Dexmedetomidine Attenuates Ferroptosis-Mediated Renal Ischemia/Reperfusion Injury and Inflammation by Inhibiting ACSL4 via α2-AR.

Ischemia-reperfusion (I/R) injury is a serious clinical pathology associated with acute kidney injury (AKI). Ferroptosis is non-apoptotic cell death that is known to contribute to renal I/R injury. Dexmedetomidine (Dex) has been shown to exert anti-inflammatory and organ protective effects. This study aimed to investigate the detailed molecular mechanism of Dex protects kidneys against I/R injury through inhibiting ferroptosis. We established the I/R-induced renal injury model in mice, and OGD/R induced HEK293T cells damage in vitro. RNA-seq analysis was performed for identifying the potential therapeutic targets. RNA-seq analysis for differentially expressed genes (DEGs) reported Acyl-CoA synthetase long-chain family member 4 (ACSL4) related to ferroptosis and inflammation in I/R mice renal, which was validated in rodent renal. Liproxstatin-1, the specific small-molecule inhibitor of ferroptosis, significantly attenuated ferroptosis-mediated renal I/R injury with decreased LPO, MDA, and LDH levels, and increased GSH level. Inhibiting the activity of ACSL4 by the Rosiglitazone (ROSI) resulted in the decreased ferroptosis and inflammation, as well as reduced renal tissue damage, with decreasing LPO, MDA and LDH level, increasing GSH level, reducing COX2 and increasing GPx4 protein expression, and suppressing the TNF-α mRNA and IL-6 mRNA levels. Dex as a α2-adrenergic receptor (α2-AR) agonist performed renal protective effects against I/R-induced injury. Our results also revealed that Dex administration mitigated tissue damage, inhibited ferroptosis, and downregulated inflammation response following renal I/R injury, which were associated with the suppression of ACSL4. In addition, ACSL4 overexpression abolishes Dex-mediated protective effects on OGD/R induced ferroptosis and inflammation in HEK293T cells, and promotion of ACSL4 expression by α2-AR inhibitor significantly reversed the effects on the protective role of Dex. This present study indicated that the Dex attenuates ferroptosis-mediated renal I/R injury and inflammation by inhibiting ACSL4 via α2-AR.

Iguratimod Alleviates Myocardial Ischemia/Reperfusion Injury Through Inhibiting Inflammatory Response Induced by Cardiac Fibroblast Pyroptosis via COX2/NLRP3 Signaling Pathway.

Background: NLRP3 inflammasome contributes a lot to sterile inflammatory response and pyroptosis in ischemia/reperfusion (I/R) injury. Cardiac fibroblasts (CFs) are regarded as semi-professional inflammatory cells and they exert an immunomodulatory role in heart. Iguratimod provides a protective role in several human diseases through exerting a powerful anti-inflammatory effect. However, it is still unclear whether iguratimod could alleviate myocardial I/R injury and whether inflammation triggered by NLRP3-related pyroptosis of CFs is involved in this process. Methods: Transcriptomics analysis for GSE160516 dataset was conducted to explore the biological function of differentially expressed genes during myocardial I/R. In vivo, mice underwent ligation of left anterior descending coronary artery for 30 min followed by 24 h reperfusion. In vitro, primary CFs were subjected to hypoxia for 1 h followed by reoxygenation for 3 h (H/R). Iguratimod was used prior to I/R or H/R. Myocardial infarct area, serum level of cardiac troponin I (cTnI), pathology of myocardial tissue, cell viability, lactate dehydrogenase (LDH) release, and the expression levels of mRNA and protein for pyroptosis-related molecules were measured. Immunofluorescence was applied to determine the cellular localization of NLRP3 protein in cardiac tissue. Results: During myocardial I/R, inflammatory response was found to be the most significantly enriched biological process, and nucleotide-binding oligomerization domain (NOD)-like receptor signaling was a crucial pathway in mediating cardiac inflammation. In our experiments, pretreatment with iguratimod significantly ameliorated I/R-induced myocardial injury and H/R-induced pyroptosis of CFs, as evidenced by reduced myocardial infarct area, serum cTnI level, and LDH release in supernatants, as well as improved pathology of cardiac tissue and cell viability. Immunofluorescence analysis showed that NLRP3 was mainly localized in CFs. Moreover, iguratimod inhibited the expression of pro-inflammatory cytokines and pyroptosis-related molecules, including NLRP3, cleaved caspase-1, and GSDMD-N. Conclusion: Our results suggested that inflammatory response mediated by NOD-like receptor signaling is of vital importance in myocardial I/R injury. Iguratimod protected cardiomyocytes through reducing the cascade of inflammation in heart by inhibiting cardiac fibroblast pyroptosis via the COX2/NLRP3 signaling pathway.

Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway.

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma.

[Correlation analysis of efficacy of yiqi chutan recipe in treating NSCLC and P4HB expression].

OBJECTIVE: To study the predicting effect of proly 4-hydroxylase beta polypeptide (P4HB) in treating non-small cell lung cancer (NSCLC) patients by Yiqi Chutan Recipe (YCR). METHODS: Totally 46 stage III and IV NSCLC patients were treated by YCR for 4 therapeutic courses. Effect was assessed by RECIST of solid tumor. P4HB expression was detected in the lung cancer tissue by immunohistochemical assay. Factors affecting disease control rates (DCR) of YCR were analyzed by Logistic regression analysis. The correlation between P4HB expression and the effect of YCR was analyzed. RESULTS: The DCR of advanced NSCLC treated by YCR was 36.96% (17/46 cases). P4HB was high expressed in advanced lung cancer tissue (6/15 cases). Gender, initial treatment, and retreatment are independent factors for affecting DCR of treating lung cancer by YCR. CONCLUSION: P4HB might be taken as a factor for predicting the effect of YCR in treating NSCLC.

[The role of secreted Wnt-antagonist genes hypermethylation in early detection of colorectal tumor].

OBJECTIVE: To investigate the functions of promoter hypermethylation of secreted Wnt-antagonist genes in colorectal tumorigenesis and progression. METHODS: Two colorectal cancer cell lines, HCT116 and SW480, were treated by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA) for demethylation. The promoter hypermethylation and expression of sFRP and WIF-1 genes in different stages of colorectal tumor and colorectal cancer cell lines were detected by methylation-specific PCR and reverse transcription PCR, respectively. RESULTS: None of the normal colorectal mucosa samples showed methylated bands of any sFRP and WIF-1genes. Hypermethylation of sFRP1, 2, 4, 5 and WIF-1 was detected in 93.1% (67/72), 83.3% (60/72), 36.1% (26/72), 52.8% (38/72) and 84.7% (61/72) of adenocarcinomas, 87.9% (29/33), 81.8% (27/33), 24.2% (8/33), 57.6% (19/33) and 72.7% (24/33) of adenomas, 52.6%, 28.9%, 2.6%, 18.4%, 23.7% of the adjacent normal mucosa. Methylation was more frequently found in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor (P < 0.05). No significant association between Wnt-antagonist genes hypermethylation and clinicopathological characteristics was found (P > 0.05). SFRP1, 2, 4, 5 and WIF-1 genes were methylated in HCT116 cell line. SFRP1, 2 and WIF-1 were methylated in SW480 cell line. The mRNA expression of sFRPs and WIF-1 genes was absent or significantly downregulated (P < 0.01) when they were methylated in two colorectal cancer cell lines. SFRP3 was expressed in two colorectal carcinoma cell lines. DAC/TSA combination treatment re-expressed the silenced sFRPs and WIF-1 genes mRNA expressions effectively. A single application of TSA could not re-express sFRPs and WIF-1 genes mRNA expressions. The influence of demethylation treatment on sFRP3 expression was minimal. CONCLUSION: Hypermethylation of Wnt-antagonist genes is a common early event in the evolution of colorectal tumor. Methylation of sFRP1, 2, 5 and WIF-1 genes might serve as biomarkers for the early detection of colorectal tumor.

Synergistic effect of triptolide combined with 5-fluorouracil on colon carcinoma.

