Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR.

BACKGROUND: Histone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). METHODS: Renal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level. RESULTS: HDAC6 was significantly upregulated in renal tubular epithelial cells (RTECs) of both AKI and CKD patients as well as mice. In the murine models of AKI induced by LPS and adenine-induced nephropathy, CAY10603 exhibited notable protective effects, including improvement in biochemical indices and pathological changes. In vivo and in vitro studies revealed that CAY10603 effectively suppressed the activation of activating transcription factor 6 (ATF6) branch of UPR triggered by thapsigargin (Tg), a commonly employed endoplasmic reticulum (ER) stressor. Consistent with these findings, CAY10603 also displayed substantial inhibition of ATF6 activation in RTECs from both murine models of LPS-induced AKI and adenine-induced nephropathy. CONCLUSIONS: Collectively, these results suggest that CAY10603 holds promise as a potential therapeutic agent for both acute and chronic kidney injury.

The transcription factor HBP1 promotes ferroptosis in tumor cells by regulating the UHRF1-CDO1 axis.

The induction of ferroptosis in tumor cells is one of the most important mechanisms by which tumor progression can be inhibited; however, the specific regulatory mechanism underlying ferroptosis remains unclear. In this study, we found that transcription factor HBP1 has a novel function of reducing the antioxidant capacity of tumor cells. We investigated the important role of HBP1 in ferroptosis. HBP1 down-regulates the protein levels of UHRF1 by inhibiting the expression of the UHRF1 gene at the transcriptional level. Reduced levels of UHRF1 have been shown to regulate the ferroptosis-related gene CDO1 by epigenetic mechanisms, thus up-regulating the level of CDO1 and increasing the sensitivity of hepatocellular carcinoma and cervical cancer cells to ferroptosis. On this basis, we constructed metal-polyphenol-network coated HBP1 nanoparticles by combining biological and nanotechnological. MPN-HBP1 nanoparticles entered tumor cells efficiently and innocuously, induced ferroptosis, and inhibited the malignant proliferation of tumors by regulating the HBP1-UHRF1-CDO1 axis. This study provides a new perspective for further research on the regulatory mechanism underlying ferroptosis and its potential role in tumor therapy.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer.

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

Citrobacter portucalensis Sb-2 contains a metalloid resistance determinant transmitted by Citrobacter phage Chris1.

Bacterial adaptation to extreme environments is often mediated by horizontal gene transfer (HGT) via genetic mobile elements. Nevertheless, phage-mediated HGT conferring bacterial arsenic resistance determinants has rarely been investigated. In this study, a highly arsenite and antimonite resistant bacterium, Citrobacter portucalensis strain Sb-2, was isolated, and genome analysis showed that several putative arsenite and antimonite resistance determinants were flanked or embedded in prophages. Furthermore, an active bacteriophage carrying one of the ars clusters (arsRDABC arsR-yraQ/arsP) was obtained and sequenced. These genes encoding putative arsenic resistance determinants were induced by arsenic and antimony as demonstrated by RT-qPCR, and one gene arsP/yraQ of the ars cluster was shown to give resistance to MAs(III) and Rox(III), thereby showing function. Here, we were able to directly show that these phage-mediated arsenic and antimony resistances play a significant role in adapting to As- and Sb-contaminated environments. In addition, we demonstrate that this phage is responsible for conferring arsenic and antimony resistances to C. portucalensis strain Sb-2.

