Vitreous microparticles contain apoptotic signals suggesting a diabetic vitreopathy

AIM: To evaluate differences in microparticle profiles in vitreous samples between diabetic and non-diabetic eyes undergoing vitrectomy. METHODS: Un-masked cross-sectional series of 34 eyes undergoing vitrectomy. Vitreous specimens were collected and processed to evaluate for membrane integrity (DAPI), apoptosis (Annexin-V), and endothelial-cell origin (V-Cadherin). A BD LSR II flow cytometer was used for analysis and standardized sub-micron-sized beads were used for size comparison. RESULTS: Thirty-four specimens underwent analysis. Greater levels of Annexin-V were found on microparticles from specimens in which blood had entered the vitreous (n=12) compared to those without blood (n=22; 52.3%±30.7% vs 19.6%±27.2%, P=0.002). Patients with diabetes having surgery with hemorrhage (n=7) had greater expression of Annexin-V than those without hemorrhage (n=8; 62.1%±31.7% vs 18.9%±20.9%, P=0.009). However, in patients with non-diabetic vitreous hemorrhage, the level of Annexin-V expression was not significantly different compared to other disease processes (38.6%±25.7%, n=5 vs 20.0%±30.9%, n=14, P=0.087). CONCLUSION: Increased expression of the apoptotic marker, Annexin-V is detected on vitreous microparticles in diabetes-related vitreous hemorrhage. When evaluating vitreous hemorrhage in patients without diabetes, the apoptotic signal is not significantly different. Vitrectomy in patients with diabetes, and improvement in visual outcomes, may be related to the removal of a serum-derived, pro-apoptotic vitreous. Further investigation is warranted in order to identify the molecular characteristics of microparticles that regulate disease.

Toxicological Profile of Biological Environment of Two Elastodontic Devices

Malocclusion and teething problems are common health problems globally, affecting people of all ages, especially children and adolescents. In addition to the pathophysiological complications associated with orthodontic problems, they also affect the well-being of the individual. Orthodontic appliances are frequently used, even from an early age, and their activity in different biological environments is very varied and incompletely described. Due to these considerations, the purpose of the study was to evaluate the toxicological profile of the biological environment (saliva at three pH values: 3, 7, and 10) of two elastodontic orthodontic appliances: Myobrace (MB) and LM TrainerTM 2 (LMD). In vitro techniques applied were conducted on human keratinocytes to evaluate cell viability (Alamar blue assay) and gene expression real-time reverse transcription–polymerase chain reaction (RT-PCR technique). In addition, it was assessed the irritating effect on the vascular plexus using as a biological model the chorioallantoic membrane of the hen’s egg by applying the hen’s egg-chorioallantoic membrane (HET-CAM) method. The obtained results showed a decrease in cell viability up to 82% in the case of LMD at pH = 3, a slight increase in mRNA expression for the anti-apoptotic marker (Bcl-2 and Bcl-xL), and a decrease in mRNA expression for the pro-apoptotic marker (Bad), and any type of toxic change at the capillary level (irritation score being below 0.9). Based on the data obtained, it can be stated that MB and LMD biological environments, at different pH values, present a safe toxicological profile.

Terretonin as a New Protective Agent against Sepsis-Induced Acute Lung Injury: Impact on SIRT1/Nrf2/NF-κBp65/NLRP3 Signaling

