Anti-EMT and anti-fibrosis effects of protocatechuic aldehyde in renal proximal tubular cells and the unilateral ureteral obstruction animal model.

CONTEXT: Protocatechuic aldehyde (PCA) is a natural product that has various benefits for fibrosis. OBJECTIVE: This study evaluated the effects of PCA on renal fibrosis. MATERIALS AND METHODS: Epithelial-mesenchymal transition (EMT) was induced by 20 ng/mL transforming growth factor-ß1 (TGF-ß1), followed by treatment with 1 and 5 µM PCA, in the rat renal proximal tubular cell line NRK-52E. Cell viability, protein expression, and scratch wound-healing assays were conducted. Sprague-Dawley (SD) rats underwent unilateral ureteral obstruction (UUO) surgery for renal fibrosis indication and were treated with 50 and 100 mg/kg PCA for 14 days. RESULTS: The IC50 of PCA was appropriately 13.75 ± 1.91 µM in NRK-52E cells, and no significant difference at concentrations less than 5 µM. PCA ameliorated TGF-ß1-induced EMT, such as enhanced E-cadherin and decreased vimentin. Fibrotic markers collagen IV and α-smooth muscle actin (α-SMA) increased in TGF-ß1-induced NRK-52E. Moreover, PCA reduced TGF-ß1-induced migration in the wound-healing assay. Analysis of rat kidneys indicated that PCA reduced UUO-induced hydronephrosis (control: 15.11 ± 1.00%; UUO: 39.89 ± 1.91%; UUO + PCA50: 18.37 ± 1.61%; UUO + PCA100: 17.67 ± 1.39%). Protein level demonstrated that PCA not only decreased vimentin expression and enhanced E-cadherin expression, but inhibited UUO-induced collagen IV and α-SMA upregulation, indicating that it could mitigate EMT in a rat model of UUO-induced renal fibrosis. DISCUSSION AND CONCLUSIONS: This study suggested that PCA decreases TGF-ß1-induced fibrosis and EMT in vitro and in vivo. These findings demonstrate pharmacological effects of PCA and might be a potential strategy for the prevention of organ fibrosis in clinics.

M2-like polarization of THP-1 monocyte-derived macrophages under chronic iron overload.

Macrophages are characterized by phenotypical and functional heterogeneity. In different microenvironments, macrophages can polarize into two types: classically activated macrophages (M1) or alternatively activated macrophages (M2). M1 macrophages are a well-known bacteriostatic macrophage, and conversely, M2 macrophages may play an important role in tumor growth and tissue remodeling. M1 macrophages have been reported to have high intracellular iron stores, while M2 macrophages contain lower intracellular iron. It has been well-described that disturbances of iron homeostasis are associated with altered immune function. Thus, it is important to investigate if chronic iron overload is capable of polarizing macrophages. Human monocytic leukemia THP-1 cells were maintained in culture medium that contained 100 µM ferrous sulfate heptahydrate (FeSO4) (I-THP-1) and differentiated into THP-1-derived macrophages (I-TDMs) by induction with phorbol 12-myristate 13-acetate (PMA). We characterized that I-TDMs not only enhanced the surface expression of CD163 and CD206 but also increased arginase and decreased iNOS protein expression. I-TDMs enhanced pSTAT6 expression and decreased pSTAT1 and NF-κB expressions. Furthermore, the gene expression profile of I-TDMs was comparable with M2 macrophages by performing human oligonucleotide DNA microarray analysis. Finally, functional assays demonstrated I-TDMs secreted higher levels of IL-10 but not M1 cytokines. Additionally, the conditional medium of I-TDMs had enhanced migration and increased invasion of A375 melanoma cells which was similar to the characteristics of tumor-associated macrophages. Taken together, we demonstrated that THP-1-derived macrophages polarized to a phenotype of M2-like characteristics when subjected to chronic iron overload.

Is combined peritoneal dialysis and hemodialysis redundant? A nationwide study from Taiwan.