HBP1 inhibits the development of type 2 diabetes mellitus through transcriptional activation of the IGFBP1 gene.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that is highly prevalent worldwide and characterized by glucose and lipid metabolism disorders. However, the pathogenic mechanisms have not been fully established. Here, we found that HMG-box transcription factor 1 (HBP1) is involved in T2DM and that its deficiency in mice aggravates the features of diabetes. In addition, we undertook screening by RNA sequencing and found that HBP1 activates the transcription of the insulin-like growth factor binding protein 1 (IGFBP1) gene. Moreover, Insulin and palmitic acid reduced HBP1 protein expression and inhibited its binding to the IGFBP1 promoter. Furthermore, HBP1 reduced the serum free insulin-like growth factor 1 (IGF-1) concentration through IGFBP1 and inhibited the PI3K/AKT signaling pathway. This forms an insulin/HBP1/IGFBP1 negative feedback regulatory loop to dynamically regulate blood glucose and insulin concentrations. These findings have elucidated a mechanism whereby HBP1 and its negative feedback regulatory loop influence the development of T2DM, thereby providing a new theoretical basis and potential therapeutic target for T2DM.

Methylation of HBP1 by PRMT1 promotes tumor progression by regulating actin cytoskeleton remodeling.

HBP1 is a sequence-specific transcription factor which generally considered as a crucial growth inhibitor. Posttranslational modification of HBP1 is vital for its function. In this study, we demonstrate that HBP1 is methylated at R378 by PRMT1, which decreases HBP1 protein stability by promoting its ubiquitination and proteasome-mediated degradation. PRMT1-mediated methylation of HBP1 alleviates the repressive effects of HBP1 on tumor metastasis and growth. GSN is identified as a novel target gene of HBP1. Methylation of HBP1 promotes actin cytoskeleton remodeling, glycolysis and tumor progression by downregulating GSN (a vital actin-binding protein) levels. The methylated HBP1-GSN axis is associated with the clinical outcomes of cancer patients. This investigation elucidates the mechanism of how methylated HBP1 facilitates actin cytoskeleton remodeling, thus attenuates its tumor-suppressive function and promotes tumor progression. Targeting methylated HBP1-GSN axis may provide a therapeutic strategy for cancer.

GP73-mediated secretion of AFP and GP73 promotes proliferation and metastasis of hepatocellular carcinoma cells.

Golgi protein 73 (GP73) and alpha fetoprotein (AFP) serve as biomarkers for the diagnosis of hepatocellular carcinoma (HCC), and their serum levels correlate with patients' outcomes. However, the mechanisms underlying these correlations are unknown. Here we show that GP73 increased the secretion of AFP through direct binding to AFP, thereby promoting the proliferation and metastasis of HCC cells that expressed AFP and its receptor (AFPR). Extracellular GP73 contributed to the proliferation and metastasis of HCC cells independent of AFP and AFPR. Moreover, extracellular AFP and GP73 synergized to enhance the malignant phenotype of HCC cells. Furthermore, extracellular GP73 and AFP inhibited the antitumor effects of sorafenib and synergistically increased the drug resistance of HCC cells. These findings, which reveal the mechanism of GP73-mediated secretion of AFP and its effects on the malignant phenotype of HCC cells, provide a comprehensive theoretical basis for the diagnosis and treatment of HCC and identify potential drug targets.

HBP1-mediated transcriptional repression of AFP inhibits hepatoma progression.

BACKGROUND: Hepatoma is a common malignancy of the liver. The abnormal high expression of alpha-fetoprotein (AFP) is intimately associated with hepatoma progress, but the mechanism of transcriptional regulation and singularly activation of AFP gene in hepatoma is not clear. METHODS: The expression of transcription factor HBP1 and AFP and clinical significance were further analyzed in hepatoma tissues from the patients who received surgery or TACE and then monitored for relapse for up 10 years. HBP1-mediated transcriptional regulation of AFP was analyzed by Western blotting, Luciferase assay, Realtime-PCR, ChIP and EMSA. After verified the axis of HBP-AFP, its impact on hepatoma was measured by MTT, Transwell and FACS in hepatoma cells and by tumorigenesis in HBP1-/- mice. RESULTS: The relative expressions of HBP1 and AFP correlated with survival and prognosis in hepatoma patients. HBP1 repressed the expression of AFP gene by directly binding to the AFP gene promoter. Hepatitis B Virus (HBV)-encoded protein HBx promoted malignancy in hepatoma cells through binding to HBP1 directly. Icaritin, an active ingredient of Chinese herb epimedium, inhibited malignancy in hepatoma cells through enhancing HBP1 transrepression of AFP. The repression of AFP by HBP1 attenuated AFP effect on PTEN, MMP9 and caspase-3, thus inhibited proliferation and migration, and induced apoptosis in hepatoma cells. The deregulation of AFP by HBP1 contributed to hepatoma progression in mice. CONCLUSIONS: Our data clarify the mechanism of HBP1 in inhibiting the expression of AFP and its suppression in malignancy of hepatoma cells, providing a more comprehensive theoretical basis and potential solutions for the diagnosis and treatment of hepatoma.

