Nrf2 knockout attenuates the astragaloside IV therapeutic effect on kidney fibrosis from liver cancer by regulating pSmad3C/3L pathways.

Fibrotic kidney injury from hepatocarcinogenesis seriously impacts treatment effect. Astragaloside IV (AS-IV), an extract of Astragalus membranaceus, has several pharmacological activities, which are useful in the treatment of edema and fibrosis. Nrf2/HO-1 is a key antioxidant stress pathway and help treatment of kidney injury. Smad3 phosphorylation is implicated in hepatocarcinogenesis. Our previous study clarified that Smad3 is differentially regulated by different phosphorylated forms of Smad3 on hepatocarcinogenesis. Therefore, we investigated the contribution of AS-IV on the therapy of kidney fibrosis from hepatocarcinogenesis. And the focus was on whether the phosphorylation of Smad3 and the regulation of Nrf2/HO-1 pathway were involved during AS-IV therapy and whether there is an effect of Nrf2 knockout on the phosphorylation of Smad3. We performed TGF-ß1 stimulation on HK-2 cells and intervened with AS-IV. Furtherly, we investigated renal injury of AS-IV on Nrf2 knockout mice during hepatocarcinogenesis and its mechanism of action. On the one hand, in vitro results showed that AS-IV reduced the ROS and α-SMA expression of HK-2 by promoting the expression pSmad3C/p21 of and Nrf2/HO-1 and suppressed the expression of pSmad3L/PAI-1. On the other hand, the in vivo results of histopathological features, serological biomarkers, and oxidative damage indicators showed that Nrf2 knockout aggravated renal injury. Besides, Nrf2 deletion decreased the nephroprotective effect of AS-IV by suppressing the pSmad3C/p21 pathway and promoting the pSmad3L/PAI-1 pathway. The experimental results were as we suspected. And we identify for the first time that Nrf2 deficiency increases renal fibrosis from hepatocarcinogenesis and attenuates the therapeutic effects of AS-IV via regulating pSmad3C/3L signal pathway.

Salvianolic acid B exerts an anti-hepatocellular carcinoma effect by regulating the Hippo/YAP pathway and promoting pSmad3L to pSmad3C simultaneously.

Salvianolic acid B (Sal B) is a component obtained from Salvia miltiorrhiza and is empirically used for liver diseases. The TGF-ß/Smad and Hippo/YAP pathways may interact with each other in hepatocellular carcinoma (HCC). Previously, we found that Sal B mediates the TGF-ß/Smad pathway in mice and delays liver fibrosis-carcinoma progression by promoting the conversion of pSmad3L to pSmad3C, but the effect of Sal B on the Hippo/YAP pathway has not been determined. Therefore, we used a DEN/CCl4/C2H5OH-induced liver cancer model in mice to analyze liver index and tumor incidence, detect AST and ALT serological markers, observe liver pathology and the number of Ki67-positive cells to evaluate the anti-HCC effect of Sal B in vivo. We used a TGF-ß1-induced HepG2 cell model, and applied an MST1/2 inhibitor, XMU-MP-1, to detect the changes in pSmad3C/pSmad3L signaling induced by MST1/2 inhibition. Sal B significantly inhibited tumorigenesis in DEN/CCl4/C2H5OH-induced mice in vivo, and suppressed the growth of HepG2 cells by inhibiting cell proliferation and migration in vitro. Here, our study also validated the role of Sal B in reversing XMU-MP-1-induced proliferation and migration of HepG2 cells in vitro. Most importantly, we elucidated for the first time the potential mechanism of Sal B against HCC via the Hippo/YAP pathway, which may be specifically related to upregulation of MST1 and inhibition of its downstream effector protein YAP. In conclusion, these findings indicate that Sal B possesses anti- HCC effects both in vivo and in vitro by regulating the Hippo/YAP pathway and promoting pSmad3L to pSmad3C synchronously.