BACKGROUND: Combined peritoneal dialysis (PD) and hemodialysis (HD) therapy (combined therapy) has numerous clinical benefits and should be emphasized for PD patients encountering technique failure. METHODS: This 12-year nationwide retrospective study was conducted to compare long-term outcomes (including admission and mortality risks) between combined therapy patients (combined group) and patients directly transferred from PD to HD (transfer group). RESULTS: All 12,407 incidental PD patients from 2000 to 2010 were enrolled and followed up until the end of 2011. A total of 688 patients in the combined group and 688 patients in the transfer group were selected after 1:1 frequency matching based on age, sex, and PD duration. The overall admission and mortality risks of the two groups were comparable in a Cox proportional hazards model (adjusted hazard ratio [HR] = 1.06 [95% confidence interval (CI) = 0.95-1.19] and 1.02 [95% CI = 0.80-1.30]), respectively). Compared with the transfer group, combined group patients with recent peritonitis or frequent hemodialysis (four HD sessions per month) had significantly higher risk of admission while combined group patients without peritonitis had significantly lower risk. The number of incidents in the combined group increased over time. On average, patients stayed on combined therapy for 2 years. CONCLUSIONS: Combined therapy (two HD sessions per month) is not redundant but a rational and cost-effective treatment, particularly for patients without recent peritonitis. Dialysis staff should be familiar with the advantages and disadvantages of combined therapy and consider it an essential part of integrated dialysis care.

Intestinal microbiota profiling and predicted metabolic dysregulation in psoriasis patients.

OBJECTIVES: The intestinal microbiota has been known to involve in obesity and host immune response. We aimed to investigate the intestinal microbiota and potential genetic function in relation to clinical presentation in psoriasis patients. METHODS: Faecal microbiota and predicted genetic function inferred from high-throughput 16S ribosomal RNA sequencing were analysed between psoriasis (n = 32) and age-, gender- and body mass index (BMI)-matched non-psoriasis subjects (n = 64), from a referral medical centre. The correlation between altered microbiota and disease activity, arthritis and systemic anti-psoriatic drugs was also investigated. RESULTS: We observed a distinct faecal microbial community structure in psoriasis patients, with an increased abundance of phylum Firmicutes and decreased abundance of phylum Bacteroidetes, across different subgroup of subjects. Ruminococcus and Megasphaera, of the phylum Firmicutes, were the top-two genera of discriminant abundance in psoriasis. A number of functional genes and metabolic pathways involving bacterial chemotaxis and carbohydrate transport were predicted over-represented, whereas genes related to cobalamin and iron transport were predicted under-represented in faecal microbiota of psoriasis patients. CONCLUSIONS: The distinct faecal microbial composition in psoriasis might be associated with altered transport of carbohydrate, cobalamin and iron, as well as chemotaxis.

Risk of inflammatory bowel disease in patients with rosacea: Results from a nationwide cohort study in Taiwan.

BACKGROUND: A link between rosacea and inflammatory bowel disease (IBD) has been proposed with unknown mechanisms. Epidemiologic evidence of this association needs to be examined. METHODS: In this nationwide cohort study, a total of 89,356 patients with rosacea and 178,712 matched patients without rosacea between 1997 and 2013 were identified in the Taiwanese National Health Insurance Research Database. Cumulative incidences of IBD were compared between these 2 cohorts. Frailty Cox proportional hazard model was used and subgroup analyses were conducted to examine the risk factors for IBD. RESULTS: The 15-year cumulative incidences of IBD were 0.036% (95% confidence interval [CI] 0.00%-1.57%) and 0.019% (95% CI 0.00%-0.83%) in rosacea and nonrosacea cohorts, respectively (P = .05). Rosacea (adjusted hazard ratio 1.94, 95% CI 1.04-3.63, P = .04) and male gender (adjusted hazard ratio 3.52, 95% CI 2.03-6.11, P < .01) were independently associated with IBD, after adjustment for major comorbidities. Multivariate subgroup analyses revealed consistent results. The incidence rates of IBD decreased with increasing antibiotic use in patients with rosacea, but without statistical significance. LIMITATION: Information related to lifestyle, diet, alcohol, and smoking was not included in the database. CONCLUSION: Patients with rosacea may have an increased risk of IBD.

Heat-shock pretreatment reduces expression and release of TSLP from keratinocytes under Th2 environment.

