The immune regulation and therapeutic potential of the SMAD gene family in breast cancer.

Breast cancer is a serious threat to human health. The transforming growth factor-ß signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-ß signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-ß signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.

Dantrolene and coenzyme Q10 as a suggested combination therapy to statin-induced myopathy in high fat diet rats: A possible interference with ROS/ TGF-ß / Smad4 signaling pathway.

One of the major hitches for statins' utilization is the development of myotoxicity. Versatile studies reported that the underlining molecular mechanisms including coenzyme Q10 (CoQ10)/ubiquinone depletion, as well as the disturbance in the cytoplasmic Ca2+ homeostasis. Therefore, we investigated the consequences of supplementing CoQ10 and dantrolene, a cytoplasmic Ca2+ reducing agent, in combination with simvastatin. This adjuvant therapy normalized the simvastatin-mediated elevation in serum ALT, AST, CK-MM, as well as tissue Ca2+ content, in addition to suppressing the simvastatin-mediated oxidative stress in simvastatin-treated rats, while having no effect upon statin-induced antihyperlipidemic effect. Additionally, the combination inhibited the simvastatin-induced TGF-ß/ Smad4 pathway activation. Collectively, the current study emphasizes on the potential utilization of dantrolene and CoQ10 as an adjuvant therapy to statins treatment for improving their side effect profile.

Cullin3 deficiency shapes tumor microenvironment and promotes cholangiocarcinoma in liver-specific Smad4/Pten mutant mice.

Cholangiocarcinoma (CC), the most lethal type of liver cancer, remains very difficult to treat due to an incomplete understanding of the cancer initiation and progression mechanisms and no effective therapeutic drugs. Thus, identification of genomic drivers and delineation of the underlying mechanisms are urgently needed. Here, we conducted a genome-wide CRISPR-Cas9 screening in liver-specific Smad4/Pten knockout mice (Smad4co/co;Ptenco/co;Alb-Cre, abbreviated as SPC), and identified 15 putative tumor suppressor genes, including Cullin3 (Cul3), whose deficiency increases protein levels of Nrf2 and Cyclin D1 that accelerate cholangiocytes expansion leading to the initiation of CC. Meanwhile, Cul3 deficiency also increases the secretion of Cxcl9 in stromal cells to attract T cells infiltration, and increases the production of Amphiregulin (Areg) mediated by Nrf2, which paracrinely induces inflammation in the liver, and promotes accumulation of exhausted PD1high CD8 T cells at the expenses of their cytotoxic activity, allowing CC progression. We demonstrate that the anti-PD1/PD-L1 blockade inhibits CC growth, and the effect is enhanced by combining with sorafenib selected from organoid mediated drug sensitive test. This model makes it possible to further identify more liver cancer suppressors, study molecular mechanisms, and develop effective therapeutic strategies.

[Regulatory effect of Qianliexiao Decoction on TGF-ß1, Smad4 and Smad 7 signaling pathways and the proliferation and apoptosis of prostate fibroblasts in mice with experimental autoimmune prostatitis].