Astragaloside IV inhibits hepatocellular carcinoma by continually suppressing the development of fibrosis and regulating pSmad3C/3L and Nrf2/HO-1 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus is a medicinal herb used in China for the prevention and treatment of diseases such as diabetes and cancer. As one of the main active ingredients of astragalus, Astragaloside IV (AS-IV) has a wide range of pharmacological effects, including anti-inflammation and anti-cancer effects. AIM OF THE STUDY: Different phosphorylated forms of Smad3 differentially regulate the progression of hepatic carcinoma. The phosphorylation of the COOH-terminal of Smad3 (pSmad3C) and activation of the Nrf2/HO-1 pathway inhibits hepatic carcinoma, while phosphorylation of the linker region of Smad3 (pSmad3L) promotes progression. Thus, pSmad3C/3L and Nrf2/HO-1 pathways are potential targets for drug of anti-cancer development. AS-IV is anti-apoptotic and can inhibit hepatocellular carcinoma cell (HCC) proliferation, invasion, and tumor growth in nude mice. However, it is not clear whether AS-IV has a therapeutic effect on inhibiting the progression of primary liver cancer by regulating the pSmad3C/3L and Nrf2/HO-1 pathway. The purpose of this study is to investigate whether AS-IV inhibits hepatocellular carcinoma by regulating pSmad3C/3L and Nrf2/HO-1 pathway. MATERIALS AND METHODS: primary liver cancer in mice induced by DEN/CCl4/C2H5OH (DCC) and HSC-T6/HepG2 cell models activated by TGF-ß1 was investigated for the mechanisms of AS-IV. In vivo assays included liver biopsy, histopathology and post-mortem analysis included immunohistochemistry, immunofluorescent, and Western blotting analysis, and in vitro assays included immunofluorescent, and Western blotting analysis. RESULTS: AS-IV significantly inhibited the development of primary liver cancer, reflecting improved liver biopsy, histopathology. The incidence and multiplicity of primary liver cancer were markedly decreased by AS-IV treatment at the 20th week. AS-IV had observable effects on the TGF-ß1/Smad and Nrf2/HO-1 expression in vivo, especially up-regulated pSmad3C, pNrf2, HO-1, and NQO1, while it down-regulated pSmad2C, pSmad2L, pSmad3L, PAI-1, and α-SMA at the 12th week and the 20th week. Furthermore, in vitro analysis further confirmed that AS-IV regulated the expression of pSmad3C/3L and Nrf2/HO-1 pathway in HSC-T6 and HepG2 cells activated by TGF-ß1. CONCLUSION: AS-IV administration delays the occurrence of primary liver cancer by continually suppressing the development of fibrosis, the mechanism of the therapeutic effect involving the regulation of the pSmad3C/3L and Nrf2/HO-1 pathways, especially in regulation reversibility and antagonism of pSmad3C and pSmad3L and promoting the phosphorylation of Nrf2.

Smad3 gene C-terminal phosphorylation site mutation exacerbates CCl4-induced hepatic fibrogenesis by promoting pSmad2L/C-mediated signaling transduction.

Current researches have confirmed that Smads, mediators of TGF-ß signaling, are strictly controlled by domain-specific site phosphorylation in the process of hepatic disease. Usually, Smad3 phospho-isoform pSmad3L and pSmad3C are reversible and antagonistic; pSmad2L/C could act together with pSmad3L by stimulating PAI-1 expression and ECM synthesis to transmit fibrogenic signals. Our recent study found that pSmad3C mutation is supposed to perform a vigorous role on the early phase of liver injury and abates salvianolic acid B's anti-hepatic fibrotic-carcinogenesis. However, whether pSmad3C mutation expedites pSmad2L/C-mediated signaling transduction during hepatic fibrogenesis remains vague. Presently, Smad3 gene C-terminal phosphorylation site mutation heterozygote (pSmad3C+/-) mice were constructed to probe if and how pSmad3C retards CCl4-induced hepatic fibrogenesis by inhibiting pSmad2L/C-mediated signaling transduction. Twelve 6-week-old pSmad3C+/- C57BL/6J mice were intraperitoneally injection with CCl4 for 6 weeks to induce liver fibrogenesis. Results showed that pSmad3C mutation aggravates the relative liver weight, biochemical parameters, collagenous fibers and fibrotic septa formation, contributes to fibrogenesis in HT-CCl4 mice. Furthermore, fibrotic-related proteins TGF-ß1, pSmad2C, pSmad2L, and PAI-1 were also increased in CCl4-induced pSmad3C+/- mice. These results suggest that pSmad3C mutation exacerbates hepatic fibrogenesis which relates to intensifying pSmad2L/C-mediated signaling transduction.

Sorafenib suppresses proliferation rate of fibroblast-like synoviocytes through the arrest of cell cycle in experimental adjuvant arthritis.

