Functional analysis of a panel of molecular markers for diagnosis of systemic lupus erythematosus in rats.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a diverse autoimmune disease that arises from a combination of complex genetic factors and environmental influences. While circRNAs and miRNAs have recently been identified as promising biomarkers for disease diagnosis, their specific expression patterns, and clinical implications in SLE are not yet fully understood. AIM OF THE WORK: The aim of the present study was to determine the role of a panel of noncoding-RNAs specifically circRNAs (circ-TubD1, circ-CDC27, and circ-Med14), along with miRNA (rno-miR-146a-5p) and mRNA (TRAF6), as novel minimally invasive diagnostic biomarkers for experimentally induced SLE. Additionally, the study involved an insilico bioinformatics analysis to explore potential pathways involved in the pathogenesis of SLE, aiming to enhance our understanding of the disease, enable early diagnosis, and facilitate improved treatment strategies. MATERIALS AND METHODS: SLE was induced in rats using single IP injection of incomplete Freund's adjuvant (IFA). The Induction was confirmed by assessing the ANA and anti-ds DNA levels using ELSA technique. qPCR analysis was conducted to assess the expression of selected RNAs in sera collected from a group of 10 rats with induced SLE and a control group of 10 rats. In addition, bioinformatics and functional analysis were used to construct a circRNA-miRNA-mRNA network and to determine the potential function of these differentially expressed circRNAs. RESULTS: SLE rats demonstrated significantly higher expression levels of circ-CDC27, circ-Med14, and rno-miR-146a-5p as well as TRAF6, with lower expression level of circ-TubD1 in sera of SLE rats relative to controls. ROC curve analysis indicated that all the selected non-coding RNAs could serve as potential early diagnostic markers for SLE. In addition, the expression level of circ-TubD1 was negatively correlated with rno-miR-146a-5p, however, rno-miR-146a-5p was positively correlated with TRAF6. Bioinformatic analysis revealed the incorporation of the circRNAs targeted genes in various immune system and neurodegeneration pathways. CONCLUSIONS: Therefore, circRNAs; circ-TubD1, circ-CDC27, and circ-Med14, in addition to the miRNA (rno-miR-146a-5p) and mRNA (TRAF6) may be involved in the development of SLE and may have promising roles for future diagnosis and targeted therapy.

Dexamethasone-tamoxifen combination exerts synergistic therapeutic effects in tamoxifen-resistance breast cancer cells.

Tamoxifen (TAM) is a key player in estrogen receptor-positive (ER+) breast cancer (BC); however, ∼30% of patients experience relapse and a lower survival rate due to TAM resistance. TAM resistance was related to the over expression of SOX-2 gene, which is regulated by the E2F3 transcription factor in the Wnt signaling pathway. It was suggested that SOX-2 overexpression was suppressed by dexamethasone (DEX), a glucocorticoid commonly prescribed to BC patients. The aim of the present study is to explore the effect of combining DEX and TAM on the inhibition of TAM-resistant LCC-2 cells (TAMR-1) through modulating the E2F3/SOX-2-mediated Wnt signaling pathway. The effect of the combination therapy on MCF-7 and TAMR-1 cell viability was assessed. Drug interactions were analyzed using CompuSyn and SynergyFinder softwares. Cell cycle distribution, apoptotic protein expression, gene expression levels of SOX-2 and E2F3, and cell migration were also assessed. Combining DEX with TAM led to synergistic inhibition of TAMR-1 cell proliferation and migration, induced apoptosis, reduced SOX-2 and E2F3 expression and was also associated with S and G2-M phase arrest. Therefore, combining DEX with TAM may present an effective therapeutic option to overcome TAM resistance, by targeting the E2F3/SOX-2/Wnt signaling pathway, in addition to its anti-inflammatory effect.

Novel fast dissolving freeze dried sublingual baicalin tablets for enhanced hepatoprotective effect: in-vitro characterization, cell viability, and in-vivo evaluation.