OBJECTIVE: To study the effect of intervening in the signal transduction pathways of TGF-ß1, Smad4 and Smad7 with Qianliexiao Decoction (QLX) on the proliferation and apoptosis of prostate fibroblasts (PrF) in mice with experimental autoimmune prostatitis (EAP). METHODS: A model of EAP with damp-heat syndrome was established in C57BL/6 mice by immunization induction combined with the TCM modeling method. The prostate tissue of the mice was harvested for isolation, culturing and purification of PrFs and detection of their purity. After modeling by stimulation with a medium containing >90%-purity or 5 ng/ml TGF-ß1, the PrFs in the logarithmic growth phase were obtained and randomly divided into a blank control (serum-free medium), a model control, a positive control (medium containing 5 ng/ml TGF-ß1), a low-dose QLX (serum containing 5% QLX), a medium-dose QLX (serum containing 10% QLX), and a high-dose QLX group (serum containing 20% QLX). After 24 hours of intervention, the proliferation of the PrFs was measured, the protein expressions of TGF-ß1, Smad4, Smad7, p-Smad4 and p-Smad7 detected by Western blot, their mRNA expressions determined by qPCR, and the apoptosis of the PrFs examined by flow cytometry. RESULTS: After induction with TGF-ß1, the proliferation of the PrFs was significantly increased in the positive control (P < 0.05), but inhibited in the medium- and low-dose QLX groups (P < 0.05) and even more significantly in the high-dose QLX group as compared with that in the model control (P < 0.01). The expressions of Smad4, p-Samd7 and TGF-ß1 proteins in the PrFs were remarkably higher in the positive control than in the model control group (P < 0.05), while those of p-Smad4 and TGF-ß1 markedly lower (P < 0.01) and that of p-Smad7 dramatically higher in the QLX intervention groups than in the positive control (P < 0.01), in an evident dose-dependent manner. In comparison with the model control group, the high-dose QLX group exhibited a significant decrease in the mRNA expression of Smad4 (P < 0.05) but all the three QLX groups showed a dramatic increase in those of Smad7 (P < 0.05) and TGF-ß1 (P < 0.01). The mRNA expression of Smad4 was markedly down-regulated in the high-dose QLX group compared with that in the positive control (P < 0.05), that of Smad7 up-regulated in the model control and QLX groups (P < 0.01), and that of TGF-ß1 down-regulated in the three QLX groups (P < 0.01). The apoptosis rate of the PrFs was significantly higher in the QLX groups than in the model control (P < 0.05) in a dose-dependent manner, but showed no statistically significant difference between the model control and the positive control groups (P > 0.05). CONCLUSIONS: TGF-ß1 can stimulate the proliferation of PrFs, up-regulate the expressions of TGF-ß1 and p-Smad4, and down-regulate that of p-Smad7, while QLX can inhibit the proliferation of PrFs in a dose-dependent manner by decreasing the expressions of TGF-ß1 and p-Smad4, increasing that of p-Smad7, and thereby suppressing TGF-ß1-induced proliferation of PrFs.

Involvement of eIF2α in halofuginone-driven inhibition of TGF-ß1-induced EMT.

Halofuginone (HF) is an extract from the widely used traditional Chinese medicine (TCM) Dichroa febrifuga that facilitates the recovery of wounds and attenuates hepatic fibrosis. However, the role of HF in the epithelial-mesenchymal transition (EMT) of IPEC-J2 cells remains unclear. The current study explored the anti-EMT effect of HF in IPEC-J2 cells and illustrates its molecular mechanism. Transforming growth factor ß1 (TGF-ß1), as a recognized profibrogenic cytokine, decreased the level of the epithelial marker E-cadherin and increased the level of the mesenchymal markers, such as N-cadherin, fibronectin (FN), vimentin (Vim), and α-smooth muscle actin (α-SMA), in IPEC-J2 cells depending on the exposure time and dose. HF markedly prevented the EMT induced by TGF-ß1. Dissection of the mechanism revealed that HF inhibited IPEC-J2 cell EMT via modulating the phosphorylation of SMAD2/3 and the SMAD2/3-SMAD4 complex nuclear translocation. Furthermore, HF could promote the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which modulates the SMAD signaling pathway. These results suggested that HF inhibits TGF-ß1-induced EMT in IPEC-J2 cells through the eIF2α/SMAD signaling pathway. Our findings suggest that HF can serve as a potential anti-EMT agent in intestinal fibrosis therapy.

A patent herbal drug Yi-Shen-Hua-Shi granule ameliorates C-BSA-induced chronic glomerulonephritis and inhabits TGFß signaling in rats.

