CRISPR/Cas9 Mediated Deletion of the Uox Gene Generates a Mouse Model of Hyperuricemia with Multiple Complications.

Hyperuricemia is a common metabolic disorder with severe complications. We aimed to develop a mouse model for spontaneous hyperuricemia. Uox-/- mouse model was generated on C57BL/6J background by deleting exon 2-4 of Uox using the CRISPR/Cas9 system. The prototypic Uox -/-mice had 5.5-fold increased serum uric acid (1351.04±276.58µmol/L) as compared to the wild type mice (P<0.0001), but died by 4 weeks. After allopurinol (3ug/g) intervention, they all survived > 8 weeks. The serum uric acid was 612.55±146.98µmol/L in the 8-week-old allopurinol-rescued Uox -/-mice, which manifested multiple complications including severe renal insufficiency, hypertension, left ventricular remodeling and systolic dysfunction, aortic endothelial dysfunction, hepatic steatosis and elevated liver enzymes, as well as hyperglycemia and hypercholesteremia. The present Uox-/- mice developed spontaneous hyperuricemia complicated with urate nephropathy, cardiovascular disease and cardiometabolic disorders, and may provide a novel tool to study hyperuricemia associated early-onset cardiovascular disorders in human.

Establishment and validation of prediction model for atherosclerotic cardiovascular disease in patients with hyperuricemia.

OBJECTIVE: To construct a risk prediction model for atherosclerotic cardiovascular disease (ASCVD) in patients with hyperuricemia. METHODS: Data in this study were obtained from the National Health and Nutrition Examination Survey (NHANES) (2007-2010). Participants from Huashan Hospital were included as an external validation. Logistic regression analysis was used to explore the relevant factors of ASCVD in patients with hyperuricemia. The discriminability of the model was evaluated using the area under the curve (AUC) statistic of the receiver operating characteristic curve. Hosmer-Lemeshow test, correction curve and decision curve analysis (DCA) were used to evaluate the model. RESULTS: A total of 389 patients collected from the NHANES were included in the final analysis. Logistic regression analysis showed that age, creatinine (Cr), glucose (Glu), serum uric acid (SUA), and history of gout were predictive factors for ASCVD in hyperuricemia (HUA) patients. These predictive factors were used to construct a nomogram. And 157 patients from NHANES were in the internal validation group and 136 patients from Huashan Hospital were in the external validation group. The AUC values of the three groups were 0.943, 0.735, and 0.664. The p values of the Hosmer-Lemeshow test were .568, .600, and .763. The calibration curve showed consistency between the nomogram and the actual observed values. The DCA curve indicated that the model has good clinical practicality. CONCLUSION: This study constructed the ASCVD risk prediction model for HUA patients, which is beneficial for medical staff to detect high-risk populations of ASCVD in the early stage.

Inhibition of ferroptosis: A new direction in the treatment of ulcerative colitis by traditional Chinese medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is a chronic idiopathic intestinal disease of unknown cause and has been classified as one of the modern intractable diseases by the World Health Organization (WHO). Ferroptosis, as an iron-ion-dependent mode of programmed cell death, is closely related to iron metabolism, lipid peroxidation, and imbalance of the antioxidant system, and plays an important role in the development of UC. In this paper, we will review the regulatory pathways of ferroptosis, the relationship between ferroptosis and the pathogenesis of UC, and the treatment of UC by TCM from the perspective of ferroptosis inhibition, and summarize the mechanism of action of the active ingredients of TCM and TCM compounds to improve UC through ferroptosis inhibition, and look forward to the prospect of the application of ferroptosis inhibition by TCM in the treatment of UC. AIM OF THIS REVIEW: This paper aims to elucidate the mechanism of action of TCM active ingredients and TCM combinations in the treatment of UC by inhibiting ferroptosis. The active ingredients of TCM have the significant advantages of multi-targets and multi-pathways, and ferroptosis is the current research hotspot in the prevention and treatment of UC, so the inhibition of ferroptosis by TCM is a key direction for future research. MATERIALS AND METHODS: The keywords "ferroptosis", "ulcerative colitis" and "TCM" were searched in Pubmed, CNKI, and Wed of Science databases. Papers related to clinical trials and pharmacological research up to August 2023 were screened for inclusion. Combined with the theory of TCM, we systematically summarized the effects of TCM active ingredients and TCM combinations in inhibiting ferroptosis and thus preventing UC. RESULTS: A large number of studies have shown that TCM active ingredients and TCM combinations inhibit the inflammatory response and oxidative stress in the course of UC mainly by interfering with iron metabolism, correcting lipid metabolism and peroxidative accumulation, and regulating the processes of glutathione (GSH) and glutathione peroxidase 4 (GPX4), to improve colonic mucosal damage and promote the repair of colonic mucosal tissue. CONCLUSION: Since the study of ferroptosis in UC is still in the exploratory stage, many issues still deserve attention in the future. This paper reviews the mechanism of ferroptosis inhibition by TCM active ingredients and TCM combinations to prevent and treat UC. In the future, we should also further increase the number of clinical experimental studies to explore whether more TCM medicines can play a therapeutic role in UC by inhibiting ferroptosis, and explore more pathways and genes targeting the inhibition of ferroptosis, to seek more TCM therapies for UC. We believe that the use of TCM active ingredients and TCM combinations to regulate ferroptosis is an important direction for future UC prevention and treatment.

