Satb2 regulates the development of dopaminergic neurons in the arcuate nucleus by Dlx1.

Dopaminergic (DA) neurons in the arcuate nucleus (ARC) of the hypothalamus play essential roles in the secretion of prolactin and the regulation of energy homeostasis. However, the gene regulatory network responsible for the development of the DA neurons remains poorly understood. Here we report that the transcription factor special AT-rich binding protein 2 (Satb2) is required for the development of ARC DA neurons. Satb2 is expressed in a large proportion of DA neurons without colocalization with proopiomelanocortin (POMC), orexigenic agouti-related peptide (AgRP), neuropeptide-Y (NPY), somatostatin (Sst), growth hormone-releasing hormone (GHRH), or galanin in the ARC. Nestin-Cre;Satb2flox/flox (Satb2 CKO) mice show a reduced number of ARC DA neurons with unchanged numbers of the other types of ARC neurons, and exhibit an increase of serum prolactin level and an elevated metabolic rate. The reduction of ARC DA neurons in the CKO mice is observed at an embryonic stage and Dlx1 is identified as a potential downstream gene of Satb2 in regulating the development of ARC DA neurons. Together, our study demonstrates that Satb2 plays a critical role in the gene regulatory network directing the development of DA neurons in ARC.

[Effects of Wenyang Huazhuo Tongluo Recipe Containing Serum on Transforming Growth Factor ß1/ Smad Signaling Pathway of Skin Fibroblasts in Systemic Sclerosis].

OBJECTIVE: To explore the effects of Wenyang Huazhuo Tongluo Recipe (WYHZTLR) containing serum on transforming growth factor ß1 (TGF-ß1)/Smad signaling pathway of skin fibroblasts in systemic sclerosis (SSc). METHODS: Totally 36 SSc patients were randomly assigned to Chinese medicine (CM) group, Western medicine (WM) group, and integrative medicine (IM) group according to random digit table, 12 in each group. Patients in the CM group took WYHZTLR decoction (one dose per day). Patients in the WM group took penicillamine tablet (0. 125 g each time, bid) and Prednisone Acetate Tablet (PAT 20 mg, qd). Patients in the IM group took penicillamine, PAT, and WYHZTLR decoction (in the same dosage of corresponding drugs as aforesaid). All patients were treated for one month to get drug containing serum. Besides, 10 untreated SSc patients' serum was taken as the control group. Healthy subjects' skin fibroblasts were originated from healthy skin tissue of the upper arms of 2 female patients undergoing plastic surgery. Corresponding serum of each group was added in the culture system of SSc patients' and healthy subjects' dermal fibroblasts respectively. Expression levels of TGF-ß1 receptor type I (TGF-ß1 RI), TGF-ß1 receptor II (TGF-ß1 RII), p-Smad2/3 and Smad7 protein were examined by Western blot. Expression levels of collagen type I and collagen type III (Col-I, Col-III) mRNA were examined by reverse transcription PCR. Contents of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the supernatant of SSc, skin fibroblasts were examined by ELISA. RESULTS: Compared with the control group, expression levels of TGF-ß1 R I and p-Smad2/3 protein significantly decreased, but expression levels of Smad7 protein significantly increased in skin fibroblasts of SSc patients and healthy subjects of WM, CM, and IM groups (P <0.05, P <0. 01). Meanwhile, the expression level of TGF-ß1 RII decreased in the IM group (P <0. 05, P <0. 01). Compared with the WM group, expression levels of TGF-ß1 RI and p-Smad2/ 3 protein significantly decreased, but that of Srnad7 protein significantly increased in IM groups (P <0. 01). mRNA expression levels of Col-I and Col-II in SSc skin fibroblasts significantly decreased more in WM, CM, and IM groups than in the control group (P <0. 05, P <0. 01). Besides, the expression level of Col-III mRNA was significantly lower in the IM group than in the WM group (P <0.01). Compared with the control group, serum levels of MMP-9 and MMP-9/TIMP-1 ratios increased more obviously in WM, CM, and IM groups (P <0.05, P <0.01). But expression levels of TIMP-1 decreased significantly in CM and IM groups (P <0.01). Expression levels of MMP-9 and MMP-9/TIMP-1 ratios increased more in the IM group than in the WM group (P <0. 01). Expression levels of TIMP-1 decreased more in CM and IM groups than in the WM group (P <0.01). CONCLUSION: WYHZTLR containing serum could reduce expression levels of Col-I and Col-III possibly through regulating key signal molecules, such as TGF-ß1 RI, p-Smad2/3, and Smad7 in TGF-ß1/Smad signaling pathway of SSc skin fibroblasts, and inhibiting transduction of TGF-ß1/Smad signaling pathway.