AIM: To investigate the inhibitive effects of triptolide (TPL) combined with 5-fluorouracil (5-FU) on colon carcinoma HT-29 cells in vitro and in vivo and their side effects. METHODS: HT-29 cells were cultured with RPMI 1640 medium. The single or combined effects of TPL and 5-FU on HT-29 cells were examined by MTT assay, flow cytometry. The combined effects were evaluated by the median-effect principle. The model of tumour xenografts was established in nude mice. TPL 0.25 mg/kg/day and 5-FU 12 mg/kg/day, either in combination or on their own, were injected into mice and the inhibitive effects and side effects were observed. RESULTS: TPL and 5-FU either combined or alone inhibited significantly the proliferation of HT-29 cells and induced obvious apoptosis. Mean (SD) growth inhibition rate reached 94.92 (2.76)% and the apoptic rate at 48 h reached 41.71 (1.38)%. The combined effects were synergistic (CI<1) at lower concentrations. TPL or 5-FU alone inhibited significantly the growth of tumour xenografts and the inhibition rates were 78.53% and 84.16%; the drugs combined had more significant effect, the tumour inhibition rate reaching 96.78%. During the course of chemotherapy, no obvious side effect was observed. CONCLUSION: The combined effects of TPL and 5-FU on the growth of colon carcinoma in vitro and in vivo were superior to the effects when the agents were used individually. TPL combined with 5-FU had synergistic effects at lower concentrations and promoted apoptosis, but did not increase the side effects of chemotherapy.

[Hypermethylation and regulation of expression of secreted frizzled-related protein genes in colorectal tumor].

OBJECTIVE: To investigate the functions of promoter hypermethylation of secreted frizzled-related protein (sFRP) genes in colorectal tumorigenesis and progression. METHODS: Three colorectal cancer cell lines, RKO, HCTll6 and SW480, were treated hy 5-aza-2'-deoxycytidine and trichostatin A for demethylation. The promoter hypermethylation and expression of sFRP genes in colorectal tumor tissue and colorectal cancer cell lines were detected hy methylation-specific PCR and reverse transcription PCR, respectively. RESULTS: None of the normal colorectal mucosa tissues showed methylation of sFRP genes. sFRP1, 2, 4 and 5 were frequently methylated in colorectal adenocarcinoma, adenoma and aberrant crypt foci (ACF) (sFRP1 > 85%, sFRP2 > 75%, sFRP5 > 50%), the differences between any two of them were not significant (P >0.05). Methylation was more frequent in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor. Hypermethylation of sFRP genes was present in three colorectal cancer cell lines. When sFRP genes were methylated, their corresponding mRNA expression was absent. After cells were treated by DAC/TSA combination, the silenced sFRP expression could be effectively re-expressed. CONCLUSION: Hypermethylation of sFRP genes is a common early event in the evolution of colorectal tumors that occurs frequently in ACF. Methylation of sFRP1, 2 and 5 genes might serve as biomarkers for the early detection of colorectal tumors. Demethylation can effectively reverse gene expression that appears possibly to be an effective way for tumor therapy.

Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor.

AIM: To investigate the functions of promoter hyper-methylation of secreted frizzled-related proteins (sFRPs) genes in colorectal tumorigenesis and progression. METHODS: The promoter hypermethylation and expression of sFRP genes in 72 sporadic colorectal carcinomas, 33 adenomas, 18 aberrant crypt foci (ACF) and colorectal cancer cell lines RKO, HCT116 and SW480 were detected by methylation-specific PCR and reverse transcription PCR, respectively. RESULTS: None of the normal colorectal mucosa tissues showed methylated bands of any of four sFRP genes. sFRP1, 2, 4 and 5 were frequently methylated in colorectal carcinoma, adenoma and ACF (sFRP1 > 85%, sFRP2 > 75%, sFRP5 > 50%), and the differences between three colorectal tissues were not significant (P > 0.05). Methylation in colorectal tumors was more frequent than in normal mucosa and adjacent normal mucosa. The mRNA of sFRP1-5 genes was expressed in all normal colorectal mucosa samples. Expression of sFRP1, 2, 4 and 5 and sFRP1, 2 and 5 was downregulated in carcinoma and adenoma, respectively. The downregulation of sFRP2, 4 and 5 was more frequent in carcinoma than in adenoma. Expression of sFRP3 which promoter has no CpG island was downregulated in only a few of colorectal tumor samples (7/105). The downregulation of sFRP1, 2, 4 and 5 expression was significantly associated with promoter hypermethylation in colorectal tumor. After cells were treated by DAC/TSA combination, the silenced sFRP mRNA expression could be effectively re-expressed in colorectal cancer cell lines. CONCLUSION: Hypermethylation of sFRP genes is a common early event in the evolution of colorectal tumor, occurring frequently in ACF, which is regarded as the earliest lesion of multistage colorectal carcinogenesis. It appears to functionally silence sFRP genes expression. Methylation of sFRP1, 2 and 5 genes might serve as indicators for colorectal tumor.