Colorectal cancer (CRC) is known as a common malignant neoplasm worldwide. The role of EGFR/HER2 in CRC is unclear. Afatinib is an irreversible EGFR/HER2 inhibitor. There were few studies of afatinib on CRC. Here, we investigated the protein levels/expressions of HER2 in sera and tumors from CRC patients and the therapeutic effect of afatinib on HER2-overexpressed CRC in vitro and in vivo. The increased HER2 levels were detected in the collected sera and tumors of patients with CRC. The serological HER2 levels were correlated with the tumor HER2 expressions in patients. Afatinib also inhibited the HER2-positive tumor cell growth and caused apoptosis in HER2-overexpressed human colorectal cancer HCT-15 cells but not in low HER2 expressed human gastric cancer MKN45 cells. In vivo study showed that afatinib reduced tumor growth in HER2-overexpressed xenografts. Moreover, afatinib-encapsulated micelles displayed higher cytotoxic activity in HCT-15 cells and were more effective for tumor growth suppression in HCT-15-induced tumor xenografts than afatinib performance alone. Taken together, these findings suggest that higher serum HER2 levels reflect the higher HER2 contents in tumors of CRC patients, and the improved afatinib-encapsulated micelles possess high therapeutic efficacy in HER2-overexpressed CRC in vitro and in vivo.

The therapeutic effect of pterosin A, a small-molecular-weight natural product, on diabetes was investigated. Pterosin A, administered orally for 4 weeks, effectively improved hyperglycemia and glucose intolerance in streptozotocin, high-fat diet-fed, and db/db diabetic mice. There were no adverse effects in normal or diabetic mice treated with pterosin A for 4 weeks. Pterosin A significantly reversed the increased serum insulin and insulin resistance (IR) in dexamethasone-IR mice and in db/db mice. Pterosin A significantly reversed the reduced muscle GLUT-4 translocation and the increased liver phosphoenolpyruvate carboxyl kinase (PEPCK) expression in diabetic mice. Pterosin A also significantly reversed the decreased phosphorylations of AMP-activated protein kinase (AMPK) and Akt in muscles of diabetic mice. The decreased AMPK phosphorylation and increased p38 phosphorylation in livers of db/db mice were effectively reversed by pterosin A. Pterosin A enhanced glucose uptake and AMPK phosphorylation in cultured human muscle cells. In cultured liver cells, pterosin A inhibited inducer-enhanced PEPCK expression, triggered the phosphorylations of AMPK, acetyl CoA carboxylase, and glycogen synthase kinase-3, decreased glycogen synthase phosphorylation, and increased the intracellular glycogen level. These findings indicate that pterosin A may be a potential therapeutic option for diabetes.

Arsenic is a ubiquitous toxic element and is known to contaminate drinking water in many countries. Several epidemiological studies have shown that arsenic exposure augments the risk of bone disorders. However, the detailed effect and mechanism of inorganic arsenic on osteoblast differentiation of bone marrow stromal cells and bone loss still remain unclear.

In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m(+) mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition.

Stroke is known as the top 10 causes of death worldwide. Development of effectively neuroprotective or preventive strategies for ischemia stroke is imperative. For the purpose of stroke prevention, we tested the neuroprotective effects of low-intensity pulsed ultrasound (LIPUS) on ischemic stroke. Adult C57BL/6 mice were used to daily treatment with LIPUS for 5 days on left hemisphere before middle cerebral artery occlusion (MCAO)-induced cerebral ischemia/reperfusion injury. Western blotting and immunohistochemistry were performed to assess the protein expressions of signaling molecules. Pretreatment with LIPUS significantly ameliorated the brain ischemic damage, including the reduction of neurological deficit score, infarct area, histopathological score, and showed a better performance in neurological and behavior functions. LIPUS pretreatment could also significantly decrease the neuronal cell apoptosis and upregulation of apoptosis-related signaling molecules and downregulation of brain-derived neurotrophic factor (BDNF) in brain tissues of MCAO-treated mice. Furthermore, LIPUS significantly prevented the decreased cell viability, the increased caspase-3 cleavage, and the decreased BDNF expression in ischemia/reperfusion-treated microglial cells. These results demonstrate that LIPUS effectively prevented the cerebral ischemia/reperfusion injury through apoptosis reduction and BDNF induction in a MCAO mouse model. The neuroprotective potential of LIPUS may provide a novel preventive strategy for ischemic stroke in high-risk patients.