Icaritin promotes apoptosis and inhibits proliferation by down-regulating AFP gene expression in hepatocellular carcinoma.

BACKGROUND: Icaritin, an active ingredient of the Chinese herb Epimedium, plays an anti-tumor role in liver cancer by inhibiting the proliferation of hepatocellular cells and promoting their apoptosis. In China, phase II and a large phase III clinical trial of icaritin reagent for the treatment of hepatocellular cancer is under-going, but the specific mechanism of icaritin action was unclear. Alpha-fetoprotein (AFP), an oncofetal protein, produced in the healthy fetal liver and yolk sac. Intracellular AFP promoted cellular proliferation and inhibited cellular apoptosis in hepatocellular carcinoma (HCC). The study was aimed to investigate the effect of icaritin on HCC through p53/AFP pathway. METHODS: Real-time RT PCR and western blot were used to detect p53 and AFP expression levels in HCC cells treated with icaritin. The mechanism of icaritin affecting p53 expression was verified by ubiquitination experiment, and the binding activity of icaritin on p53 in AFP promoter region was verified by luciferase experiment. EdU, MTT and flow cytometry were used to determine whether icaritin affected HCC cellular proliferation and apoptosis through p53/ AFP pathway. Expression levels of p53 and AFP in xenograft mouse model were determined by western blotting. RESULTS: Our results showed icaritin inhibited AFP expression at mRNA and protein level. AFP was also identified as the target gene of the p53 transcription factor. Icaritin abrogated murine double minute (Mdm) 2-mediated p53 ubiquitination degradation to improve the stability of p53. Up-regulated p53 protein levels then transcriptionally inhibited the AFP promoter. Icaritin-mediated decrease of AFP through Mdm2/p53 pathways inhibited HCC cellular proliferation and promoted HCC cellular apoptosis. CONCLUSION: Our findings revealed the mechanism of icaritin in promoting apoptosis and inhibiting proliferation in liver cancer cells. The regulatory mechanism of icaritin in AFP protein down-regulation provides a theoretical and experimental basis for further research into new drugs for the treatment of liver cancer.

Downregulation of miR-34a Promotes Proliferation and Inhibits Apoptosis of Rat Osteoarthritic Cartilage Cells by Activating PI3K/Akt Pathway.