BACKGROUND: Atopic dermatitis is a chronic, relapsing inflammatory disease of the skin. Current therapy is not curative, and recalcitrant disease is a big stress and challenge for parents and physicians. This study explored the potential role of heat-shock protein 70 (HSP-70) and its anti-inflammatory effects on keratinocyte under TH2 environment. METHODS: Human keratinocyte cell line (HaCa T) was stimulated with IL-4, IL-13, and TNF-α to synthesize and secrete thymic stromal lymphopoietin (TSLP), an important cytokine of immunopathogenesis in atopic dermatitis. Heat shock was performed by immersing the cell-contained flash into a water bath of 45°C for 20 min. Cell viability, TSLP expression, and secretion of HaCa T cells were measured and compared. Possible regulatory mechanisms influencing the expression of TSLP, such as the STAT6 and NF-κB signal pathways, were investigated. RESULTS: Heat-shock treatment induced intracellular HSP-70 expression in HaCa T cells without affecting cell viability. The induced expression and secretion of TSLP in HaCa T cells were suppressed by heat shock. The NF-κB signal pathway was inhibited by heat shock, leading to decreased TSLP expression and secretion. CONCLUSION: Heat stress-induced HSPs can significantly reduce the production and secretion of TSLP from HaCaT cells under Th2 environment. Thus, the evidence highlights the potential role of HSP-70 for atopic dermatitis in the future.

Association between antidiabetic drugs and psoriasis risk in diabetic patients: results from a nationwide nested case-control study in Taiwan.

BACKGROUND: The risk of psoriasis in diabetic patients has rarely been explored. OBJECTIVES: We sought to investigate the association between antidiabetic therapies and psoriasis. METHODS: The incidence of psoriasis was compared between a representative diabetic cohort and a matched nondiabetic cohort. We next conducted a nationwide cohort study with 1,659,727 diabetic patients using the National Health Insurance Research Database of Taiwan 1997 through 2011. Multivariate conditional logistic regression was used for nested case-control analyses. RESULTS: Incidence rates of psoriasis among diabetic patients and nondiabetic matched control subjects were 70.2 (95% confidence interval [CI] 59.5-80.9) and 42.3 (95% CI 39.5-45.5) per 100,000 person-years, respectively (P < .0001). Frequent insulin use was associated with higher risk of incident psoriasis (adjusted odds ratio 1.29, 95% CI 1.18-1.42) after adjusting for comorbidities, disease duration, and number of hospital visits. Among diabetic patients without history of insulin use, frequent use of thiazolidinedione was associated with lower risk of psoriasis (adjusted odds ratio 0.87, 95% CI 0.77-0.99). LIMITATIONS: The National Health Insurance Research Database did not contain information regarding disease severity, diet, body mass index, lifestyle, or family history. CONCLUSION: Among diabetic patients, regular insulin use is associated with psoriasis development. Frequent use of thiazolidinedione may be associated with modest reduction in psoriasis risk.

Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies.

OBJECTIVE: Type 2 diabetes mellitus is associated with a higher risk of hepatocellular carcinoma (HCC), which is attenuated by the use of metformin. However, there are no studies addressing the effect of metformin on hepatocarcinoma cells from the antitumoural perspective. DESIGN: In the nationwide case-control study, the authors recruited 97,430 HCC patients and 19,860 age-, gender- and physician visit date-matched controls. The chemopreventive effects of metformin were examined by multivariate analysis and stratified analysis. The in vitro effects of metformin on cell proliferation and cell cycle were studied in HepG2 and Hep3B hepatoma cell lines. RESULTS: The OR of diabetes in HCC patients was 2.29 (95% CI 2.25 to 2.35, p<0.001). Each incremental year increase in metformin use resulted in 7% reduction in the risk of HCC in diabetic patients (adjusted OR=0.93, 95% CI 0.91 to 0.94, p<0.0001). In the multivariate stratified analysis, metformin use was associated with a reduced risk of HCC in diabetic patients in nearly all subgroups. Cell line studies showed that metformin inhibits hepatocyte proliferation and induces cell cycle arrest at G0/G1 phase via AMP-activated protein kinase and its upstream kinase LKB1 to upregulate p21/Cip1 and p27/Kip1 and downregulate cyclin D1 in a dose-dependent manner, but independent of p53. Combined treatment of oral metformin with doxorubicin functioned more efficiently than either agent alone, in vivo. CONCLUSIONS: Use of metformin is associated with a decreased risk of HCC in diabetic patients in a dose-dependent manner, via inhibition of hepatoma cells proliferation and induction of cell cycle arrest at G0/G1 phase.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway.