Diabetes-associated advanced glycation end-products (AGEs) can increase extracellular matrix (ECM) expression and induce renal fibrosis. Calbindin-D28k, which plays a role in calcium reabsorption in renal distal convoluted tubules, is increased in a diabetic kidney. The role of calbindin-D28k in diabetic nephropathy still remains unclear. Here, calbindin-D28k protein expression was unexpectedly induced in the renal tubules of db/db diabetic mice. AGEs induced the calbindin-D28k expression in human renal proximal tubule cells (HK2), but not in mesangial cells. AGEs induced the expression of fibrotic molecules, ECM proteins, epithelial-mesenchymal transition (EMT) markers, and endoplasmic reticulum (ER) stress-related molecules in HK2 cells, which could be inhibited by a receptor for AGE (RAGE) neutralizing antibody. Calbindin-D28k knockdown by siRNA transfection reduced the cell viability and obviously enhanced the protein expressions of fibrotic factors, EMT markers, and ER stress-related molecules in AGEs-treated HK2 cells. Chemical chaperone 4-Phenylbutyric acid counteracted the AGEs-induced ER stress and ECM and EMT markers expressions. Calbindin-D28k siRNA in vivo delivery could enhance renal fibrosis in db/db diabetic mice. These findings suggest that inducible calbindin-D28k protects against AGEs/RAGE axis-induced ER stress-activated ECM induction and cell injury in renal proximal tubule cells.

Silicon dioxide nanoparticles (SiO2NPs) are widely applied in industry, chemical, and cosmetics. SiO2NPs is known to induce pulmonary toxicity. In this study, we investigated the molecular mechanisms of SiO2NPs on pulmonary toxicity using a lung alveolar epithelial cell (L2) model. SiO2NPs, which primary particle size was 12 nm, caused the accumulation of intracellular Si, the decrease in cell viability, and the decrease in mRNAs expression of surfactant, including surfactant protein (SP)-A, SP-B, SP-C, and SP-D. SiO2NPs induced the L2 cell apoptosis. The increases in annexin V fluorescence, caspase-3 activity, and protein expression of cleaved-poly (ADP-ribose) polymerase (PARP), cleaved-caspase-9, and cleaved-caspase-7 were observed. The SiO2NPs induced caspase-3 activity was reversed by pretreatment of caspase-3 inhibitor Z-DEVD-FMK. SiO2NPs exposure increased reactive oxygen species (ROS) production, decreased mitochondrial transmembrane potential, and decreased protein and mRNA expression of Bcl-2 in L2 cells. SiO2NPs increased protein expression of cytosolic cytochrome c and Bax, and mRNAs expression of Bid, Bak, and Bax. SiO2NPs could induce the endoplasmic reticulum (ER) stress-related signals, including the increase in CHOP, XBP-1, and phospho-eIF2α protein expressions, and the decrease in pro-caspase-12 protein expression. SiO2NPs increased phosphoinositide 3-kinase (PI3K) activity and AKT phosphorylation. Both ROS inhibitor N-acetyl-l-cysteine (NAC) and PI3K inhibitor LY294002 reversed SiO2NPs-induced signals described above. However, the LY294002 could not inhibit SiO2NPs-induced ROS generation. These findings demonstrated first time that SiO2NPs induced L2 cell apoptosis through ROS-regulated PI3K/AKT signaling and its downstream mitochondria- and ER stress-dependent signaling pathways.

Nobiletin (Nob), a critical active flavonoid of citrus fruits, has received attention for its superior physical functions, which have shown to improve the progression of diseases. Chronic kidney disease (CKD) is recognized as a global health problem, and its mortality and morbidity rates are worsened with an increased risk of accompanying disorders. In this study, we aimed to elucidate whether Nob treatment ameliorates kidney fibrosis and also to identify the potential signaling networks in a unilateral ureteral obstructive (UUO) mouse model, which was used to mimic the progression of CKD. Six-week-old C57BL/6J mice were orally treated with 50 mg/kg of Nob for 14 constitutive days after UUO surgery. We found that the administration of Nob diminished kidney fibrosis and the expression of EMT markers, ameliorated oxidative stress and ferroptosis-associated injury, and mitigated the inflammatory response in the kidneys of UUO mice. Our results suggested that Nob treatment has antiferroptosis, anti-inflammatory, and antifibrotic effects, improving the progression of CKD in UUO mice. Nob may serve as a potential therapeutic candidate for the improvement of progressive CKD in further studies.

