Smad3 Mediates Diabetic Dyslipidemia and Fatty Liver in db/db Mice by Targeting PPARδ.

Transforming growth factor-ß (TGF-ß)/Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases. However, the role of Smad3 in dyslipidemia and non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes remains unclear, and whether targeting Smad3 has a therapeutic effect on these metabolic abnormalities remains unexplored. These topics were investigated in this study in Smad3 knockout (KO)-db/db mice and by treating db/db mice with a Smad3-specific inhibitor SIS3. Compared to Smad3 wild-type (WT)-db/db mice, Smad3 KO-db/db mice were protected against dyslipidemia and NAFLD. Similarly, treatment of db/db mice with SIS3 at week 4 before the onset of type 2 diabetes until week 12 was capable of lowering blood glucose levels and improving diabetic dyslipidemia and NAFLD. In addition, using RNA-sequencing, the potential Smad3-target genes related to lipid metabolism was identified in the liver tissues of Smad3 KO/WT mice, and the regulatory mechanisms were investigated. Mechanistically, we uncovered that Smad3 targeted peroxisome proliferator-activated receptor delta (PPARδ) to induce dyslipidemia and NAFLD in db/db mice, which was improved by genetically deleting and pharmacologically inhibiting Smad3.

Functional analysis of miRNAs combined with TGF-ß1/Smad3 inhibitor in an intrauterine rat adhesion cell model.

In this study, we aimed to study the role of miRNAs in intrauterine adhesion (IUA) disease. An IUA cell model was constructed by TGF-ß1. Smad3 inhibitor (SIS3) can inhibit the Smad3 signaling pathway and affect the role of TGF-ß1; thus, it was used to identify the role of Smad3 and related miRNAs in IUA. Cell number significantly increased in the TGF-ß1 group after 72 h and 96 h, respectively, compared with that in the control group (P < 0.05). However, cell proliferation was significantly decreased in the TGF-ß1 + SIS3 group (P < 0.0001). Cell apoptosis was increased in the TGF-ß1 + SIS3 group compared with that in the TGF-ß1 group. Western Blot (WB) analysis suggested that TGF-ß1 treatment could effectively increase the expression of α-SMA, COL1, Smad3, and p-Smad3, which could be inhibited by SIS3 treatment. A total of 235 and 530 differentially expressed miRNAs in the TGF-ß1 + SIS3 group were significantly up- and downregulated compared with those in the TGF-ß1 group, respectively. These differentially expressed miRNAs were enriched in the MAPK and PI3K-AKT pathways. The ten most differentially expressed miRNAs were selected to verify their expressions using quantitative real-time polymerase chain reaction (qPCR). Furthermore, overexpression of rno-miR-3586-3p and rno-miR-455-5p can promote cell proliferation and exacerbate the IUA pathogenic process. However, overexpression of rno-miR-204-3p and rno-miR-3578 can inhibit cell behavior and IUA progression. The above results can provide detailed information for the understanding of IUA molecular mechanisms.

Discovery of a novel selective water-soluble SMAD3 inhibitor as an antitumor agent.

Targeting the SMAD3 protein is an attractive therapeutic strategy for treating cancer, as it avoids the potential toxicities due to targeting the TGF-ß signaling pathway upstream. Compound SIS3 was the first selective SMAD3 inhibitor developed that had acceptable activity, but its poor water solubility limited its development. Here, a series of SIS3 analogs was created to investigate the structure-activity relationship for inhibiting the activation of SMAD3. On the basis of this SAR, further optimization generated a water-soluble compound, 16d, which was capable of effectively blocking SMAD3 activation in vitro and had similar NK cell-mediated anticancer effects in vivo to its parent SIS3. This study not only provided a preferable lead compound, 16d, for further drug discovery or a potential tool to study SMAD3 biology, but also proved the effectiveness of our strategy for water-solubility driven optimization.

Transforming growth factor-ß1 mediates psoriasis-like lesions via a Smad3-dependent mechanism in mice.

Transforming growth factor (TGF)-ß1 signals through downstream Smad-dependent and -independent pathways to exert its biological actions. It has been reported that overexpression of TGF-ß1 results in the development of psoriasis-like lesions in a mouse model of K5.TGF-ß(WT) transgenic mice. However, the signalling mechanisms by which TGF-ß1 mediates the development of psoriasis-like lesions remain unknown. The aim of the present study was to investigate the hypothesis that TGF-ß1 mediates the development of psoriasis-like lesions via a Smad3-dependent mechanism. This was tested in a mouse model of K5.TGF-ß(WT) transgenic mice by blocking TGF-ß signalling with a specific Smad3 inhibitor. Topical treatment with a Smad3 inhibitor markedly blocked TGF-ß/Smad3 signalling and progressive psoriasis-like lesions in K5.TGF-ß(WT) transgenic mice, as evidenced by decreased skin severity scores, double skin fold thickness (DSFT) scores, infiltration of CD3(+) T cells and F4/80(+) macrophages and the degree of fibrosis in the dermis. This was associated with a marked reduction in TGF-ß1, interleukin (IL)-6, IL-23 and IL-17A both locally in skin plaque lesions and systemically in the plasma, resulting in inhibition of both the T helper (Th) 17 cell transcription factor RORγt and accumulation of CD4(+) IL-17A(+) cells within the skin plaque lesions. In conclusion, TGF-ß1 mediates the development of psoriasis-like lesions via a Smad3-dependent, Th17-mediated mechanism. Targeting TGF-ß/Smad3 signalling with a Smad3 inhibitor may represent a novel and effective therapy for psoriasis.