Endophytic fungi are proving to be an excellent source of chemical entities with unique structures and varied bioactivities. Terretonin (TE) and its structurally related derivatives are a class of meroterpenoids, possessing the same unique tetracyclic core skeleton, which have been reported from the Aspergillus genus. This study was carried out to assess the potential protective effects of TE separated from the endophytic fungus A. terreus against LPS (lipopolysaccharide)-induced ALI (acute lung injury) in mice. The results revealed that TE alleviated pulmonary edema as it lowered both the W/D lung ratio and protein content. The inflammatory response represented by inflammatory cell infiltration into the lung tissues was greatly repressed by TE. That was supported by the improved histopathological results and also by the reduced level of myeloperoxidase in the lung. TE showed a potent antioxidant activity as it attenuated lipid peroxidative markers (malondialdehyde, 4-hydroxynonenal, and protein carbonyl) and enhanced endogenous antioxidants (reduced glutathione, superoxide dismutase, and catalase) in lung tissues. Similarly, TE increased the mRNA expression of SIRT1, Nrf2, and its genes (HO-1, NQO1, and GCLm). On the other hand, TE restrained the activation of NF-κB (nuclear factor-κB) in the lung. Consequently, TE depressed the pro-inflammatory cytokines: nitric oxide (NOx), TNF-α (tumor necrosis factor-α), and interleukins (IL-6 and -1β). Additionally, TE inhibited NLRP3 signaling and interrupted apoptosis by decreasing the levels of proapoptotic markers (Bax and caspase-3) and increasing the level of an anti-apoptotic marker (Bcl-2). In conclusion, TE had a remarkable protective potential on LPS-induced lung damage via antioxidant and anti-inflammatory mechanisms. This finding encourages further investigations on this promising candidate.

Effect of a novel piperazine compound on cancer cells

Many drugs have been developed for anticancer chemotherapy. However, more anti-cancer drugs should be developed from potential chemicals to circumvent the disadvantages of existing drugs. Most anti-cancer chemicals induce apoptosis in cancer cells. This study tested the efficiency of a new chemical, the piperazine derivative 1-[2-(Allylthio) benzoyl]-4-(4-methoxyphenyl) piperazine (CB01), on glioblastoma (U87) and cervix cancer (HeLa) cells. CB01 was highly cytotoxic to these cells (IC50S < 50 nM) and induced the traditional apoptotic symptoms of DNA fragmentation and nuclear condensation at 40 nM. Western-blot analysis of the cell lysates revealed that the intracellular apoptotic marker proteins, such as cleaved caspase-3, cytochrome c, and Bax, were highly upregulated in the CB01-treated cells. Furthermore, increased activities of caspase-3 and -9, but not caspase-8, were observed. Therefore, these results suggest that CB01 can act as an anticancer chemotherapeutic by stimulating the intrinsic mitochondrial signaling pathway to induce cytotoxicity and apoptosis in cancer cells.

Betulinic Acid Modulates the Expression of HSPA and Activates Apoptosis in Two Cell Lines of Human Colorectal Cancer

Betulinic acid (BA) is a pentacyclic triterpene usually isolated from botanical sources. Numerous studies have reported the inhibitory effect of BA against human colorectal cancer cells (CRC). However, its effect on the expression of the molecular chaperone HSPA is unclear. The aim of this research is to investigate the anti-cancer activities of BA purified from Piper retrofractum and study its effect on the expression of HSPA in colorectal cancer HCT116 and SW480 cells. The viability of both cancer cells was reduced after they were treated with an increasing dosage of BA. Flow cytometry assay revealed that levels of cell apoptosis significantly increased after incubation with BA in both cancer cells. Pro-apoptotic markers including Bax, cleaved-caspase-3 and cleaved-caspase-9 were increased while anti-apoptotic marker Bcl-2 was decreased after BA treatment. Western blot also showed that the expression of HSPA fluctuated upon BA treatment, whereby HSPA was increased at lower BA concentrations while at higher BA concentrations HSPA expression was decreased. Preliminary molecular docking assay showed that BA can bind to the nucleotide binding domain of the HSP70 at its ADP-bound state of the HSP70. Although further research is needed to comprehend the BA-HSPA interaction, our findings indicate that BA can be considered as potential candidate for the development of new treatment for colorectal cancer.

LncRNA KASRT Serves as a Potential Treatment Target by Regulating SRSF1-Related KLF6 Alternative Splicing and the P21/CCND1 Pathway in Osteosarcoma: An In Vitro and In Vivo Study