Heat shock protein 60 (HSP60) is a mitochondrial chaperone. Advanced glycation end products (AGEs) have been shown to interfere with the β-cell function. We hypothesized that AGEs induced β-cell hypertrophy and dysfunction through a HSP60 dysregulation pathway during the stage of islet/β-cell hypertrophy of type-2-diabetes. We investigated the role of HSP60 in AGEs-induced β-cell hypertrophy and dysfunction using the models of diabetic mice and cultured β-cells. Hypertrophy, increased levels of p27Kip1, AGEs, and receptor for AGEs (RAGE), and decreased levels of HSP60, insulin, and ATP content were obviously observed in pancreatic islets of 12-week-old db/db diabetic mice. Low-concentration AGEs significantly induced the cell hypertrophy, increased the p27Kip1 expression, and decreased the HSP60 expression, insulin secretion, and ATP content in cultured β-cells, which could be reversed by RAGE neutralizing antibody. HSP60 overexpression significantly reversed AGEs-induced hypertrophy, dysfunction, and ATP reduction in β-cells. Oxidative stress was also involved in the AGEs-decreased HSP60 expression in β-cells. Pancreatic sections from diabetic patient showed islet hypertrophy, increased AGEs level, and decreased HSP60 level as compared with normal subject. These findings highlight a novel mechanism by which a HSP60-correlated signaling pathway contributes to the AGEs-RAGE axis-induced β-cell hypertrophy and dysfunction under diabetic hyperglycemia.

Renal tubulointerstitial fibrosis is an important pathogenic feature in chronic kidney disease and end-stage renal disease, regardless of the initiating insults. A recent study has shown that CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is involved in acute ischemia/reperfusion-related acute kidney injury through oxidative stress induction. However, the influence of CHOP on chronic kidney disease-correlated renal fibrosis remains unclear. Here, we investigated the role of CHOP in unilateral ureteral obstruction (UUO)-induced experimental chronic tubulointerstital fibrosis. The CHOP knockout and wild type mice with or without UUO were used. The results showed that the increased expressions of renal fibrosis markers collagen I, fibronectin, α-smooth muscle actin, and plasminogen activator inhibitor-1 in the kidneys of UUO-treated wild type mice were dramatically attenuated in the kidneys of UUO-treated CHOP knockout mice. CHOP deficiency could also ameliorate lipid peroxidation and endogenous antioxidant enzymes depletion, tubular apoptosis, and inflammatory cells infiltration in the UUO kidneys. These results suggest that CHOP deficiency not only attenuates apoptotic death and oxidative stress in experimental renal fibrosis, but also reduces local inflammation, leading to diminish UUO-induced renal fibrosis. Our findings support that CHOP may be an important signaling molecule in the progression of chronic kidney disease.

The human epidermal growth factor receptor 2 (HER2) involved proliferation, angiogenesis, and reduced apoptosis in gastric cancer (GC), which is a common target for tumor therapy. HER2 is usually overexpressed in more than 15% GC patients, developing a reliable diagnostic tool for tumor HER2 detection is important. In this study, we attend to use polyethylene glycol (PEG) linked anti-HER2/neu peptide (AHNP-PEG) as a nuclear imaging agent probe for HER2 detection in GC xenograft animal model.

Hypoxic microenvironment is a common situation in solid tumors. Carbonic anhydrase IX (CA9) is one of the reliable cellular biomarkers of hypoxia. The role of CA9 in colorectal cancer (CRC) remains to be clarified. CA9 inhibitor such as sulfonamides is known to block CA9 activation and reduce tumor growth consequently. Here, we aimed to investigate the CA9 expression in serum and tumor from different stages of CRC patients and utilize sulfonamide derivative with indium-111 labeling as a probe for CRC nuclear imaging detection in vivo. The serum CA9 was correlated with the tumor CA9 levels in different stages of CRC patients. Hypoxia increased cell viability and CA9 expression in colorectal cancer HCT-15 cells. Sulfonamide derivative 5-(2-aminoethyl)thiophene-2-sulfonamide (ATS) could bind with CA9 in vitro under hypoxia. Moreover, tumor tissues in HCT-15-induced xenograft mice possessed higher hypoxic fluorescence signal as compared with other organs. We also found that the radioisotope signal of indium-111 labeled ATS, which was utilized for CRC detection in HCT-15-induced xenograft mice, was markedly enhanced in tumors as compared with non-ATS control. Taken together, these findings suggest that CA9 is a potential hypoxic CRC biomarker and measurement of serum CA9 can be as a potential tool for diagnosing CA9 expressions in CRC clinical practice. The radioisotope-labeled sulfonamide derivative (ATS) may be useful to apply in CRC patients for nuclear medicine imaging.