Treatment for type 2 diabetes and diabetic nephropathy by targeting Smad3 signaling.

TGF-ß/Smad3 signaling plays a critical role in type 2 diabetes (T2D) and type 2 diabetic nephropathy (T2DN), but treatment by specifically targeting Smad3 remains unexplored. To develop a new Smad3-targeted therapy for T2D and T2DN, we treated db/db mice at the pre-diabetic or established diabetic stage with a pharmacological Smad3 inhibitor SIS3. The therapeutic effect and mechanisms of anti-Smad3 treatment on T2D and T2DN were investigated. We found that anti-Smad3 treatment on pre-diabetic db/db mice largely attenuated both T2D and T2DN by markedly reducing blood glucose levels, and inhibiting the elevated serum creatinine, microalbuminuria, and renal fibrosis and inflammation. Unexpectedly, although SIS3 treatment on the established diabetic db/db mice inhibited T2DN but did not significantly improve T2D. Mechanistically, we uncovered that inhibition of T2DN in SIS3-treated db/db mice was associated with effectively restoring the balance of TGF-ß/Smad signaling by inhibiting Smad3 while increasing Smad7, thereby suppressing Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation via lncRNA Erbb4-IR and LRN9884-dependent mechanisms. We also revealed that inhibition of islet ß cell injury by preventing the loss of islet Pax 6 could be the mechanism through which the pre-diabetic treatment, rather than the late SIS3 treatment on db/db mice significantly improved the T2D phenotype.

Co-delivery of doxorubicin and SIS3 by folate-targeted polymeric micelles for overcoming tumor multidrug resistance.

Multidrug resistance (MDR) is considered as a critical limiting factor for the successful chemotherapy, which is mainly characterized by the overexpression of ATP-binding cassette (ABC) transporter ABCB1 or ABCG2. In this study, folate-targeted polymeric micellar carrier was successfully constructed to co-delivery of doxorubicin (DOX) and SIS3 (FA/DOX/SIS3 micelles), a specific Smad3 inhibitor which sensitizes ABCB1- and ABCG2-overexpressing cancer cells to chemotherapeutic agents. The ratio of DOX to SIS3 in polymeric micelles was determined based on the anti-tumor activity against resistant breast cells. In addition, FA/DOX/SIS3 micelles exhibited a much longer circulation time in blood and were preferentially accumulated in resistant tumor tissue. Pharmacodynamic studies showed that FA/DOX/SIS3 micelles possessed superior anti-tumor activity than other DOX-based treatments. Overall, FA/DOX/SIS3 micelles are a promising formulation for the synergistic treatment of drug-resistant tumor.

SIS3, a good candidate for the reverse of type 2 diabetes mellitus in mice.

TGF-ß signaling plays an extremely important role in the occurrence and development of type 2 diabetes mellitus (T2DM), and the blockade of TGF-ß/Smad3 pathway protests against the high-fat diet-induced obesity and diabetes. As a specific small molecule inhibitor of Smad3 protein, the biological activities of compound SIS3 were evaluated by high-fat diet-induced T2DM model mice. In vivo results indicated that SIS3 can not only significantly reduce the body weight, fat mass, and fasting blood glucose in high-fat diet-induced T2DM model mice, but also improve insulin sensitivity and oral glucose tolerance of high-fat diet-induced T2DM model mice after the injection of SIS3 with 5 mg/kg for 45 days.

Tangshen formula promotes cellular cholesterol efflux in HG-induced mTECs by suppression of TGF-β1/Smad3 pathway / 中国临床药理学与治疗学

AIM: To observe the effect of Tangshen formula (TSF) treatment on TGF-β1/Smad3 pathway and cellular cholesterol efflux and explore its potential mechanism in HG-induced mouse tubular epithelial cells (mTECs). METHODS: After 25 mmol/L high glucose induced mTECs, TSF and Smad3 inhibitor SIS3 were given to intervene respectively. The lipid content in the cells was detected by ELISA kit; TGF-β1/Smad3 pathway and PGC-1α, LXR, ABCA1, ABCG1 were detected by Western blot and real-time PCR. RESULTS: TSF diminished the levels of TC, TG, LDL-C and increased the levels of HDL-C in HG-induced mTECs. Western blot and real-time PCR analysis showed that expression levels of TGF-β1, Smad3, Collagen and Fibronectin were significantly downregulated in the HG-induced mTECs with TSF and SIS3 treatment. And PGC-1α, LXR, ABCA1, ABCG1 expression levels were significantly upregulated in the HG-induced mTECs with TSF and SIS3 treatment. CONCLUSION: TSF can promote the cellular cholesterol efflux in HG-induced mTECs vis suppression of TGF-β1/Smad3 pathway.