ETHNOPHARMACOLOGY RELEVANCE: Yi-Shen-Hua-Shi (YSHS) granule is a modern Chinese patent drug for treating chronic glomerulonephritis (CGN). It is derived from a traditional Chinese medicine formula Sheng-Yang-Yi-Wei decoction that is used to treat CGN in ancient China. Pharmacological activities of YSHS granule have not been reported. In this work, we investigated the anti-CGN effects and TGFß signaling-related mechanism of action of this herbal drug. MATERIALS AND METHODS: The rat model of CGN was established by injection of cationization-bovine serum albumin (C-BSA) for five weeks. After finishing C-BSA injection, drugs were intragastrically administered to the rats once daily for four weeks. Clinical signs were recorded daily. Serum and urine biochemical parameters were analyzed by respective kits. Protein levels were examined by Western blotting. Pathological changes of renal tissues were evaluated by HE and Masson's trichrome staining. RESULTS AND CONCLUSIONS: No significant differences in clinical signs and body weights were found among normal, model and drug treatment groups. Proteinuria; albuminuria; increased urine volume; elevated urea nitrogen, creatinine, total cholesterol and triglyceride levels in sera; decreased serum total protein and albumin; as well as renal pathological damage and fibrosis were observed in CGN model rats. YSHS granule ameliorated all the abnormal behavioral and biochemical changes in the model rats. Mechanical investigations showed that YSHS granule down-regulated proteins levels of TGFß1, phospho-Smad2/3 (Thr 8) and Smad4 in rat renal tissues. In conclusion, YSHS granule demonstrates therapeutic effects in a rat model of CGN, and inhibition of the TGFß/Smad signaling pathway is involved in the mechanism of action of the granule. This study provides a pharmacological basis for the use of modern YSHS granule and ancient Sheng-Yang-Yi-Wei decoction in treating CGN.

Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle Fibrosis.

Geniposide is the main bioactive constituent of gardenia fruit. Skeletal-muscle fibrosis is a common and irreversibly damaging process. Numerous studies have shown that geniposide could improve many chronic diseases, including metabolic syndrome and tumors. However, the effects of geniposide on skeletal-muscle fibrosis are still poorly understood. Here, we found that crude extracts of gardenia fruit pomace could significantly decrease the expression of profibrotic genes in vitro. Moreover, geniposide could also reverse profibrotic-gene expression induced by TGF-ß and Smad4, a regulator of skeletal-muscle fibrosis. In addition, geniposide treatment could significantly downregulate profibrotic-gene expression and improve skeletal-muscle injuries in a mouse model of contusion. These results together suggest that geniposide has an antifibrotic effect on skeletal muscle through the suppression of the TGF-ß-Smad4 signaling pathway.

Curcumin induces apoptosis and cell cycle arrest via the activation of reactive oxygen species-independent mitochondrial apoptotic pathway in Smad4 and p53 mutated colon adenocarcinoma HT29 cells.

Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53.

Hedyotis diffusa Willd suppresses metastasis in 5­fluorouracil­resistant colorectal cancer cells by regulating the TGF­ß signaling pathway.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract, and threatens the survival and health of patients with CRC. Chemotherapy remains one of the main therapeutic approaches for patients with CRC; however, drug resistance limits the long­term use. CRC cells with multi­drug resistance (MDR) exhibit increased survival times and metastatic potential, which may lead to the recurrence and metastasis of CRC. In addition, MDR is one of the major causes of chemotherapy failure in clinical treatment. Hedyotis diffusa Willd (HDW) has been used in the treatment of inflammation­associated diseases and malignant tumors, including CRC. The authors previously demonstrated that HDW could reverse MDR in CRC cells; however, its underlying mechanism, particularly in MDR­associated metastasis, remains to be elucidated. In the present study, the drug­resistant CRC cell line HCT­8/5­fluorouracil (5­FU) was used to investigate the effect of HDW on the growth and metastasis of cancer cells. Cell viability was assessed using the MTT assay. Cell adhesion potential was evaluated using adhesion experiments. Cell migration was assessed using wound healing and Transwell assays. The mRNA and protein expression levels of crucial factors in the transforming growth factor­ß (TGF­ß) signaling pathway, including TGF­ß, Mothers against decapentaplegic homolog 4 (SMAD4), neural (N)­cadherin, and epithelial (E)­cadherin, were analyzed using the reverse transcription­semi­quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that the HCT­8/5­FU cell line was more resistant to 5­FU and thus can be used as the resistant cell model. HDW was able to inhibit the viability, and adhesive, migratory and invasion potential of the HCT­8/5­FU cells. In addition, HDW was able to downregulate the expression of TGF­ß, SMAD4 and N­cadherin, and upregulate E­cadherin, at the gene and protein level. In conclusion, the results demonstrated that HDW may suppress the metastasis of 5­FU­resistant CRC cells via regulation of the TGF­ß signaling pathway, which was also considered to be one of the underlying mechanisms of its anti­CRC effect.