OBJECTIVES: Rheumatoid arthritis, a recurrent incendiary autoimmune joint syndrome, features by prominent synovial hyperplasia. Fibroblast-like synoviocytes are the executive components in the pathogenesis of rheumatoid arthritis. It is generally accepted that excessive proliferation and reduced apoptosis of fibroblast-like synoviocytes lead to synovial hyperplasia. Our previously studies found that sorafenib could inhibit adjuvant arthritis in rats and induced adjuvant arthritis fibroblast-like synoviocytes apoptosis. Presently, we aim to investigate the inhibitory effect with mechanisms of action of sorafenib on adjuvant arthritis fibroblast-like synoviocytes proliferation. METHODS: Cell counting kit-8 and flow cytometry detection were conducted to monitor FLSs proliferation and cell cycle. Western blotting and qPCR assays were performed to detect P21, P53, CDK4, CyclinD1 and proliferating cell nuclear antigen content levels. KEY FINDINGS: Sorafenib significantly inhibited adjuvant arthritis fibroblast-like synoviocytes proliferation with an IC50 value of 4 µmol/L by a concentration-dependent pattern, which accompanies by G1 cell cycle arrest. Also, sorafenib significantly decreased the levels of P21, CyclinD1, CDK4 and proliferating cell nuclear antigen, as well as up-regulated P53 expression in adjuvant arthritis fibroblast-like synoviocytes. CONCLUSIONS: Sorafenib could inhibit adjuvant arthritis fibroblast-like synoviocytes proliferation via arresting G1/S cell cycle progression, which was partially through CDK4/CyclinD1-mediated pathway, as well as up-regulating P53 and down-regulating proliferating cell nuclear antigen expressions. These results suggest that sorafenib may provide a new paradigm for rheumatoid arthritis treatment.

Scar-reducing effects of gambogenic acid on skin wounds in rabbit ears.

Hypertrophic scar (HS) is a dermal fibroproliferative disease that often occurs following abnormal wound healing. To date, there is no satisfied treatment strategies for improvement of scar formation with few side effects. The effects of gambogenic acid (GNA) on scar hypertrophy has not been studied previously. The present study was undertaken to find out the scar-reducing effects of GNA (0.48, 0.96 or 1.92 mg/ml) on skin wounds in rabbit ears. Scar evaluation index (SEI), collagen I (Col1) and collagen III (Col3), microvascular density (MVD), CD4+T cells and macrophages, vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), phospho-VEGFR 2 (p-VEGFR2) and p-FGFR1, interleukin (IL)-1ß, IL-6, IL-10 and tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and connective tissue growth factor (CTGF) in scar tissue were detected using various methods, respectively. Our data showed that GNA significantly reduced SEI, and the expression of Col1 and Col3 in scar tissue in a concentration-dependent manner. Also, it decreased MVD, the infiltration of CD4+T cells and macrophages, and the levels of VEGFR2, p-VEGFR2, FGFR1, p-FGFR1, TGF-ß1, CTGF, IL-1ß, IL-6, TNF-α, in addition to upregulated IL-10 in scar tissue. As a result, this study revealed that GNA reduced HS formation, which was associated with the inhibition of neoangiogenesis, local inflammatory response and growth factor expression in scar tissue during wound healing. These findings suggested that GNA may be considered as a preventive and therapeutic candidate for HS.

Smad3 C-terminal phosphorylation site mutation attenuates the hepatoprotective effect of salvianolic acid B against hepatocarcinogenesis.

Smad3 phosphorylation is implicated in hepatic fibro-carcinogenesis. Moreover, Smad3 phospho-isoform pSmad3L and pSmad3C are reversible and antagonistic, and the balance could shift from carcinogenesis to tumor-suppression. pSmad3C has recently assigned to perform a preventative effect against primary liver injury. Salvianolic acid B (Sal B), a component derived from Salvia miltiorrhiza, is empirically used for hepatic diseases. Our prior study clarified that Sal B could delay hepatic fibrosis-carcinoma progression by converting pSmad3L/3C in mice. However, the roles of Smad3 phospho-isoform conversion and antagonism in the anti-hepatocarcinogenic effects of Sal B in pSmad3C- or/and pSmad3L-mutated mice/cells remain vague. Currently, corresponding doses/concentrations of Sal B was co-administrated to pSmad3C+/- mutational mice/plasmids-transfected HepG2 cells. Notably, in vivo functional studies revealed that pSmad3C mutation attenuates Sal B-induced ameliorative effects on histopathological characteristics and decreased serological biomarkers, and potential mechanism involves attenuation of increases in pSmad3C/p21 and decreases in pSmad3L/PAI-1/c-Myc expression. Expectedly, in vitro results showed that up-regulating pSmad3C enhances the inhibitory effects on proliferation, migration and contributes to apoptosis accompanied by a shift of pSmad3L/PAI-1/c-Myc oncogenic to pSmad3C/p21 tumour-suppressive signalling; however, opposite effects occur when upregulated pSmad3L. This study is the first to identify pSmad3C as a key target by which Sal B prevents hepatocarcinogenesis.