Baicalin (BG), a natural product, has been used in the prevention and treatment of drug-induced liver injury (DILI); however, its poor solubility and extensive liver metabolism limit its pharmacological use. The aim of the present study was the formulation of fast-dissolving freeze-dried sublingual tablets (FFSTs) to increase BG dissolution, avoid first-pass metabolism, and overcome swallowing difficulties. FFSTs were prepared following a 23 factorial design. The effect of three independent variables namely matrix former, Maltodextrin, concentration (4%, and 6%), binder concentration (2%, and 3%), and binder type (Methocel E5, and Methocel E15) on the FFSTs' in-vitro disintegration time and percentage dissolution was studied along with other tablet characteristics. Differential scanning calorimetry, scanning electron microscopy, in-vitro HepG2 cell viability assay, and in-vivo characterization were also performed. F8 (6% Maltodextrin, 2% Mannitol, 2% Methocel E5), with desirability of 0.852, has been furtherly enhanced using 1%PEG (F10). F10 has achieved an in-vitro disintegration time of 41 secs, and 60.83% in-vitro dissolution after 2 min. Cell viability assay, in-vivo study in rats, and histopathological studies confirmed that pretreatment with F10 has achieved a significant hepatoprotective effect against acetaminophen-induced hepatotoxicity. The outcome of this study demonstrated that FFSTs may present a patient-friendly dosage form against DILI.

Favipiravir, an antiviral drug, in combination with tamoxifen exerts synergistic effect in tamoxifen-resistant breast cancer cells via hTERT inhibition.

Tamoxifen (TAM) is one of the most successful treatments for breast cancer; however, TAM resistance continues to be a significant barrier. TAM resistance has been reported to be associated with increased expression of human telomerase reverse transcriptase (hTERT). This enzyme shares structural similarity with RNA-dependent RNA polymerase (RdRp) enzyme of RNA viruses, suggesting that RdRp inhibitors may also inhibit hTERT. Favipiravir (FAV) is an antiviral drug that inhibits RdRp of RNA viruses. Thus, we propose that FAV may also elicit an antitumor effect by suppressing hTERT. This study aimed to investigate the effect of FAV and TAM on TAM-resistant breast cancer (TAMR-1). The cell viabilities were determined. The levels of CDK1/ hTERT, in addition to regulators of hTERT-targeted signaling pathways were measured. Apoptosis, migration, and cell cycle distribution were also determined. Our data revealed that the combination of TAM and FAV suppressed cell proliferation synergistically (CI < 1) and resulted in a significant change in cell migration and apoptosis. Indeed, this was associated with reduced levels of hTERT and CDK1 and shift in the cell cycle distribution. Our findings suggest that the TAM/FAV combination exhibits synergistic effects against TAMR-1 human breast cancer cells by targeting hTERT.

The Potential Role of Circulating Long Miscellaneous RNAs in the Diagnosis and Prognosis of Hepatitis C Related Hepatocellular Carcinoma.

Ribonucleic acids (RNAs) are important regulators of gene expression and crucial for the progression of hepatocellular carcinoma (HCC). This study was designed to determine the diagnostic and prognostic utility of the circulating long miscellaneous RNAs; LINC01419, AK021443, and AF070632 in HCV-related HCC patients. Real-time PCR was used to measure their relative expression levels in the plasma of 194 HCV patients, 120 HCV-related HCC patients and 120 healthy controls. LINC01419 and AK021443 expression levels had significantly increasing linear trend estimates while AF070632 was dramatically downregulated in HCC compared to HCV. Interestingly, LINC01419 and AK021443 served as more significant diagnostic biomarkers for HCC than AF070632 and AFP. Multivariate analysis with cox regression revealed that the high expression of AK021443 [HR = 10.06, CI95%: 3.36-30.07], the high expression of LINC01419 [HR 4.13, CI95%: 1.32-12.86], and the low expression of AF070632 [HR = 2.70, CI95%: 1.07-6.81] were significant potential prognostic factors for HCC. Besides, the Kaplan-Meier analysis showed that HCC patients with high LIN01419 and AK021443 and low AF070632 expression levels had shorter OS. The circulating LINC01419 and AK021443 can be used as noninvasive potential biomarkers for diagnosis and prognosis of HCV-related HCC patients than AF070632 providing new targets for limiting the progression of the disease.

