.

Reduced microRNA (miRNA) let-7a expression and the activation of insulin-like growth factor-1 receptor (IGF1R) signalling are both involved in prostate cancer and progression. In the present study, we demonstrated that the growth inhibitory effect of let-7a1 is directly related to targeting IGF1R gene expression in PC-3 cells. TargetScan predicted three potential target sites (T1, T2 and T3) of let-7a in the 3' untranslational region (3' UTR) of IGF1R mRNA. Real-time PCR, Western blot and luciferase reporter assays were used to detect the effects of let-7a1 overexpression or let-7a1 inhibitor on the IGF1R gene expression in PC-3 cells. The results indicated that let-7a1 could inhibit IGF1R expression by directly targeting the T1 and T2 sites in the 3' UTR of the IGF1R mRNA. We then used RT-PCR, luciferase reporter assays, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry and Hoechst 33342 staining to examine whether let-7a1-mediated inhibition of IGF1R expression also affects the IGF1R-mediated signalling events, including Elk1 activity and c-fos gene expression, proliferation, apoptosis and cell cycle. We demonstrated that let-7a1-mediated IGF1R downregulation was accompanied by attenuation of Elk1 activity and c-fos expression, inhibition of cell proliferation, enhanced apoptosis and cell cycle arrest, and that loss function of let-7a1 via inhibition can upregulate IGF1R accompanied by an increase of Elk1 activity and c-fos expression, thereby enhancing cell proliferation. Altogether, these findings suggest that let-7a may be novel therapeutic candidate for prostate cancer.

The inhibitory effects of NKX3.1 on IGF-1R expression and its signalling pathway in human prostatic carcinoma PC3 cells.

NKX3.1, which is a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as a tumour suppressor gene. In this study, we investigated the inhibitory effect of NKX3.1 on insulin-like growth factor (IGF)-1R expression and its downstream signalling pathway in PC3 cells. PC3 cells were stably transfected with NKX3.1 expression plasmid (pcDNA3.1-NKX3.1) or vector plasmid (pcDNA3.1+). The IGF-IR mRNA and protein expression levels were assessed in PC3-NKX3.1 transfectants by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The expression and activation of IGF-1/IGF-1R downstream signalling targets were examined by Western blotting and luciferase reporter assay. The cells were subsequently treated with relevant concentrations of IGF-1. The effect of IGF-1 on cell growth was examined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay and flow cytometry analysis. A significant suppression of IGF-1R mRNA and protein expression was observed after forced expression of NKX3.1 in PC3 cells. Correspondingly, the forced expression of NKX3.1 decreased IGF-1-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) and activation of the Elk-1 transcription factor and downregulated the expression of the downstream target genes c-fos and cyclin D1. Furthermore, the forced expression of NKX3.1 inhibited IGF-1-induced cell growth. In conclusion, NKX3.1 could downregulate IGF-1R expression and could inhibit IGF-1R-mediated mitogen-activated protein kinase (MAPK)/ERK and AKT signalling pathways, which might partially leads to the inhibition of IGF-1-induced cell growth. This study provides new insights into the molecular mechanisms that NKX3.1 exerts against prostate cancer and ultimately expands the scope of alternative approaches in advanced prostate cancer therapy.

Emodin ameliorates high-glucose induced mesangial p38 over-activation and hypocontractility via activation of PPARgamma.

Early stage diabetic nephropathy is characterized by elevated glomerular filtration. Recent studies have identified high-glucose induced p38 MAPK (p38) over-activation in mesangial cells. Mesangial hypocontractility is the major underlying mechanism, however, no ameliorating agents are currently available. We investigated the protective effects of emodin on high-glucose induced mesangial cell hypocontractility. Mesangial cells were cultured under normal (5.6 mM) and high glucose (30 mM) conditions. Emodin was administrated at doses of 50 mg/l and 100 mg/l. Angiotension II stimulated cell surface reductions were measured to evaluate cell contractility. p38 activity was detected using Western blotting. To further explore the possible mechanism of emodin, expression of the peroxisome proliferator- activated receptorgamma (PPARgamma) was measured and its specific inhibitor, gw9662, was administrated. Our results showed: (1) high-glucose resulted in a 280% increase in p38 activity associated with significant impairment of mesangial contractility; (2) emodin treatment dose-dependently inhibited high-glucose induced p38 over-activation (a 40% decrease for 50 mg/l emodin and a 73% decrease for 100 mg/l emodin), and mesangial hypocontractility was ameriolated by emodin; (3) both the PPARgamma mRNA and protein levels were elevated after emodin treatment; (4) inhibition of PPARgamma using gw9662 effectively blocked the ameliorating effects of emodin on high-glucose induced p38 over-activation and mesangial hypocontractility. Emodin effectively ameliorated p38 over-activation and hypocontractility in high-glucose induced mesangial cells, possibly via activation of PPARgamma.

Curcumin downregulates homeobox gene NKX3.1 in prostate cancer cell LNCaP.

AIM: To elucidate the effect and the mechanisms of curcumin on the expression of the human homeobox gene NKX3.1 in the prostate cancer cell LNCaP. METHODS: The expression change of NKX3.1 in cells incubated with varying concentrations of curcumin was observed by Western blotting and RT-PCR. A dual luciferase reporter assay was used to test the effect of curcumin on the activity of the NKX3.1 1040 bp promoter. Curcumin-treated cells disposed to a designated amount of androgen analog R1881 and the androgen receptor (AR) antagonist flutamide, then the expression of NKX3.1 or the activity of the NKX3.1 promoter were investigated by Western blotting or reporter gene assay, respectively. Finally, Western blotting and electrophoretic mobility shift assay were performed to demonstrate the effect of curcumin on the expression of AR and its binding activity to the androgen response element (ARE). RESULTS: Curcumin downregulated the expression of NKX3.1 and the activity of the NKX3.1 1040 bp promoter in LNCaP cells. R1881 increased the expression of NKX3.1, and the AR antagonist flutamide decreased the expression of NKX3.1 in LNCaP cells, while curcumin could inhibit androgen-AR mediated induction of NKX3.1 expression. Curcumin decreased the expression of AR and the binding activity to ARE directly. CONCLUSION: Curcumin could downregulate NKX3.1 expression in LNCaP cells. It could also inhibit the androgen-AR mediated induction of NKX3.1 expression by downregulating AR expression and blocking its DNA binding activity.