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines.

MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 µM Zn), or with 15 or 50 µM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 µM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-ß/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.

Emodin inhibits angiogenesis in pancreatic cancer by regulating the transforming growth factor-ß/drosophila mothers against decapentaplegic pathway and angiogenesis-associated microRNAs.

Emodin is a traditional Chinese medicine, which has been demonstrated to inhibit the growth of pancreatic cancer cells. However, the underlying molecular mechanisms remain to be elucidated. The present study investigated whether emodin suppresses angiogenesis in pancreatic cancer. A nude mouse pancreatic cancer xenograft model was established using SW1990 human pancreatic cancer cells by surgical orthotopic implantation. Different doses of emodin were injected into the abdominal cavities of the tumor­bearing mouse models and controls three times each week for 2 weeks. The tumors were measured and weighed, the expression of cluster of differentiation 34 was detected using immunochemistry, and microvessel densities were calculated. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting were performed to determine the mRNA and protein expression levels of transforming growth factor (TGF)­ß and drosophila mothers against decapentaplegic (Smad) homologs. The angiogenesis­associated microRNAs (miR), miR­20, miR­155 and miR­210 were assessed by RT­qPCR. A negative dose­dependent association was revealed between treatment with emodin and the volume and weight of tumors and microvessel density. Emodin was associated with lower mRNA and protein expression levels of TGF­ß1 and its downstream target, angiopoietin­like 4, and higher mRNA and protein expression levels of TGF­ß receptor (TßR)I, TßRII and Smad4. Notably, treatment with emodin was associated with lower expression levels of miR­155 and miR­210 and higher expression levels of miR­20b. The present study suggested that treatment with emodin may repress angiogenesis in pancreatic cancer by altering the activities of the TGF-ß/Smad pathway and angiogenesis-associated miR-20b, miR-155, and miR-210.

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

UNLABELLED: The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE: KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

[Qianlongtong capsule elevates the Smad4 gene expression in prostate stromal cells].

OBJECTIVE: To investigate the effects of the plasma containing Qianlongtong Capsule (QLT)-containing plasma on the expression of the Smad4 gene in prostate stromal cells in vitro and provide some experimental evidence for the treatment of benign prostatic hyperplasia (BPH) with Chinese medicinal compound. METHODS: Fifteen cases of BPH were equally randomized to three groups to be treated with QLT at a high dose (6 capsules once), a medium dose (3 capsules once), and a low dose (1.5 capsules once), tid, for 7 days consecutively. QLT-containing plasma was collected from the patients. Prostate stromal cells were identified by immunofluorescence when they became monolayered and cultured in the QLT-containing plasma for 24 hours, followed by detection of the expression of the Smad4 gene by real-time quantitative PCR and that of the Smad4 protein by Western blot. RESULTS: After treatment with the QLT-containing plasma, the expression of the Smad4 gene in the stromal cells was significantly increased in a dose-dependent manner as compared with the blank control and no-QLT groups (P < 0.01). The expression of the Smad4 protein was also markedly elevated after treatment. The differences were statistically significant between the blank control and medium-dose groups (P < 0.01), low-dose and medium-dose groups (P < 0.05), and high-dose and the other groups (P < 0.01), but not between the blank control and low-dose groups (P > 0.05). CONCLUSION: QLT-containing plasma could inhibit the proliferation and improve the apoptotic index of prostate stromal cells in vitro, which was related to the elevation of the mRNA and protein expressions of Smad4.