PurposeLong non-coding RNA KLF6 alternative splicing regulating transcript (lnc-KASRT) locates within the intronic region of SRSF1, possessing the potential to regulate KLF6 alternative splicing to promote carcinogenicity. Then, the current in vitro and in vivo study aimed to investigate the effect of lnc-KASRT on regulating tumor malignant behaviors, and the implication of its interaction with KLF6 alternative splicing in osteosarcoma.MethodsLnc-KASRT overexpression or knockdown plasmid was transfected into U-2OS and Saos-2 cells. Then, KLF6-SV1 knockdown plasmid with or without lnc-KASRT overexpression plasmid was transfected into these cells for compensative experiments. In vivo, lnc-KASRT overexpression or knockdown Saos-2 cells were injected in mice for tumor xenograft construction.ResultsLnc-KASRT expression was increased in most osteosarcoma cell lines compared to control cell line. Lnc-KASRT overexpression promoted cell viability, mobility, and anti-apoptotic marker expression, while reducing apoptosis rate and pro-apoptotic marker expression; meanwhile, it regulated SRSF1, KLF6 alternative splicing (increased KLF6-splice variant 1 (KLF6-SV1), decreased KLF6-wild type (KLF6-WT)), and followed P21/CCND1 pathway in U-2OS/Saos-2 cells. The lnc-KASRT knockdown exhibited opposite trends. Subsequent compensative experiments disclosed that KLF6-SV1 knockdown attenuated most of the tumor-promoting effects of lnc-KASRT overexpression in U-2OS/Saos-2 cells. In vivo experiments further validated that lnc-KASRT enhanced tumor growth and reduced tumor apoptosis; meanwhile, it also increased tumor KLF6-SV1, MMP-1, and MMP-9 expressions but decreased tumor SRSF1 and KLF6-WT expressions in xenograft mice.ConclusionLnc-KASRT serves as a potential treatment target via regulating SRSF1-related KLF6 alternative splicing and following P21/CCND1 pathway in osteosarcoma.

CORRELATION OF BCL 2 ONCOPROTEIN EXPRESSION AND ITS PROGNOSTIC SIGNIFICANCE IN COLORECTAL CARCINOMA WITH AGE, SITE GRADE AND STAGE

Background & Objective: Colorectal cancer is the third most prevalent malignancy with high mortality rate, necessitating markers that predict survival and guide the treatment. Previous studies have examined the immunohistochemical expression of Bcl-2, anti apoptotic marker, in colorectal carcinoma, but results have been contradictory.To evaluate the histopathological features of colorectal carcinoma, immunohistochemical expression of Bcl-2 must be analyzed to find out statistical association of Bcl-2 expression with certain prognostic factors histopathologic type, grade and TNM staging and also clinical parameters like age gender, site. Methods: This prospective study was conducted on the colectomy specimens of colorectal carcinoma,over a period of 2 years 6months From may 2018 to November 2020 The tumor morphology and Bcl-2 status were evaluated by immunohistochemistry in each case,with the inclusion criteria of resected intestinal specimens among which only malignant epithelial lesions and exclusion of all benign lesions. Results:The study included 40 cases,with age group of patients 51-60(37.5%) years and male:female ratio of 1.2:1.Bcl-2 positivity was seen in 37.5% of the cases.Weak,moderate,and strong expression of Bcl-2 was seen in 62.5%, 25%, and 12.5% of cases respectively. Even though early stages of colorectal carcinoma showed greater frequency of Bcl-2 expression than advanced stages (25% versus 12.5%), however this association was not statistically significant. Conclusion: The expression of bcl 2 out of 40 resected specimens of colorectal carcinoma correlated with various histological and clinical paramaters .bcl 2 positivity was assessed by semiquantitaive method and its expression was decreased with increase in grade and stage of the tumour and increased with the early stages of colorectal cancer

Protocatechuic Acid Abrogates Oxidative Insults, Inflammation and Apoptosis in Liver and Kidney Associated With Monosodium Glutamate Intoxication in Rats