Stroke, which is the second leading cause of mortality in the world, is urgently needed to explore the medical strategies for ischemic stroke treatment. Both icariin (ICA) and icaritin (ICT) are the major active flavonoids extracted from Herba epimedii that have been regarded as the neuroprotective agents in disease models. In this study, we aimed to investigate and compare the neuroprotective effects of ICA and ICT in a middle cerebral artery occlusion (MCAO) mouse model. Male ICR mice were pretreated with both ICA and ICT, which ameliorated body weight loss, neurological injury, infarct volume, and pathological change in acute ischemic stroke mice. Furthermore, administration of both ICA and ICT could also protect against neuronal cell apoptotic death, oxidative and nitrosative stress, lipid peroxidation, and extracellular matrix (ECM) accumulation in the brains. The neuroprotective effects of ICT are slightly better than that of ICA in acute cerebral ischemic stroke mice. These results suggest that pretreatment with both ICA and ICT improves the neuronal cell apoptosis and responses of oxidative/nitrosative stress and counteracts the ECM accumulation in the brains of acute cerebral ischemic stroke mice. Both ICA and ICT treatment may serve as a useful therapeutic strategy for acute ischemic stroke.

Acute kidney injury (AKI) is known as a sudden episode of kidney injury, which happens suddenly within a few hours or a few days. Quercetin (3,3',4',5,7-pentahydroxyflavone) is a flavonoid found in plants. Quercetin is known to have several biological activities, such as anti-oxidant, anti-inflammatory, and anti-carcinogenic effects. However, low water solubility and bioavailability are the limitations of quercetin for its clinical applications. Moreover, ischemia/reperfusion (I/R) injury is a common cause of AKI. There are no satisfactory strategies for I/R-induced AKI. Developing suitable preventive or therapeutic intervention for AKI is an important and urgent issue. We investigated the benefit effect of synthesized polyethylene glycol (PEG) conjugated polyethyleneimine (PEI) nanoparticles for targeted delivery of quercetin on AKI in a mouse model. An I/R-induced AKI mouse model was used to evaluate the therapeutic effect of quercetin polymeric nanoparticles by intravenous injection. Biochemical changes for renal function in blood samples were analyzed. Histological and immunohistochemical changes were also analyzed. The biochemical changes of blood urea nitrogen (BUN), creatinine, and cystatin C were significantly increased in I/R-induced AKI mice, which could be significantly reversed by quercetin polymeric nanoparticles. Quercetin polymeric nanoparticles could also significantly decrease the histological lesions, positive staining for 3-nitrotyrosine and cyclooxygenase-2, and lipid peroxidation in the kidneys of I/R-induced AKI mice. These results demonstrate for the first time that quercetin polymeric nanoparticles possess therapeutic potential for the treatment of I/R-induced AKI in vivo.

Nootkatone is one of the major active ingredients of Alpiniae oxyphyllae, which has been used as both food and medicinal plants for the treatment of diarrhea, ulceration, and enuresis. In this study, we aimed to investigate whether nootkatone treatment ameliorated the progression of chronic kidney diseases (CKD) and clarified its underlying mechanisms in an obstructive nephropathy (unilateral ureteral obstructive; UUO) mouse model. Our results revealed that nootkatone treatment preventively decreased the pathological changes and significantly mitigated the collagen deposition as well as the protein expression of fibrotic markers. Nootkatone could also alleviate oxidative stress-induced injury, inflammatory cell infiltration, and renal cell apoptotic death in the kidneys of UUO mice. These results demonstrated for the first time that nootkatone protected against the progression of CKD in a UUO mouse model. It may serve as a potential therapeutic candidate for CKD intervention.