BACKGROUND: Melanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. OBJECTIVE: To explore whether IMQ could induce melanogenesis in melanoma cells. METHODS: The mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. RESULTS: We demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. CONCLUSIONS: Low dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells.

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status.

Sodium-Glucose Transport Protein 2 Inhibitor Use for Type 2 Diabetes and the Incidence of Acute Kidney Injury in Taiwan.

Importance: The association between sodium-glucose transport protein 2 inhibitor (SGLT2i) use and the incidence of acute kidney injury (AKI) remains controversial. The benefits of SGLT2i use in patients to reduce AKI requiring dialysis (AKI-D) and concomitant diseases with AKI as well as improve AKI prognosis have not yet been established. Objective: To investigate the association between SGLT2i use and AKI incidence in patients with type 2 diabetes (T2D). Design, Setting, and Participants: This nationwide retrospective cohort study used the National Health Insurance Research Database in Taiwan. The study analyzed a propensity score-matched population of 104 462 patients with T2D treated with SGLT2is or dipeptidyl peptidase 4 inhibitors (DPP4is) between May 2016 and December 2018. All participants were followed up from the index date until the occurrence of outcomes of interest, death, or the end of the study, whichever was earliest. Analysis was conducted between October 15, 2021, and January 30, 2022. Main Outcomes and Measures: The primary outcome was the incidence of AKI and AKI-D during the study period. AKI was diagnosed using International Classification of Diseases diagnostic codes, and AKI-D was determined using the diagnostic codes and dialysis treatment during the same hospitalization. Conditional Cox proportional hazard models assessed the associations between SGLT2i use and the risks of AKI and AKI-D. The concomitant diseases with AKI and its 90-day prognosis, ie, the occurrence of advanced chronic kidney disease (CKD stage 4 and 5), end-stage kidney disease, or death, were considered when exploring the outcomes of SGLT2i use. Results: In a total of 104 462 patients, 46 065 (44.1%) were female patients, and the mean (SD) age was 58 (12) years. After a follow-up of approximately 2.50 years, 856 participants (0.8%) had AKI and 102 (<0.1%) had AKI-D. SGLT2i users had a 0.66-fold risk for AKI (95% CI, 0.57-0.75; P < .001) and 0.56-fold risk of AKI-D (95% CI, 0.37-0.84; P = .005) compared with DPP4i users. The numbers of patients with AKI with heart disease, sepsis, respiratory failure, and shock were 80 (22.73%), 83 (23.58%), 23 (6.53%), and 10 (2.84%), respectively. SGLT2i use was associated with lower risk of AKI with respiratory failure (hazard ratio [HR], 0.42; 95% CI, 0.26-0.69; P < .001) and shock (HR, 0.48; 95% CI, 0.23-0.99; P = .048) but not AKI with heart disease (HR, 0.79; 95% CI, 0.58-1.07; P = .13) and sepsis (HR, 0.77; 95% CI, 0.58-1.03; P = .08). The 90-day AKI prognosis for the risk of advanced CKD indicated a 6.53% (23 of 352 patients) lower incidence in SGLT2i users than in DPP4i users (P = .045). Conclusions and Relevance: The study findings suggest that patients with T2D who receive SGLT2i may have lower risk of AKI and AKI-D compared with those who receive DPP4i.

Terpinen-4-ol Induces Apoptosis in Human Nonsmall Cell Lung Cancer In Vitro and In Vivo.

Terpinen-4-ol, a monoterpene component of the essential oils of several aromatic plants, exhibits antitumor effects. In this study, the antitumor effects of terpinen-4-ol and the cellular and molecular mechanisms responsible for it were evaluated and studied, respectively on human nonsmall cell lung cancer (NSCLC) cells. Our results indicated that terpinen-4-ol elicited a dose-dependent cytotoxic effect, as determined by MTT assay. Increased sub-G1 population and annexin-V binding, activation of caspases 9 and 3, cleavage of poly(ADPribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated involvement of the mitochondrial apoptotic pathway in terpinen-4-ol-treated A549 and CL1-0 cells. Elevation of the Bax/Bcl-2 ratio and a decrease in IAP family proteins XIAP and survivin were also observed following terpinen-4-ol treatment. Notably, terpinen-4-ol was able to increase p53 levels in A549 and CL1-0 cells. Diminution of p53 by RNA interference induced necrosis instead of apoptosis in A549 cells following terpinen-4-ol treatment, indicating that terpinen-4-ol-elicited apoptosis is p53-dependent. Moreover, intratumoral administration of terpinen-4-ol significantly suppressed the growth of s.c. A549 xenografts by inducing apoptosis, as confirmed by TUNEL assay. Collectively, these data provide insight into the molecular mechanisms underlying terpinen-4-ol-induced apoptosis in NSCLC cells, rendering this compound a potential anticancer drug for NSCLC.