Adjuvant activity of cordycepin, a natural derivative of adenosine from Cordyceps militaris, on an inactivated rabies vaccine in an animal model.

Vaccination is the most feasible way of preventing rabies, an ancient zoonosis that remains a major public health concern globally. However, administration of inactivated rabies vaccination without adjuvants is always inefficient and necessitates four to five injections. In the current study, we explored the adjuvant characteristics of cordycepin, a major bioactive component of Cordyceps militaris, to boost immune responses against a commercially available rabies vaccine. We found that cordycepin could stimulate stronger phenotypic and functional maturation of dendritic cells (DCs). For animal experiments, mice were immunized 3 times with rabies vaccine in the presence or absence of cordycepin at 1-week interval. Analysis of T cell differentiation and serum antibody isotypes showed that humoral immunity was dominant with a Th2 biased immune response. These results were also supported by the raised ratio of follicular helper T cells (TFH) and germinal center B cells (GCB). Thus, titer of rabies virus neutralizing antibody (RVNAb) and rabies virus-specific memory B cells were both raised as a result. Furthermore, administration of cordycepin did not cause pathological phenomena or body weight loss. The findings indicate that cordycepin could be used as a promising adjuvant for rabies vaccines to get a higher range of protection without any side effects.

Intestinal uric acid excretion contributes to serum uric acid decrease during acute gout attack.

OBJECTIVE: Spontaneous serum uric acid (SUA) decrease has been found in many patients during acute gout attacks, but its mechanism remains unclear. METHODS: The spontaneous regulation of SUA during a gout attack and its possible causes were evaluated in patients with gout. The mechanism of the spontaneous SUA decrease was further studied in Caco2 cells and a monosodium urate (MSU)-induced gout model of wild-type mice and ABCG2-/- mice. The urate transport function of intestinal epithelial cells was detected by transwell culture of Caco2 cells. Expression of ATP-binding cassette super-family G member 2 (ABCG2), IL-1ß and phosphoinositide 3-kinase (PI3K)/Akt was analysed using real-time PCR, western blotting, or immunofluorescence assays. RESULTS: SUA decreased during acute gout attacks in both the gout patients and MSU-induced gouty mice. Increased serum CRP and IL-1ß levels were correlated with the SUA decrease. Intestinal uric acid excretion and expression of ABCG2 were upregulated in the mice during acute gout attacks. In the ABCG2-/- mice, intestinal uric acid excretion significantly decreased during gout attacks. In an in vitro study of a transwell culture, ABCG2 and its upstream PI3K/Akt pathway were significantly upregulated in intestinal epithelial cells. However, ABCG2 expression and its associated intestinal uric acid transport were inhibited when PI3K/Akt was blocked by a PI3K inhibitor, LY294002. CONCLUSIONS: Increased intestinal urate excretion resulted in spontaneous SUA downregulation during acute gout attacks. Inflammation-induced PI3K/Akt activation and ABCG2 expression in epithelial cells might contribute to the upregulation of intestinal uric acid excretion.

P-Selectin Glycoprotein Ligand-1 Deficiency Protects Against Aortic Aneurysm Formation Induced by DOCA Plus Salt.

PURPOSE: P-selectin glycoprotein ligand-1 (PSGL-1) acts as a crucial regulator for the inflammatory cells infiltration by mediating the adhesion of leukocytes. However, the role of PSGL-1 in aortic aneurysm remains elusive. Here, we investigated the role of PSGL-1 in aortic aneurysm (AA) development. METHODS: We first detected PSGL-1 expression in samples from aortic aneurysm patients and mouse AA models via western blotting, immunofluorescence, and flow cytometry, and then we used global PSGL-1 knockout mice and their wild type controls to establish an aortic aneurysm model induced by deoxycorticosterone acetate (DOCA) plus high salt (HS). The incidence, fatality rates, and the pathological changes of aortic aneurysm were analyzed in each group. The inflammation, adhesion molecules expression, and PSGL-1 mediated leukocyte-endothelial adhesion and their underlying mechanisms were explored further. RESULTS: Increased PSGL-1 levels were observed in human and mouse aortic aneurysm, and on leukocytes of mice treated with DOCA+HS. PSGL-1 deficiency reduced the incidence and severity of aortic aneurysm significantly, as well as decreased elastin fragmentation, collagen accumulation, and smooth muscle cells degeneration. Mechanistically, the protective effect of PSGL-1 inhibition was mediated by the reduced adhesion molecules, and the subsequently reduced leukocyte-endothelial adhesion through the NF-κB pathway, which finally led to reduced inflammatory cells infiltration and decreased inflammatory factors expression. CONCLUSION: PSGL-1 deficiency is protective against inflammatory cells migration and recruitment in the condition of AA through attenuation of leukocyte-endothelial adhesion. Inhibition of PSGL-1 may be a potential therapeutic target for the prevention and treatment of human AA.