LncRNA NEAT1 and MALAT1 are involved in polycystic ovary syndrome pathogenesis by functioning as competing endogenous RNAs to control the expression of PCOS-related target genes.

Accumulating evidence has shown an abnormal expression of several non-coding RNAs in ovarian tissues which might be closely linked with the pathogenesis of PCOS. The aim of this study was to identify competing endogenous (ce) RNA network: long non-coding RNA (lncRNA), microRNA (miRNA) and their target genes: androgen receptor (AR), follistatin (FST) and insulin receptor substrate-2 (IRS-2), which are relevant to PCOS, to underline the molecular pathogenesis of PCOS and assist in early diagnosis and treatment. Bioinformatic analysis was performed to retrieve a ceRNA network: [lncRNA (NEAT1 and MALAT1) - miRNA (miR-30a-5p and miR-30d-5p) - mRNA (AR, FST and IRS-2)] linked to PCOS. Expression of the selected RNAs was examined by qPCR in peripheral blood leukocytes obtained from 73 PCOS patients (41 obese and 32 non-obese) and 31 healthy controls. PCOS patients showed significantly higher expression levels of NEAT1, miR-30a-5p, AR, FST and IRS-2, with significantly lower expression levels of MALAT1 and miR-30d-5p relative to controls especially in obese versus non-obese patients. Receiver operating characteristic (ROC) curve analysis indicated that most of the selected RNAs could serve as potential early diagnostic markers for PCOS with the highest efficiency obtained upon combining NEAT1 and miR-30d-5p or MALAT1 and miR-30a-5p with either of PCOS target genes. Moreover, all addressed RNAs had been proved as potential predictors of PCOS. The obtained data of ceRNA network raised the possibility that NEAT1 overexpression may increase the expression levels of AR, FST and IRS-2 by sponging miR-30d-5p, while low expression of MALAT1 may allow higher expression of the above genes via increasing miR-30a-5p, suggesting their involvement in PCOS pathogenesis and promising role for future diagnosis and targeted therapy.

Diagnostic significance of hsa_circ_0000146 and hsa_circ_0000072 biomarkers for Diabetic Kidney Disease in patients with type 2 diabetes mellitus.

Background: Diabetic Kidney Disease (DKD) is a significant challenge in healthcare. However, there are currently no reliable biomarkers for renal impairment diagnosis, prognosis, or staging in DKD patients. CircRNAs and microRNAs have emerged as noninvasive and efficient biomarkers. Methods: We explored Cannabinoid receptor 1 (CNR1), C reactive protein (CRP), hsa_circ_ 0000146 and 0000072, and hsa-miR-21 and 495 as diagnostic biomarkers in DKD. The serum concentrations of CRP and CNR1 were measured using ELISA. Rt-qPCR was used to evaluate the expression levels of CNR1, circRNAs, and miRNAs in 55 controls, 55 type 2 diabetes mellitus patients, and 55 DKD patients. Their diagnostic value was determined by their ROC curve. KEGG pathway was used to predict the functional mechanism of the circRNA's target genes. Results: DKD patients exhibited a significant increase in CRP and CNR1 levels and the expression of miR-21 and 495. The expression levels of circ_0000146 and 0000072 decreased in DKD patients. ROC analysis revealed that circRNAs and miRNAs alone or CNR1 and CRP have significant diagnostic potential. The functional prediction results showed the involvement of hsa_circ_0000146 and 0000072 in various pathways that regulate DKD. Conclusions: Therefore, the examined circRNAs and miRNAs may represent a novel noninvasive biomarker for diagnosing and staging DKD.