Association of SGLT2 inhibitors with lower incidence of death in type 2 diabetes mellitus and causes of death analysis.

Sodium-glucose cotransporter 2 inhibitor (SGLT2i) potentially decrease all-cause and cardiovascular death, however, associations with non-cardiovascular death remain unclear. Therefore, we investigated SGLT2i associations with death and the cause of death. We used the Taiwanese National Health Institutes Research database linked to the National Register of Deaths (NRD). Incident type 2 diabetes mellitus (T2DM) patients and propensity score matched T2DM SGLT2i and Dipeptidyl peptidase 4 inhibitor (DPP4i) users were investigated. The index year was the SGLT2i or DPP4i prescription date from May 2016. Patients were followed-up until death or December 2018. Deaths verified by the NRD and grouped accordingly. Multiple Cox proportional hazards models were used. In total, 261,211 patients were included in the population; 47% of the patients were female and the average age was 62 years. The overall incidence of all-cause death was 8.67/1000 patient-years for SGLT2i and 12.41 for DPP4i users during follow-up. After adjusting for potential risk factors in the propensity score matched population, SGLT2i users were associated with lower risks of all-cause death, cardiovascular death, cancer death, and non-cancer, non-vascular death compared with DPP4i-users. For specific death causes, significantly lower death risks from heart disease, cerebrovascular disease, and accidents were associated with SGLT2i-use. SGLT2i benefits for T2DM patients were not different across subgroups. Compared with DPP4i-use, SGLT2i-use for T2DM was associated with lower disease and death risk.

Impact of peritoneal dialysis-related peritonitis on PD discontinuation and mortality: A population-based national cohort study.

BACKGROUND: The impact of peritoneal dialysis-associated peritonitis (PD peritonitis) on long-term outcomes is uncertain. This nationwide retrospective study was conducted in Taiwan to understand the incidence, risk factors and long-term outcomes of PD peritonitis. METHODS: A total of 11,202 incident adult peritoneal dialysis (PD) patients from 2000 to 2010 were collected from a Longitudinal Health Insurance Database and followed up until the end of 2011. Definition of peritonitis, the primary outcome, simultaneously met the diagnosis of peritonitis (International Classification of Diseases, Ninth Revision, Clinical Modification 567) and antibiotic use. Secondary outcomes included the impact of peritonitis on PD discontinuation and survival. Cox proportional hazards models with and without time-dependent variables were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: There were 7634 peritonitis episodes in 4245 patients during the follow-up period. The overall incidence of peritonitis was 0.18 episodes per patient-year. Peritonitis-associated risk factors included older age, female gender, chronic heart failure, cerebrovascular disease, liver cirrhosis and lower monthly income. In an adjusted Cox hazard proportional regression with the time-dependent model, peritonitis patients had a higher risk of PD discontinuation (HR 2.71, 95% CI 2.52-2.92) and mortality (HR 1.68, 95% CI 1.57-1.81) compared to patients without peritonitis. The adjusted HRs for mortality increased with each prior episode: one episode, two episodes and more than two episodes (all p < 0.05). The adjusted HRs for PD discontinuation also increased with the frequency of peritonitis. These negative effects were greatest during the first year and persisted significantly after 5 years. In a sensitivity analysis in which peritonitis within 30 days of death or PD discontinuation was excluded, peritonitis patients still had significantly increased risk of PD discontinuation and mortality compared to patients without peritonitis. CONCLUSIONS: Although peritonitis incidence was low, our findings reveal that peritonitis carried acute and long-term sequelae of higher PD discontinuation and lower patient survival.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells.

BACKGROUND: Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. OBJECTIVE: To determine whether lysosomes are involved in IMQ-induced apoptosis. METHODS: The human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. RESULTS: IMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. CONCLUSIONS: IMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

EGR-1 plays a protective role in AMPK inhibitor compound C-induced apoptosis through ROS-induced ERK activation in skin cancer cells.