Systemic approaches identify a garlic-derived chemical, Z-ajoene, as a glioblastoma multiforme cancer stem cell-specific targeting agent.

Glioblastoma multiforme (GBM) is one of the most common brain malignancies and has a very poor prognosis. Recent evidence suggests that the presence of cancer stem cells (CSC) in GBM and the rare CSC subpopulation that is resistant to chemotherapy may be responsible for the treatment failure and unfavorable prognosis of GBM. A garlic-derived compound, Z-ajoene, has shown a range of biological activities, including anti-proliferative effects on several cancers. Here, we demonstrated for the first time that Z-ajoene specifically inhibits the growth of the GBM CSC population. CSC sphere-forming inhibition was achieved at a concentration that did not exhibit a cytotoxic effect in regular cell culture conditions. The specificity of this inhibitory effect on the CSC population was confirmed by detecting CSC cell surface marker CD133 expression and biochemical marker ALDH activity. In addition, stem cell-related mRNA profiling and real-time PCR revealed the differential expression of CSC-specific genes, including Notch, Wnt, and Hedgehog, upon treatment with Z-ajoene. A proteomic approach, i.e., reverse-phase protein array (RPPA) and Western blot analysis, showed decreased SMAD4, p-AKT, 14.3.3 and FOXO3A expression. The protein interaction map (http://string-db.org/) of the identified molecules suggested that the AKT, ERK/p38 and TGFß signaling pathways are key mediators of Z-ajoene's action, which affects the transcriptional network that includes FOXO3A. These biological and bioinformatic analyses collectively demonstrate that Z-ajoene is a potential candidate for the treatment of GBM by specifically targeting GBM CSCs. We also show how this systemic approach strengthens the identification of new therapeutic agents that target CSCs.

Compound Astragalus and Salvia miltiorrhiza extract suppresses rabbits' hypertrophic scar by modulating the TGF-ß/Smad signal.

BACKGROUND: Hypertrophic scar is a fibro-proliferative disease. Our previous studies demonstrate that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits proliferation and invasion in keloid fibroblasts. OBJECTIVE: To investigate the effects of CASE on hypertrophic scar. METHODS: Rabbits were divided into the control, model and three dosage groups of CASE (0.94, 1.88, 3.76%). An animal model of hypertrophic scar was established and treated with CASE ointment or ointment base. The histopathological detection by hematoxylin & eosin and Masson's trichrome staining and protein expression of scars by Western blot were performed. RESULTS: The hydroxyproline content was decreased under CASE treatment. Transforming growth factor beta 1 (TGF-ß1) protein expression increased in the model group while it decreased under CASE treatment. The elevated expression of Smad4 protein was decreased under CASE treatment. Additionally, CASE promoted Smad7 protein expression. CONCLUSION: CASE could inhibit formation of hypertrophic scar by modulating TGF-ß/Smad signal and may be useful for the treatment of hyperplastic scars.

[Renal protection of Tangke Decoction on rats with diabetes and its effect on the expression of TGF-beta1/Smad4].