Tributyltin (TBT), an endocrine disrupting chemical, can be found in food (particular in fish and seafood) and drinking water by contamination. Here, we elucidated the effects and possible mechanisms of low-dose TBT on the growth and function of pancreatic β-cells and glucose metabolism in mice. Submicromolar-concentration of TBT significantly induced β-cell cytotoxicity and apoptosis, which were accompanied by poly (ADP-ribose) polymerase cleavage and mitogen-activated protein kinases-JNK and ERK1/2 phosphorylation. TBT could also suppress the glucose-stimulated insulin secretion in β-cells and isolated mouse islets. TBT increased reactive oxygen species production. TBT-induced β-cell cytotoxicity and apoptosis were significantly prevented by antioxidant N-acetylcysteine (NAC) and JNK inhibitor SP600125, but not ERK1/2 inhibitor PD98059 and p38 inhibitor SB203580. Both NAC and SP600125 inhibited JNK phosphorylation and reduced cell viability in TBT-treated β-cells. Four-week exposure of TBT (0.25 mg/kg) to mice revealed the decreased plasma insulin, increased blood glucose and plasma malondialdehyde, suppressed islet insulin secretion, and increased islet caspase-3 activity, which could be reversed by NAC treatment. After removing the TBT exposure for 2 weeks, the TBT-induced glucose metabolism alteration was significantly reversed. These results suggest that low-dose TBT can induce β-cell apoptosis and interfere with glucose homeostasis via an oxidative stress-related pathway.

The therapeutic effects of low intensity pulsed ultrasound (LIPUS) on renal ischemia/reperfusion injury (IRI) with acute kidney injury (AKI) are still unclear. A renal tubule cell model under H2O2 or hypoxia/reoxygenation (H/R) conditions with or without LIPUS pre-treatment (1 MHz, 30 and 100 mW/cm2, 15 min) was used to test the in vitro effects of LIPUS. An AKI mouse model of unilateral IRI with nephrectomy of the contralateral kidney for 48 h with or without LIPUS treatment (3 MHz, 100 mW/cm2, 20 min/day) 5 day before IRI were used to investigate the in vivo effects of LIPUS. LIPUS significantly protected the renal tubule cell viability and prevented inflammatory signals against H2O2 challenge. LIPUS could inhibit the apoptosis-related molecular signals and increase the protein levels of endogenous antioxidant enzymes, α-Klotho, and Sirt1 in renal tubule cells after H/R challenge. LIPUS alleviated the increases in the serum levels of blood urea nitrogen, creatinine, and cystatin C, renal pathological changes and apoptosis-related molecular signals, and impaired antioxidant enzymes in AKI mice. The IRI-induced inflammatory responses in the kidneys and spleens could be reversed by LIPUS. These findings suggest that LIPUS treatment displays the benefits for renal protection in IRI-induced AKI mice.

Cancer stem cells (CSCs) are involved in drug resistance, metastasis, and relapse of cancers, which can significantly affect tumor therapy. Hence, to develop specifically therapeutic target probe at CSCs for improvement of survival and quality of life of cancer patients is urgently needed. The CD166 protein has been suggested to be involved in colorectal cancer (CRC) tumorigenesis and to be considered a marker for colorectal CSCs (CRCSCs) detection. In this study, therefore, we attend to apply a nuclear imaging agent probe, Glycine18-Cystine-linked CD166-targeted peptides (CD166tp-G18C), to detect the changes of CD166 level in a CRC xenograft mouse model.

The incidence of stroke recurrence is still higher despite the advanced progression of therapeutic treatment and medical technology. Low intensity pulsed ultrasound (LIPUS) has been demonstrated to possess therapeutic effects on neuronal diseases and stroke via brain-derived neurotrophic factor (BDNF) induction. In this study, we hypothesized that LIPUS treatment possessed therapeutic benefits for the improvement of stroke recurrence. Adult male C57BL/6J mice were subjected to a middle cerebral artery occlusion (MCAO) surgery and then followed to secondary MCAO surgery as a stroke recurrence occurred after nine days from the first MCAO. LIPUS was administered continuously for nine days before secondary MCAO. LIPUS treatment not only decreased the mortality but also significantly moderated neuronal function injury including neurological score, motor activity, and brain pathological score in the recurrent stroke mice. Furthermore, the administration of LIPUS attenuated the apoptotic neuronal cells and increased Bax/Bcl-2 protein expression ratio and accelerated the expression of BDNF in the brain of the recurrent stroke mice. Taken together, these results demonstrate for the first time that LIPUS treatment arouses the expression of BDNF and possesses a therapeutic benefit for the improvement of stroke recurrence in a mouse model. The neuroprotective potential of LIPUS may provide a useful strategy for the prevention of a recurrent stroke.

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.