Skin cancer is caused by abnormal proliferation, gene regulation and mutation of epidermis cells. Compound C is commonly used as an inhibitor of AMP-activated protein kinase (AMPK), which serves as an energy sensor in cells. Recently, compound C has been reported to induce apoptotic and autophagic death in various skin cancer cell lines via an AMPK-independent pathway. However, the signaling pathways activated in compound C-treated cancer cells remain unclear. The present oligodeoxynucleotide-based microarray screening assay showed that the mRNA expression of the zinc-finger transcription factor early growth response-1 (EGR-1), which helps regulate cell cycle progression and cell survival, was significantly upregulated in compound C-treated skin cancer cells. Compound C was demonstrated to induce EGR-1 mRNA and protein expression in a time and dose-dependent manner. Confocal imaging showed that compound C-induced EGR-1 protein expression was localized in the nucleus. Compound C was demonstrated to activate extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of this compound C-induced ERK phosphorylation downregulated the mRNA and protein expression of EGR-1. In addition, removal of compound C-induced reactive oxygen species (ROS) not only decreased ERK phosphorylation, but also inhibited compound C-induced EGR-1 expression. A functional assay showed that knock down of EGR-1 expression in cancer cells decreased the survival rate while also increasing caspase-3 activity and apoptotic marker expression after compound C treatment. However, no difference in autophagy marker light chain 3-II protein expression was observed between compound C-treated control cells and EGR-1-knockdown cells. Thus, it was concluded that that EGR-1 may antagonize compound C-induced apoptosis but not compound C-induced autophagy through the ROS-mediated ERK activation pathway.

Evaluation of the Therapeutic Effects of Protocatechuic Aldehyde in Diabetic Nephropathy.

Diabetic nephropathy (DN) is one of the most severe chronic kidney diseases in diabetes and is the main cause of end-stage renal disease (ESRD). Protocatechuic aldehyde (PCA) is a natural product with a variety of effects on pulmonary fibrosis. In this study, we examined the effects of PCA in C57BL/KS db/db male mice. Kidney morphology, renal function indicators, and Western blot, immunohistochemistry, and hematoxylin and eosin (H&E) staining data were analyzed. The results revealed that treatment with PCA could reduce diabetic-induced renal dysfunction, as indicated by the urine albumin-to-creatinine ratio (db/m: 120.1 ± 46.1µg/mg, db/db: 453.8 ± 78.7 µg/mg, db/db + 30 mg/kg PCA: 196.6 ± 52.9 µg/mg, db/db + 60 mg/kg PCA: 163.3 ± 24.6 µg/mg, p < 0.001). However, PCA did not decrease body weight, fasting plasma glucose, or food and water intake in db/db mice. H&E staining data revealed that PCA reduced glomerular size in db/db mice (db/m: 3506.3 ± 789.3 µm2, db/db: 6538.5 ± 1818.6 µm2, db/db + 30 mg/kg PCA: 4916.9 ± 1149.6 µm2, db/db + 60 mg/kg PCA: 4160.4 ± 1186.5 µm2p < 0.001). Western blot and immunohistochemistry staining indicated that PCA restored the normal levels of diabetes-induced fibrosis markers, such as transforming growth factor-beta (TGF-ß) and type IV collagen. Similar results were observed for epithelial-mesenchymal transition-related markers, including fibronectin, E-cadherin, and α-smooth muscle actin (α-SMA). PCA also decreased oxidative stress and inflammation in the kidney of db/db mice. This research provides a foundation for using PCA as an alternative therapy for DN in the future.

Association of Sodium-Glucose Transport Protein 2 Inhibitor Use for Type 2 Diabetes and Incidence of Gout in Taiwan.