OBJECTIVE: To elucidate the expression and function of miR-34a in rat osteoarthritic cartilage cells, and further to explore its mechanism. MATERIAL AND METHODS: Rat model of osteoarthritis was constructed and knee joint cartilage cells were isolated in vitro. Immunocytochemical staining was used for identification. qRT-PCR was used to detect the expression of miR-34a in cartilaginous tissues and cartilage cells. Cartilage cells were divided into blank control (BC), negative control (NC), miR-34a inhibitor (34aI), osteoarthritis model (OA), osteoarthritis model + negative control (OA + NC) and osteoarthritis model + miR-34a inhibitor (OA + 34aI) groups. Cell proliferation was detected by CCK-8 and colony formation assays. Cell apoptosis was studied by flow cytometry and Western blot. PI3K/AKT-pathway-related proteins were also analyzed by Western blot. To further validate the effect of miR-34a on the PI3K/Akt pathway, the cartilage cells were divided into blank control (BC), osteoarthritis model (OA), osteoarthritis model + miR-34a inhibitor (OA + 34aI), osteoarthritis model + PI3K activator (OA + IGF-1) and osteoarthritis model + miR-34a inhibitor + PI3K inhibitor (OA + 34aI + LY) groups, the experiments above were repeated. RESULTS: The expression of miR-34a in cartilaginous tissues and cells of osteoarthritis model was significantly higher than that in normal (p < 0.05). After silencing miR-34a gene, the cell proliferation and proteins expression of PI3K/Akt pathway were increased, while the apoptosis rate and expression of apoptosis-related proteins were decreased. Addition of PI3K activator also evidently promoted proliferation and inhibited apoptosis. The protein expression of Bax, Cleaved caspase-3 and Cleaved caspase-9 were dramatically decreased, while the ratios of p-PI3K/PI3K and p-Akt/Akt were increased in OA + IGF-1 group. CONCLUSION: Downregulation of miR-34a regulated proliferation and apoptosis of cartilage cells by activating PI3K/Akt pathway, providing a potential therapeutic approach for the treatment of osteoarthritis.

Acetylation of alpha-fetoprotein promotes hepatocellular carcinoma progression.

Alpha-fetoprotein (AFP) is a well-established biomarker for hepatocellular carcinoma (HCC). Here, we investigated the acetylation state of AFP in vivo. AFP acetylation was regulated by the acetyltransferase CBP and the deacetylase SIRT1. Acetylation of AFP at lysines 194, 211, and 242 increased the stability of AFP protein by decreasing its ubiquitination and proteasomal degradation. AFP acetylation promoted its oncogenic role by blocking binding to the phosphatase PTEN and the pro-apoptotic protein caspase-3, which increased signaling for proliferation, migration, and invasion and decreased apoptosis. High levels of acetylated AFP in HCC tissues were associated with HBV infection and correlated with poor prognosis and decreased patient survival. In HCC cells, hepatitis B virus X protein (HBx) and palmitic acid (PA) increased the level of acetylated AFP by disrupting SIRT1-mediated deacetylation. AFP acetylation plays an important role in HCC progression and provides a new potential prognostic marker and therapeutic target for HCC.

Solid-phase extraction based on a molecularly imprinted polymer nanoshell at the surface of silica nanospheres for the specific enrichment and identification of alkaloids from Crinum asiaticum L. var. sinicum.

A molecularly imprinted nanoshell on the surface of silica nanospheres was prepared for specific enrichment and identification of alkaloids from Crinum asiaticum L. var. sinicum. The nanoshell was synthesized by surface polymerization using lycorine as the template, acrylamide as the functional monomer, ethylene glycol dimethacrylate as the cross-linker, 2',2-azobisisobutyronitrile as the initiator and acetonitrile as the pore-forming agent. The core-shell nanospheres were characterized by transmission electron microscopy and infrared spectroscopy, and the results show that the nanoshell layer was homogeneously attached to the surface of vinyl-modified SiO2 nanospheres. The adsorption capacity of the nanospheres was estimated by binding equilibrium and adsorption kinetics experiments. The maximum adsorption amount of lycorine on the nanospheres was 6.68 µmol/g and the imprinting factor was nearly 2.5, indicating a good imprinting effect. The nanospheres were successfully applied in solid-phase extraction for lycorine from Crinum asaticum L. var. sinicum and detection of target molecule in rat metabolites. The average recoveries of lycorine in Crinum asaticum L. var. sinicum extraction and rat metabolites were 93.5 ± 0.6% (n = 3) and 91.6 ± 1.9% (n = 3), respectively. This work provides a simple approach for the fabrication of a molecularly imprinted nanoshell at the surface of silica nanospheres-based solid-phase extraction for drug analysis.

Clinical effects of joint application of ß-sodium aescinate and mannitol in treating early swelling after upper limb trauma surgery.