Hepatocyte-specific TAZ deletion downregulates p62/ Sqstm1 expression in nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is characterized by inflammation, hepatocellular injury, and different degrees of fibrosis. Previous studies have indicated that the transcriptional coactivator with PDZ-binding motif TAZ (WWTR1) is correlated with the increased level of liver cholesterol which suppresses TAZ proteasomal degradation and promotes fibrotic NASH by activating soluble adenylyl cyclase -calcium-RhoA pathway. However, the exact mechanism by which TAZ promotes inflammatory and hepatocyte injury has not yet been fully addressed. Reportedly, p62/Sqstm1plays a pivotal role in inflammatory and hepatocyte injury during NASH development. Here, we demonstrated that p62/Sqstm1 was overexpressed in the livers of mouse NASH models in a TAZ-dependent manner. In addition, hepatocyte-specific TAZ deletion reduced p62/Sqstm1 both in vitro and in vivo. Strikingly, luciferase reporter data demonstrated that p62/Sqstm1 is a TAZ/TEAD target gene and can be transcriptionally regulated by TAZ, indicating that hepatocyte-specific TAZ deletion downregulates p62/Sqstm1 expression in NASH.

Inhibition of miR-122 reduced atherosclerotic lesion formation by regulating NPAS3-mediated endothelial to mesenchymal transition.

AIMS: Endothelial to mesenchymal transition (EndMT) is closely related to atherosclerosis. Herein, we aim to determine whether miR-122 is involved in EndMT and the underlying mechanism in atherosclerosis. MAIN METHODS: qRT-PCR was performed to detect miR-122 expression in ApoE-/- mice and cellular EndMT model induced by H2O2. MiR-122 expression in vivo was modulated by lenti-virus injection and by genetic manipulation. Hematoxylin and eosin (HE) and Oil-red O staining were used to observe the plaque size and lipid accumulation in the aortic roots. F4/80 staining, elastin staining, and masson staining were used to observe the components of atherosclerotic lesions. MiR-122 expression in endothelial cells was modulated by transfection of miR-122 mimic and inhibitor. Western blotting and co-localization of endothelial markers (VE-cadherin, CD31) and mesenchymal markers (Vimentin, α-SMA) were carried out to determine EndMT. KEY FINDINGS: MiR-122 was upregulated in the aortic intima and serum of ApoE-/- mice induced by HFD and in cellular EndMT model. Inhibition of miR-122 repressed the atherosclerotic plaque progression and vulnerable plaque formation in ApoE-/- mice. In vitro, endothelial cells acquired a spindle-shaped morphology accompanying decrease of the endothelial markers (VE-cadherin, CD31) and increase of the mesenchymal markers (Vimentin, α-SMA) in the presence of H2O2, which was inhibited by miR-122 inhibitor. Furthermore, NPAS3 functions as a target of miR-122, and NPAS3 silencing abolished the anti-EndMT effect of miR-122 inhibitor. SIGNIFICANCE: Inhibition of miR-122 prevents atherosclerosis and regulates NPAS3-mediated EndMT, suggesting that miR-122 may be a novel target in the treatment of EndMT-associated diseases including atherosclerosis.

SULT2B1b promotes epithelial-mesenchymal transition through activation of the ß-catenin/MMP7 pathway in hepatocytes.

Epithelial-mesenchymal transition (EMT) occurs in the progression of liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The hydroxysteroid sulfotransferase 2B1b (SULT2B1b) promotes the proliferation of hepatocarcinoma cells both in vitro and in vivo. However, the correlation between SULT2B1b and the EMT in hepatocytes has not yet been addressed. The present study demonstrated that the SULT2B1b overexpression promoted the EMT process in mouse primary hepatocytes in the absence or presence of TGF-ß1 treatment. Moreover, SULT2B1b interference suppressed the EMT and attenuated the migration and invasion abilities of human hepatocarcinoma BEL-7402 cells by inhibiting the activation of the ß-catenin/MMP-7 pathway. In summary, SULT2B1b enhanced the EMT of hepatocytes and promoted the migration and invasion abilities of BEL-7402 cells by activing the ß-catenin/MMP-7 pathway. Therefore, inhibition of SULT2B1b has therapeutic potential for the treatment of HCC.