Effects of sorafenib on fibroblast-like synoviocyte apoptosis in rats with adjuvant arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by synovial inflammation and hyperplasia resulting from an imbalance between the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs). Our previous study found that sorafenib had inhibitory effects in rats with adjuvant arthritis (AA). The present study investigated the role of sorafenib in the induction of AA FLS apoptosis in vitro. FLSs obtained from AA rats were cultured in vitro and identified. Cell apoptosis was detected using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. Real-time PCR and Western blotting assays were used to quantify the expression levels of Fas, Caspase-3, Mcl-1, NF-κB and C-jun gene products in AA FLSs. Our data revealed that sorafenib (4 µmol/L) induced apoptosis in AA FLSs, and flow cytometry analysis showed that AA FLSs treated with sorafenib (4 µmol/L) in vitro accumulated in early and late apoptosis. There were significant increases in the expression levels of Fas, Caspase-3 and Mcl-1, and significant decreases in NF-κB and C-jun expression in AA FLSs treated with sorafenib. In summary, these results demonstrate that sorafenib promotes AA FLS apoptosis, which may be related to the upregulation of Fas and Caspase-3 and downregulation of NF-κB and C-jun. All of these findings suggest that sorafenib exerts an inhibitory effect on AA rats in vivo via AA FLS apoptotic induction, which has potential therapeutic implications for RA.

Antiarthritic Effects of Sorafenib in Rats with Adjuvant-Induced Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the synovial membrane of joints. In this study, we aimed to investigate whether sorafenib exerts antiarthritic effects on RA in vivo. Adjuvant arthritis (AA) was induced (day 0) in male Sprague-Dawley rats by intradermal injection of 0.1 mL of complete Freund's complete adjuvant into the left hind paw. Sorafenib (10, 20, or 40 mg/kg/day) was administered intragastrically from day 10 to 24. Body weight, paw volume, synovial inflammation, and tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-10, and IL-17 serum levels were detected. In addition, microvascular density (MVD) and the expression of vascular endothelial growth factor receptor 2 (VEGFR-2) and fibroblast growth factor receptor 1 (FGFR-1) in synovial tissues were analyzed. Our data revealed that sorafenib administration led to significant body weight gain in AA rats but suppressed paw swelling, synovial hyperplasia, and inflammatory infiltration. Furthermore, it decreased TNF-α, IL-1ß, and IL-17 serum levels and upregulated IL-10. MVD and VEGFR-2 and FGFR-1 expression in synovial tissues were significantly reduced. Thus, this study shows that sorafenib exerts anti-arthritic effects in AA rats and therefore has potential in RA treatment. Anat Rec, 301:1519-1526, 2018. © 2018 Wiley Periodicals, Inc.

Effect of recombinant human endostatin on hypertrophic scar fibroblast apoptosis in a rabbit ear model.

Hypertrophic scar (HS) is a dermal fibroproliferative disorder characterized by the excessive proliferation of fibroblasts and is thought to result from a cellular imbalance caused by the increased growth and reduced apoptosis of hypertrophic scar fibroblasts (HSFs). Our recent study demonstrated that recombinant human endostatin (rhEndostatin) plays a key role in the inhibition of HSF proliferation in vitro, with a resulting decrease in dermal thickness and scar hypertrophy. However, the effect of this protein on HSF apoptosis is unknown. The present study was undertaken to directly examine the effect of rhEndostatin on HSF apoptosis in the rabbit ear model. Transmission electron microscopy and flow cytometry were used to investigate HSF apoptosis in scar tissues and cultured HSFs in vitro, respectively. The expression levels of the c-jun, c-fos, NF-κB, fas, caspase-3, and bcl-2 gene products in HSFs were quantified using real-time PCR and Western blotting assays. Our data reveal that rhEndostatin (2.5 or 5mg/ml) induces HSF apoptotic cell death in scar tissue. Additionally, HSFs treated with rhEndostatin (100mg/L) in vitro accumulated in early and late apoptosis and displayed significantly decreased expression of c-jun, c-fos, NF-κB, fas, caspase-3 and bcl-2. In sum, these results demonstrate that rhEndostatin induces HSF apoptosis, and this phenotypeis partially due to downregulation of NF-κB and bcl-2. These findings suggest that rhEndostatin may have an inhibitory effect on scar hypertrophy in vivo via HSF apoptotic induction and therefore has potential therapeutic use for the treatment of HS.