A Group of ent-Kaurane Diterpenoids Inhibit Hedgehog Signaling and Induce Cilia Elongation.

The Hedgehog (Hh) signaling pathway plays important roles in the tumorigenesis of multiple cancers and is a key target for drug discovery. In a screen of natural products extracted from Chinese herbs, we identified eight ent-Kaurane diterpenoids and two triterpene dilactones as novel Hh pathway antagonists. Epistatic analyses suggest that these compounds likely act at the level or downstream of Smoothened (Smo) and upstream of Suppressor of Fused (Sufu). The ent-Kauranoid-treated cells showed elongated cilia, suppressed Smo trafficking to cilia, and mitotic defects, while the triterpene dilactones had no effect on the cilia and ciliary Smo. These ent-Kaurane diterpenoids provide new prototypes of Hh inhibitors, and are valuable probes for deciphering the mechanisms of Smo ciliary transport and ciliogenesis.

[Effect of Wenyang Huazhuo Tongluo Recipe on Peripheral Blood Thl7/Treg Cell Balance in Systemic Sclerosis Patients].

OBJECTIVE: To observe the effect of Wenyang Huazhuo Tongluo Recipe (WYHZTLR) on the proportion of T helper 17 cells (Thl7)/regulatory T cells (Treg), and serum levels of IL-17 and IL-10 in peripheral blood of systemic sclerosis (SSc) patients with yang qi insufficiency and turbidity induced collaterals blockage syndrome (YQITICBS). METHODS: Totally 82 SSc patients were randomly assigned to the Western medicine group (as the control group) and the integrated Chinese and Western medicine group (as the treatment group), 41 cases in each group. All patients took methotrexate (MTX) tablet and prednisone tablet. Patients in the treatment group additionally took WYHZTLR. The treatment course for all was six consecutive months. Besides, another 70 healthy volunteers were recruited as a healthy control group (as the healthy group). Percentages of Th17 and Treg in peripheral blood were detected by flow cytometry. Serum levels of IL-17, IL-10, von Willebrand factor (vWF), aminoterminal propeptide of type l procollagen (PIIINP), and cross-linked carboxyterminal telopeptide of type I collagen ( I CTP) were measured by double antibody sandwich ELISA. The correlations between Th17/Treg and levels of vWF, PIIINP, I CTP, skin score, and disease activity index were observed by Pearson correlation analysis. RESULTS: The percentage of Th17 in peripheral blood, ratios of Th17/Treg, and the serum level of IL-17 were significantly higher, but the percentage of Treg and the serum level of IL-10 were significantly lower in SSc patients, when compared with those of the healthy group (P <0. 05, P <0. 01). Compared with the same group before treatment, the percentage of Thl7, ratios of Thl7/Treg, and levels of IL-17, vWF, and PIIINP all decreased in the two groups after treatment (P <0.05, P <0.01), but the percentage of Treg and the IL-10 level increased (P <0. 05, P <0. 01). Meanwhile,the level of I CTP was higher in the treatment group after treatment (P <0. 05). The improvement of all indices except the percentage of Th17 was more obvious in the treatment group than in the control group (P <0. 05, P <0. 01). The ratio of Th17/Treg was positively correlated with levels of vWF, PIIINP, skin score, and disease activity index before and after treatment respectively (P <0. 01), but with no obvious correlation with the level of I CTP (P >0. 05). CONCLUSION: WYHZTLR could achieve its therapeutic effect on SSc patients by regulating Th17/Treg imbalance, lowering levels of vWF and PIIINP, and elevating the level of I CTP.