Protective effects of butylated hydroxytoluene on the initiation of N-nitrosodiethylamine-induced hepatocellular carcinoma in albino rats.

Diethylnitrosamine (DEN), a hepatocarcinogen, is found in a variety of smoked and fried foods and was reported to be hepatotoxic in mice. Butylated hydroxytoluene (BHT) is a potent antioxidant used in cosmetic formulations and as a food additive and preservative. As a result, BHT was studied as a potential inhibitor in the early stages of diethylnitrosamine (DEN)-induced HCC. Male Wistar albino rats (n = 24) were equally subdivided. Group 1 was the negative control; Group 2 and 3 administered BHT and DEN, respectively; Group 4 received BHT followed by DEN. Blood samples and rat livers were taken for biochemical and histological investigation. Hepatotoxicity was assessed by increased liver enzymes and HCC indicators, along with reduced antioxidant and pro-apoptotic factors. AFP, AFPL3, GPC3, GSH, SOD, MDA, CASP3 and BAX expression increased significantly after DEN treatment. DEN also reduced GPx, CAT, and CYP2E1 activity, and BCl-2 expression. Moreover, in the hepatic parenchyma, the DEN caused histological alterations. Pretreatment with BHT enhanced antioxidant status while preventing histopathological and most biochemical alterations. BHT pretreatment suppresses DEN-initiated HCC by decreasing oxidative stress, triggering intrinsic mitotic apoptosis, and preventing histopathological changes in liver tissue.

Thymoquinone Suppresses Angiogenesis in DEN-Induced Hepatocellular Carcinoma by Targeting miR-1-3p.

Hepatocellular carcinoma (HCC) is characterized by its high vascularity and metastasis. Thymoquinone (TQ), the main bio-active constituent of Nigella sativa, has shown anticancer and hepatoprotective effects. TQ's anticancer effect is mediated through miRNA regulation. miR-1-3p plays a significant role in various cancers but its role in HCC invasiveness remains poorly understood. Bio-informatics analysis predicted that the 3'-UTR of TIMP3 is a target for miR-1-3p; Rats were equally divided into four groups: Group 1, the negative control; Group 2 received TQ; Group 3 received DEN; and Group 4 received DEN after pretreatment with TQ. The expression of TIMP3, MMP2, MMP9, and VEGF in rats' liver was determined immunohistochemically. RT-qPCR was used to measure the miR-1-3p level in rats' liver, and TIMP3, MMP2, MMP9, and VEGF in the HepG2 cells after being transfected with miR-1-3p mimic or inhibitor; In rats pretreated with TQ, a decreased expression of MMP2, MMP9 and VEGF, and increased expression levels of TIMP3 and miR-1-3p were detected. Treating the HepG2 cells with miR-1-3p mimic led to the upregulation of TIMP3 and downregulation of MMP2, MMP9, and VEGF, and showed a significant delay in wound healing; These results suggested that the anti-angiogenic effect of TQ in HCC may be mediated through the regulation of miR-1-3p.

Suppressive effects of thymoquinone on the initiation stage of diethylnitrosamine hepatocarcinogenesis in rats.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death globally. Chemoprevention is the most effective technique for reducing HCC incidence. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, exhibits anti-inflammatory and antineoplastic activities against various cancers. Therefore, TQ was tested as an inhibitor of the initial phase of diethylnitrosamine (DEN)-induced HCC in rats. Twenty-four male Wistar albino rats were randomly placed into four equal groups. Group 1 received saline and acted as the negative control; Group 2 received TQ; Group 3 received DEN; and Group 4 received TQ for 7 days and DEN on the 8th day. After 24 h of fasting, blood samples were taken from the slaughtered rats. Additionally, each rat's liver was dissected and separated into two halves for histological and biochemical investigation. DEN-induced hepatotoxicity was detected by elevated hepatic enzymes and HCC biomarkers reduced antioxidant and proapoptotic statuses. DEN administration caused a significant increase in the levels of glutathione, superoxide dismutase, malondialdehyde, caspase-3, alpha-fetoprotein (AFP), AFPL3, glypican 3, and the expression of BAX. However, DEN significantly decreased glutathione peroxidase, catalase, and CYP2E1 and the expression of BCl-2. Furthermore, it caused histological changes and showed a strong positive GSH S-transferase P expression in the hepatic parenchyma. Pretreatment with TQ prevented the histopathological and most of the biochemical changes and improved the antioxidant status. TQ supplementation appears to suppress the development of DEN-initiated liver cancer by reducing oxidative stress, activating the intrinsic mitotic apoptosis pathway, and retaining the antioxidant enzymes.