OBJECTIVE: To observe the effect of Tangke Decoction (TD) on the expression of TGF-beta1/Smad4 of rats with early diabetes and to explore the effect and mechanism of TD against the renal injury induced by diabetes. METHODS: SD rats were randomly divided into the normal control group (n = 12), the model group (n = 10), the Chinese herbs prevented group (n =10), the Chinese herbs treated group (n = 10), and the Western medicine control group (n = 10). TD (18 mg/kg) was given by gastrogavage to rats in the Chinese herbs prevented group immediately after successful modeling for 12 weeks, once daily. At the 4th week of successful modeling, rats in the rest 4 groups were administered by gastrogavage. Equal volume of normal saline was given to rats in the model group and the normal control group. Benazepril suspension (1 mg/kg) was administered by gastrogavage to rats in the Western medicine control group for 8 weeks, once daily. TD (18 mg/kg) was given by gastrogavage to rats in the Chinese herbs treated group for 8 weeks, once daily. The body weight, kidney weight, index of kidney weight, fasting blood sugar, 24 h urinary albumin excretion rate were examined after experiment. The pathological changes of the renal tissue were observed by HE staining, Masson staining, and electron microscope. The expression of renal transforming growth factor-beta1, (TGF-beta1) and Smad4 were detected using immunohistochemical assay. RESULTS: Compared with the normal control group, the body weight of rats decreased significantly; the kidney weight, index of kidney weight, blood sugar, 24 h urinary protein excretion, the urinary albumin excretion rate,TGF-beta1 and Smad4 expression increased significantly in the model group (all P < 0.01). Compared with the model group, the aforesaid indices were improved in each treatment group with statistical difference (P < 0.05, P < 0.01). Compared with the Western medicine control group, the kidney weight, index of kidney weight, blood sugar, 24 h urinary protein excretion, and the urinary albumin excretion rate were obviously improved in the Chinese herbs prevented group (P < 0.01). The renal pathological changes were most obvious in the model group significantly, but they were improved in all treatment groups. CONCLUSION: TD could obviously improve the symptoms of diabetes and down-regulate the expression of renal TGF-beta1 and Smad4 of early diabetic nephropathy rats, which suggested that TD had certain preventive effect on early diabetic nephropathy.

Quercetin prevents ethanol-induced iron overload by regulating hepcidin through the BMP6/SMAD4 signaling pathway.

Emerging evidence has demonstrated that chronic ethanol exposure induces iron overload, enhancing ethanol-mediated liver damage. The purpose of this study was to explore the effects of the naturally occurring compound quercetin on ethanol-induced iron overload and liver damage, focusing on the signaling pathway of the iron regulatory hormone hepcidin. Adult male C57BL/6J mice were pair-fed with isocaloric-Lieber De Carli diets containing ethanol (accounting for 30% of total calories) and/or carbonyl iron (0.2%) and treated with quecertin (100 mg/kg body weight) for 15 weeks. Mouse primary hepatocytes were incubated with ethanol (100 mM) and quercetin (100 µM) for 24 h. Mice exposed to either ethanol or iron presented significant fatty infiltration and iron deposition in the liver; these symptoms were exacerbated in mice cotreated with ethanol and iron. Quercetin attenuated the abnormity induced by ethanol and/or iron. Ethanol suppressed BMP6 and intranuclear SMAD4 as well as decreased hepcidin expression. These effects were partially alleviated by quercetin supplementation in mice and hepatocytes. Importantly, ethanol caused suppression of SMAD4 binding to the HAMP promoter and of hepcidin messenger RNA expression. These effects were exacerbated by anti-BMP6 antibody and partially alleviated by quercetin or human recombinant BMP6 in cultured hepatocytes. In contrast, co-treatment with iron and ethanol, especially exposure of iron alone, activated BMP6/SMAD4 pathway and up-regulated hepcidin expression, which was also normalized by quercetin in vivo. Quercetin prevented ethanol-induced hepatic iron overload different from what carbonyl iron diet elicited in the mechanism, by regulating hepcidin expression via the BMP6/SMAD4 signaling pathway.

[Effect of bushen tiaojing recipe on the expressions of Smad1, Smad5, Smad8, and Smad4 in human mural granulosa cells].