Henryin, an ent-kaurane diterpenoid, inhibits Wnt signaling through interference with ß-catenin/TCF4 interaction in colorectal cancer cells.

Aberrant Wnt/ß-catenin signaling has been strongly associated with the tumorigenesis of human colorectal cancer. Inhibitors of this pathway may then offer therapeutic strategies as well as chemoprevention for this malignant disease. Henryin is an ent-kaurane diterpenoid isolated from Isodonrubescens var. lushanensis, a plant long been used in folk medicine to prevent inflammation and gastrointestinal disease. In the present study, we report that henryin selectively inhibits the proliferation of human colorectal cancer cells with a GI50 value in the nano-molar range. Microarray analysis and reporter assays showed that henryin worked as a novel antagonist of Wnt signaling pathway. Henryin reduced the expression of Cyclin D1 and C-myc, and induced G1/S phase arrest in HCT116 cells. Concurrently, henryin did not affect the cytosol-nuclear distribution of soluble ß-catenin, but impaired the association of ß-catenin/TCF4 transcriptional complex likely through directly blocking the binding of ß-catenin to TCF4. We also then analyzed the structure-activity relationship among the ent-kaurane type diterpenoids. Our data suggests that henryin, as a novel inhibitor of Wnt signaling, could be a potential candidate for further preclinical evaluation for colon cancer treatment, and as such warrants further exploration.

[Effect of Wenyang Huazhuo Tongluo recipe contained serum on proliferation and cell cycle of systemic sclerosis skin fibroblasts].

OBJECTIVE: To investigate the effect of "Wenyang Huazhuo Tongluo recipe" (WYHZTLR) on proliferation and cell cycle of systemic sclerosis (SSc) skin fibroblasts in vitro by serum pharmacologic technique. METHODS: Prednisone, D-penicillamine and WYHZTLR contained serum from patients with SSc were respectively added into cultured normal dermal fibroblast cell-line and SSc lesional dermal fibroblast cell-line. Then the cells were incubated for 48 hours. Cell proliferation and cell cycle were examined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. RESULTS: Western medicine Chinese medicine and integrated Chinese and western medicine could markedly inhibit the proliferation of normal dermal fibroblasts and SSc lesional dermal fibroblasts in vitro compared with control group (P < 0.05 or P < 0.01). But 20% serum in integrated Chinese and western medicine group could markedly inhibit the proliferation of fibroblasts compared with western medicine group (P < 0.01). Different drug-contained serum of WYHZTLR could markedly increase the percentage of G0-G1 stage cells (P < 0.01) and depress the percentage of sphase cells and G2-M stage cells compared with control group (P < 0.05 or P < 0.01). CONCLUSION: WYHZTLR can block the progression of SSc skin fibroblasts into sphase and G2-M stage and inhibit the proliferation of SSc skin fibroblasts, and WYHZTLR was unselectively interfered with normal and SSc fibroblasts in vitro.

[Clinical trial and evaluation on comprehensive treatment on attack in acute stage: report of 522 cases].

OBJECTIVE: To investigate the clinical effect of the comprehensive treatment to acute stage of attack. METHODS: On the basis of the previous observation, the study of the randomization control with general treatment, treatment on acupuncture and western medical treatment were carried out. A comprehensive treatment on overall traditional Chinese medical differentiation according to the superiority of every treatment was assessed and evaluated in 522 patients with attack. RESULTS: The comprehensive treatment of cerebral infarction was superior to the western medicine treatment. General treatment, treatment on traditional Chinese medical differentiation, acupuncture group revealed different improvement on neural function, daily viability, cognitive function in various extent. CONCLUSION: The comprehensive treatment that based on overall traditional Chinese medical differentiation has advantage and characteristic. It has positive combined action to the attack and relevant to clinical setting, easier to popularization and application. Various appraising amount form has different evaluating effects in different stage.