In Vitro Cytotoxic Activity and Phytochemical Characterization (UPLC/T-TOF-MS/MS) of the Watermelon (Citrullus lanatus) Rind Extract.

Reusing food waste is becoming popular in pharmaceutical industries. Watermelon (Citrullus lanatus) rind is commonly discarded as a major solid waste. Here, the in vitro cytotoxic potential of watermelon rind extracts was screened against a panel of human cancer cell lines. Cell cycle analysis was used to determine the induction of cell death, whereas annexin V-FITC binding, caspase-3, BAX, and BCL-2 mRNA expression levels were used to determine the degree of apoptosis. VEGF-promoting angiogenesis and cell migration were also evaluated. Moreover, the identification of phytoconstituents in the rind extract was achieved using UPLC/T-TOF-MS/MS, and a total of 45 bioactive compounds were detected, including phenolic acids, flavonoids aglycones, and their glycoside derivatives. The tested watermelon rind extracts suppressed cell proliferation in seven cancer cell lines in a concentration-dependent manner. The cytotoxicity of the rind aqueous extract (RAE) was higher compared with that of the other extracts. In addition to a substantial inhibitory effect on cell migration, the RAE triggered apoptosis in HCT116 and Hep2 cells by driving the accumulation of cells in the S phase and elevating the activity of caspase-3 and the BAX/BCL-2 ratio. Thus, a complete phytochemical and cytotoxic investigation of the Citrullus lanatus rind extract may identify its potential potency as an anticancer agent.

Combination of imatinib and clotrimazole enhances cell growth inhibition in T47D breast cancer cells.

Imatinib mesylate (IM), a tyrosine kinase inhibitor, is used as targeted cancer therapy. However, mono-targeting by IM does not always achieve full tumor eradication and thus it is recommended to combine IM with other anticancer agents. Clotrimazole (CLT) is an antifungal azole derivative with promising anticancer effects due to inhibiting the activity of glycolytic enzymes. The present study aimed to evaluate the effect of combining CLT with IM on breast cancer cell line in an attempt to establish effective new combination. T47D human breast cancer cell line was treated with different concentrations of IM and/or CLT for 48 h. IM-CLT interaction was determined by isobologram equation and combination index. Cell viability was confirmed by measuring LDH activity. As indicators of glycolysis inhibition, the expression of hexokinase-2 (HK-2) and 6-phosphofructo-1-kinase (PFK-1) plus the activity of intracellular lactate dehydrogenase (LDH) and pyruvate kinase (PK) were determined. In addition, glucose consumption and adenosine triphosphate (ATP) production were measured. Moreover, nitric oxide (NO), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-α (HIF-α) were also determined as they are modulators for glycolysis. This study demonstrated that IM or CLT synergistically inhibited cell growth in T47D as shown by combination and dose reduction indices. The combination of 15 µM IM and 20 µM CLT significantly decreased glucose consumption, activity of both PK and intracellular LDH, while increased leaked LDH, VEGF and NO in the medium compared to each drug alone. Furthermore the combination decreased gene expression of HK-2, PFK-1 and ATP content compared to the control. In conclusion, the synergistic effect of CLT on IM cytotoxicity in T47D cell line maybe mediated through inhibition of glycolysis and increasing both NO and VEGF. Further studies are required to confirm the efficiency and safety of this combination.