The aim of the present study was to examine the clinical merits of joint application of ß-sodium aescinate and mannitol for the treatment of early swelling of upper limb trauma after surgery. We verified whether the expression of serum aquaporin 1 (AQP-1) was involved in swelling mechanism. A total of 102 patients with swelling after upper limb trauma surgery were enrolled into the study and divided randomly into 3 groups (n=34 cases per group). Group A was treated with ß-sodium aescinate; group B was treated with with mannitol and group C was treated with both ß-sodium aescinate and mannitol. The expression level of AQP-1, and clinical effects and complications before and after treatment were compared§. The time of swelling subsidence in group C was significantly shorter than that of the other two groups and differences were statistically significant (P<0.05). The recovery ratio and total efficiency in group C were significantly higher than those in other two groups and differences were statistically significant (P<0.05). Three and seven days after treatment, the AQP-1 levels in group A and group C were decreased and AQP-1 level decreased further with time. Differences of comparison within groups were statistically significant (P<0.05), although the differences of comparison between the groups showed no statistical significance (P>0.05). We also compared the AQP-1 level in group B before and after treatment, and the differences were not statistically significant (P>0.05). When the complication incidence in the 3 groups was compared, no statistical significance was detected (P>0.05). We concluded that the joint use of ß-sodium aescinate and mannitol in treating early swelling after upper limb trauma surgery produced satisfactory outcomes. This might be related to reduction of the AQP-1 level.

[Effects of mannose-6-phosphate on transforming growth factor beta and transforming growth factor beta receptor expression of flexor tendon cells].

OBJECTIVE: By culturing tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes of rabbits' tendon in vitro, to study the effects of mannose-6-phosphate on transforming growth factor beta (TGF-beta) peptide and receptor expression, and to provide the experimental basis for preventing the tendon healing adhesion by mannose-6-phosphate. METHODS: Eight adult New Zealand white rabbits, regardless of their gender and weighing 4.0-4.5 kg, were selected. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All 3 cells were divided into 2 groups at random after cells were adjusted to a concentration of 4 x 10(4) per well and 1 x 10(4)/mL. The first was the control group without supplementation. The experimental group was supplemented with mannose-6-phosphate. The expressions of TGF-beta and TGF-beta receptor were quantified with enzyme-linked immunosorbent assay. The expression of TGF-beta1 mRNA was also assessed with in situ hybridization and the expression of TGF-beta1 was assessed with immunohistochemistry. RESULTS: The expressions of TGF-beta and TGF-beta receptor in experimental group were significantly lower than that in control group (P < 0.05). The expression levels of TGF-beta1 and TGF-beta2 decreased in descending order of tendon sheath fibroblasts (36.1%, 37.9%), epitenon tenocytes (31.0%, 32.1%), and endotenon tenocytes (31.2%, 27.0%). The expression levels of TGF-beta3 decreased in descending order of endotenon tenocytes (42.5%), tendon sheath fibroblasts (41.2%), and epitenon tenocytes (33.3%). The expression levels of TGF-beta receptor 1 and TGF-beta receptor 2 decreased in descending order of epitenon tenocytes (29.9%, 26.2%), endotenon tenocytes (27.8%, 23.5%), and tendon sheath fibroblasts (23.1%, 20.0%). The expression levels of TGF-beta receptor 3 decreased in descending order of endotenon tenocytes (26.1%), epitenon tenocytes (19.2%), and tendon sheath fibroblasts (15.8%). In experimental group, the positive expression of TGF-beta1 mRNA and the expression level of intracellular TGF-beta1 mRNA in all 3 tendon cells were significantly lower than those in the control group (P < 0.05). Immunohistochemical staining showed the expressions of TGF-beta1 in all 3 tendon cells were significantly lower in the experimental group than in the control group. CONCLUSION: Mannose-6-phosphate can significantly decrease the expressions of TGF-beta peptide, TGF-beta receptor, and TGF-beta1 mRNA. Modulation of mannose-6-phosphate levels may provide a mean of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.