Monosodium glutamate (MSG), a commonly used flavor enhancer, has been reported to induce hepatic and renal dysfunctions. In this study, the palliative role of protocatechuic acid (PCA) in MSG-administered rats was elucidated. Adult male rats were assigned to four groups, namely control, MSG (4 mg/kg), PCA (100 mg/kg), and the last group was co-administered MSG and PCA at aforementioned doses for seven days. Results showed that MSG augmented the hepatic (AST and ALT) and renal (urea and creatinine) functions markers as well as glucose, triglycerides, total cholesterol and LDL levels. Moreover, marked increases in MDA levels accompanied by declines in GSH levels and notable decreases in the activities of SOD, CAT, GPx, and GR were observed in MSG-treated group. The MSG-mediated oxidative stress was further confirmed by down-regulation of Nfe2l2 gene expression levels in both tissues. In addition, MSG enhanced the hepatorenal inflammatory response as witnessed by increased inflammatory cytokines (IL-1b and TNF-α) and elevated NF-κB levels in both tissues. Further, significant increases in Bax (pro-apoptotic biomarker) levels together with decreases in Bcl-2 (anti-apoptotic marker) levels were observed in MSG administration. Hepatic and renal histopathological screening supported the biochemical and molecular findings. On the contrary, co-treatment of rats with PCA resulted in remarkable enhancement of the antioxidant cellular capacity, suppression of inflammatory mediators and apoptosis. These effects are possibly endorsed for activation of Nrf-2 and suppression of NF-kB signaling pathways. Collectively, addition of PCA counteracted MSG-induced hepatic and renal injurious effects through modulation of oxidative, inflammatory and apoptotic alterations.

Antitumor Potential of Lippia citriodora Essential Oil in Breast Tumor-Bearing Mice

Lippia citriodora is a flowering plant cultivated for its lemon-scented leaves and used in folk medicine for the preparation of tea for the alleviation of symptoms of gastrointestinal disorders, cold, and asthma. The oil extracted from the plant leaves was shown to possess antioxidant potential and to exert antiproliferative activity against breast cancer. The aim of this study was to further investigate potential antitumor effects of L. citriodora oil (LCO) on breast cancer. The in vitro antiproliferative activity of LCO was examined against murine DA3 breast cancer cells by the sulforhodamine B assay. We further explored the LCO’s pro-apoptotic potential with the Annexin-PI method. The LCO’s anti-migratory effect was assessed by the wound-healing assay. LCO was found to inhibit the growth of DA3 cells in vitro, attenuate their migration, and induce apoptosis. Finally, oral administration of LCO for 14 days in mice inhibited by 55% the size of developing tumors in the DA3 murine tumor model. Noteworthy, in the tumor tissue of LCO-treated mice the apoptotic marker cleaved caspase-3 was elevated, while a reduced protein expression of survivin was observed. These results indicate that LCO, as a source of bioactive compounds, has a very interesting nutraceutical potential.

Predictive Value of Apoptotic Factor M30 for Negative Left Ventricular Remodeling in Patients Undergoing Primary Percutaneous Coronary Intervention

Background: Left Ventricular Negative Remodeling (LVNR) following Primary Percutaneous Coronary Intervention (PPCI) is an important cause of LV systolic dysfunction due to Irreversible Myocardial Injury (IMI). Both necrosis and apoptosis contribute to IMI and LVNR. We assessed the role of specific apoptotic marker M30 in predicting LVNR in patients of anterior wall ST Elevation Myocardial Infarction (STEMI) undergoing PPCI within 12 hours of symptom onset. Methods: This prospective study was done on 100 consecutive patients of anterior wall STEMI (87 men and 13 women, mean age 52.15±12.08 years) meeting our inclusion and exclusion criteria. Blood sample for M30 was drawn at 24 hours after symptom onset, when it reaches peak level. Transthoracic echo was done in each patient at 24 hours after PPCI and at 6 months. LVNR was defined as ≥20% increase in LV end diastolic volume at 6 months after PPCI. Results: 44 patients (44%) developed LVNR at 6 months post PPCI. Diabetes mellitus (p=0.032), symptom onset to balloon time (p=0.059), CPK-MB (p=0.007) and M30 level (p=0.012) were independent predictors of LVNR. The cutoff value of M30 for predicting LVNR was 81.18u/ml with positive predictive value of 70.4% (AUC 85.3, p<0.001). Conclusion: In patients of anterior wall STEMI undergoing PPCI, the apoptotic marker M30 is useful for early prediction of LVNR. This can assist in better risk stratification of patients after successful PPCI and identify the subgroup of patients who require more intensive medical follow up with antiremodeling drugs to attenuate the development of LVNR.