Importance: The use of sodium-glucose transport protein 2 (SGLT2) inhibitors is currently a standard intervention in patients with type 2 diabetes (T2DM) and exerts favorable pleiotropic effects to consistently lower blood urate levels. However, to date, no association between SGLT2 inhibitor use and the incidence of gout have been established. Objective: To investigate whether prescribed SGLT2 inhibitors are associated with lower gout incidence in patients with T2DM. Design, Setting, and Participants: In a cohort study, all patients with incident T2DM in Taiwan National Health Institution databases between May 1, 2016, and December 31, 2018, were retrospectively analyzed. As a comparator, patients using dipeptidyl peptidase 4 (DPP4) inhibitors were included. A total of 47 905 individuals receiving an SGLT2 inhibitor and 183 303 receiving a DPP4 inhibitor were evaluated, along with 47 405 pairs of patients using an SGLT2 inhibitor or DPP4 inhibitor in 1:1 propensity score-matched analyses. Data analysis was conducted from April 1 to June 30, 2021. Main Outcomes and Measures: A gout diagnosis was based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) and the International Statistical Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM). Multiple Cox proportional hazards regression models were used to calculate hazard ratios (HRs) and 95% CIs. Results: In total, 231 208 patients with T2DM were included in the population; 113 812 individuals (49.22%) were women, and the mean (SD) age was 61.53 (12.86) years. The overall gout incidence was 20.26 per 1000 patient-years for SGLT2 inhibitor users and 24.30 per 1000 patient-years for DPP4 inhibitor users. When potential risk factors were adjusted in the propensity score-matched population, use of SGLT2 inhibitors was associated with a lower risk of gout (HR, 0.89; 95% CI, 0.82-0.96) compared with DPP4 inhibitors, particularly for patients receiving dapagliflozin (HR, 0.86; 95% CI, 0.78-0.95). A sensitivity analysis, performed when a gout diagnosis was ascertained using the ICD-9-CM or ICD-10-CM code with gout-related medication, also showed a significantly lower risk for gout incidence of 15% with SGLT2 inhibitors (HR, 0.85; 95% CI, 0.74-0.97). Subgroup analysis indicated that SGLT2 inhibitor benefits in patients with T2DM to achieve a lower gout risk were not different across subgroups. Conclusions and Relevance: The findings of this study suggest that patients with T2DM who are receiving SGLT2 inhibitors may have a lower risk for gout compared with those receiving DPP4 inhibitors.

Primary aldosteronism is associated with risk of urinary bladder stones in a nationwide cohort study.

We analyzed database from the Taiwan National Health Insurance to investigate whether primary aldosteronism (PA) increases the risk of bladder stones. This retrospective nationwide population-based cohort study during the period of 1998-2011 compared patients with and without PA extracted by propensity score matching. Cox proportional hazard models and competing death risk model were used to estimate the hazard ratios (HRs), sub-hazard ratios (SHRs) and corresponding 95% confidence intervals (CIs). There were 3442 patients with PA and 3442 patients without PA. The incidence rate of bladder stones was 5.36 and 3.76 per 1000 person-years for both groups, respectively. In adjusted Cox hazard proportional regression models, the HR of bladder stones was 1.68 (95% CI 1.20-2.34) for patients with PA compared to individuals without PA. Considering the competing risk of death, the SHR of bladder stones still indicates a higher risk for PA than a comparison cohort (SHR, 1.79; 95% CI 1.30-2.44). PA, age, sex, and fracture number were the variables significantly contributing to the formation of bladder stones. In conclusion, PA is significantly associated with risk of bladder stones.

Imiquimod Accelerated Antitumor Response by Targeting Lysosome Adaptation in Skin Cancer Cells.

Lysosomal adaptation is a cellular physiological process in which the number and function of lysosomes are regulated at the transcriptional and post-transcriptional levels in response to extracellular and/or intracellular cues or lysosomal damage. Imiquimod (IMQ), a synthetic toll-like receptor 7 ligand with hydrophobic and weak basic properties, exhibits both antitumor and antiviral activity against various skin malignancies as a clinical treatment. Interestingly, IMQ has been suggested to be highly concentrated in the lysosomes of plasmacytoid dendritic cells, indicating that IMQ could modulate lysosome function after sequestration in the lysosome. In this study, we found that IMQ not only induced lysosomal membrane permeabilization and dysfunction but also increased lysosome biogenesis to achieve lysosomal adaptation in cancer cells. IMQ-induced ROS production but not lysosomal sequestration of IMQ was the major cause of lysosomal adaptation. Moreover, IMQ-induced lysosomal adaptation occurred through lysosomal calcium ion release and activation of the calcineurin/TFEB axis to promote lysosome biogenesis. Finally, depletion of TFEB sensitized skin cancer cells to IMQ-induced apoptosis in vitro and in vivo. In summary, a disruption of lysosomal adaptation might represent a therapeutic strategy for synergistically enhancing the cytotoxicity of IMQ in skin cancer cells.