OBJECTIVE: To explore the effect of Bushen Tiaojing Recipe (BTR) on the expressions of drosophila mothers against decapentaplegic protein (Smadl), Smad5, Smad8, and Smad4 on human mural granulosa cells. METHODS: Sixty-six patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to two groups in the ratio of 1:2, the treatment group and the control group. Twenty-three patients in the treatment group were treated with BTR and GnRHa/FSH/hCG, while forty-three patients in the control group were treated with GnRHa/FSH/hCG. The mRNA expressions of Smad1, Smad5, Smad8, and Smad4 on mural granulosa cells of the mature follicle were detected by real-time PCR on the ovum retrieval day. The expressions of Smad1, Smad5, Smad8, and Smad4 at the protein level were observed using cell immunofluorescence method. RESULTS: The mRNA and protein expressions of Smadl in the granulosa cells were significantly higher in the treatment group than in the control group (P <0.05). There was no statistical difference in the mRNA and protein expressions of Smad5, Smad8, and Smad4 between the two groups. CONCLUSION: The mechanisms of BTR for improving the pregnancy rate and the ovarian functions might be correlated with up-regulating mRNA and protein expressions of Smadl of human mural granulosa cells.

TGFß/Smad signalling in psoriatic epidermis models exposed to salt water soaks and narrowband ultraviolet B radiation.

The role of transforming growth factor-ß1 (TGFß1) and Smad signalling has not been established in psoriasis treatment. We aimed to investigate the effect of combined treatment with salt water soaks and ultraviolet radiation on the expression of TGFß1/Smad signalling proteins in a psoriatic model. We studied mRNA expression (real-time RT-PCR) of TGFß1, TGFß receptor type I (TGFßRI), Smad2, Smad3, Smad4, Smad7, minichromosome maintenance protein 7, and involucrin in normal as well as psoriatic epidermis models (PEM) which were treated for three consecutive days with differently concentrated salt water solutions [(3% NaCl; 30% NaCl, 30% Dead Sea salt water (DSSW)] and subsequent narrowband ultraviolet B (NB-UVB). In PEM, TGFß1 and Smad3 was significantly increased as compared to normal epidermis models. By contrast, TGFßRI mRNA was significantly decreased in PEM. Significant increase of mRNA levels of TGFß1, TGFßRI, Smad2 and Smad3 was predominantly observed in non-irradiated and irradiated PEM pre-treated with 30% NaCl and/or DSSW which was paralleled by increase of involucrin mRNA. In PEM pre-treated with DSSW, TGFßRI, Smad2, Smad3, Smad4, and Smad7 mRNA was significantly higher in irradiated PEM when compared to non-irradiated samples. It has been shown that TGFß1/Smad signalling is altered in a psoriatic model and may play a role in the mode of action of salt water soaks and NB-UVB phototherapy of psoriasis.

Maohuoside A acts in a BMP-dependent manner during osteogenesis.

There has been a strong interest in searching for natural therapies for osteoporosis. Epimedium koreanum Nakai is an herb that is commonly used in East Asia to treat osteoporosis, and most studies of its activity have focused on its major ingredient, icariin. In this study, maohuoside A (MHA), a single compound isolated from the E. koreanum, was found to promote osteogenesis in mouse bone marrow-derived mesenchymal stem cells. We hypothesise that, if MHA potently induces osteogenic differentiation in a bone morphogenetic protein-dependent manner, it may be used to broaden the sources for cell transplantation and thereby establish more efficient bone regeneration systems. Real-time polymerase chain reaction and western blot were used to detect the expression of SMAD4, a marker of bone formation. Microcomputed tomography and histomorphometric techniques showed that oral treatment with MHA was followed by an increase in the bone mineral density of the lumbar vertebrae in mice. This result also indicates that MHA may directly activate osteopontin gene transcription. In conclusion, MHA seems to enhance the osteogenesis of bone marrow-derived mesenchymal stem cells at least partly via bone morphogenetic protein signalling.