The aim of this study was to elucidate the role of PPARγ in interleukin-1β- (IL-1β-) induced cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) expression through ROS generation in OA chondrocytes.  Reactive oxygen species (ROS) induced by extracellular cytokines trigger the expression of inflammatory mediators in osteoarthritis (OA) chondrocyte. Peroxisome proliferator-activated receptor gamma (PPARβ-induced ROS generation and chondrocyte apoptosis were determined by flow cytometry. Contents of NADPH oxidase (NOX), caspase-3, and caspase-9 were evaluated by biochemical detection. The involvement of NOX2 and mitogen-activated protein kinases (MAPKs) in IL-1β-induced COX-2 and PGE2 expression was investigated using pharmacologic inhibitors and further analyzed by western blotting. Activation of PPARγ was performed by using a pharmacologic agonist and was analyzed by western blotting.  IL-1β-induced COX-2 and PGE2 expression was mediated through NOX2 activation/ROS production, which could be attenuated by N-acetylcysteine , GW1929 (PPARγ agonist), DPI , SB203580 (p38MAPK inhibitor), PD98059 , and SP600125 . ROS activated p38MAPK to enter the nucleus, which was attenuated by PPARγ.  IL-1β induced COX-2 and PGE2 expression via activation of NOX2, which led to ROS production and MAPK activation. The activation of PPARγ exerted protective roles in the pathogenesis of OA. In OA chondrocytes, IL-1 Osteoarthritis (OA) is a chronic degenerative arthritis. The clinical manifestations are joint pain, stiffness, dysfunction, and deformity, which often seriously affect the life of patients , 2. Its γ) is a ligand-induced transcription factor known to play a role in normal cell function. After activation, PPARγ binds to a specific response element (PPRE) with retinol X receptor (RXR) heterodimer and promotes the expression of the target gene [γ level is elevated during preadipocyte differentiation and plays a central role in lipid metabolism, glucose homeostasis, inflammation, and cell proliferation [γ is a key regulator of cartilage health, and the lack of PPARγ leads to the acceleration of spontaneous OA [γ is thought to have protective effects on cell apoptosis and inflammation in OA.Peroxisome proliferator-activated receptor gamma  and p22phox, cytoplasmic subunits p47phox, p67phox, and p40phox, and small molecule GTPase-binding protein Rac. Its catalytic subunit gp91phox and its homologues NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2 are known as the NOX family, which exists in almost all organs, tissues, and cells [NADPH oxidase (NOX) is an enzyme complex composed of membrane subunits gp91nd cells .ROS is considered to be an important homeostatic factor in the development of OA. ROS produced by mitochondrial electron transfer is beneficial to maintain the redox balance of glycolysis, so as to maintain the dynamic balance of metabolism of articular chondrocytes . ROS hasMitogen-activated protein kinases (MAPKs) are a class of serine/threonine protein kinases widely existing in mammals. They exist in most cells and are important transmitters of extracellular signal transduction to the nucleus . MAPKs pβ to induce OA in vitro. We found that PPARγ activity decreased and chondrocyte apoptosis increased. We inferred that increasing PPARγ activity could reduce the production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) through the NOX2/ROS/p38MAPK signal axis, thus reducing chondrocyte apoptosis and alleviating OA progress.In this study, we used IL-1μg streptomycin and 10% fetal bovine serum (FBS) was from Gibco . Cell Counting Kit-8 (CCK-8), FITC Annexin V Apoptosis Detection Kit, 0.25% trypsin, ROS assay kit, PGE2 ELISA kit, NOX, caspase-9 and caspase-3 colorimetric assay kits, and BCA protein assay kit were purchased from Beyotime Biotechnology . TRIzol was from Invitrogen . The High-Capacity cDNA Reverse Transcription kit was obtained from Applied Biosystems . SYBR Select Master Mix was obtained from Applied Biosystems . Collagenase II was dissolved to 2 mg/ml in DMEM to digest articular cartilage. N-acetylcysteine (NAC), PPARγ agonist (GW1929), NOX2 inhibitor (DPI), p38MAPK inhibitor (SB203580), ERK inhibitor (PD98059), and JNK inhibitor (SP600125) were purchased from Cell Signaling Technology  and dissolved in dimethyl sulfoxide (DMSO).Collagenase II was obtained from Sigma . Dulbecco's modified Eagle's medium (DMEM) supplied with 100 U penicillin and 100 μm nylon cell strainer . The primary chondrocytes were cultured in DMEM supplemented with 10% FBS, 100 U penicillin, and 100 μg streptomycin in a 5% CO2 incubator at 37°C. Confluent chondrocytes were split in 1 : 2 ratios up to passages 2-3 and used for subsequent experiments. This study was reviewed and approved by the ethics committee of the No.2 People's Hospital of Changzhou, Jiangsu, China.The rat chondrocytes were isolated and cultured as described previously . In brieCultured chondrocytes were seeded on the gelatin precoated slices and grew to 50%-60% confluence. Then, the slices were fixed with 4% formaldehyde for 30 min, penetrated with 0.5% Triton X-100 for 10 min, and blocked with 1% BSA for 1 h at room temperature. Rabbit anti-rat collagen II antibody and rabbit anti-rat SOX9 antibody (antibodies were purchased from Abcam and diluted in 1 : 1000) were added and incubated in a wet box at 4°C overnight. The fluorescent second antibody was added, incubated in a wet box, and placed at room temperature for 1 h. Antiquenching tablets and DAPI 1 : 500 diluted slices were stored in a refrigerator at -20°C and photographed under a fluorescence microscope .β in the experiment, the cell viability was evaluated by the CCK-8 assay. Chondrocytes were plated in 96-well plates at a density of 5 × 103 cells/well to adhere overnight and treated with 0 ng/ml, 1 ng/ml, 10 ng/ml, or 30 ng/ml IL-1β for 0 min, 10 min, 30 min, 1 h, 2 h, 12 h, 24 h, and 48 h. After incubation time, 10 μl of CCK-8 solution was added to each well and further incubated for 1 h at 37°C in 5% CO2. Absorbance at 450 nm was measured using a microplate reader .To assess the time and concentration-response relationship of IL-1μM GW1929, 10 μM DPI, 10 μM SB203580, 10 μM PD98059, or 10 μM SP600125 for 1 h and then treated with 10 ng/ml IL-1β, cell proliferation was detected by the CCK-8 assay , cell apoptosis (24 h) and ROS level (24 h) were detected by flow cytometry, PGE2 content in cell supernatant was detected by ELISA (24 h), and COX-2 expression was detected by qRT-PCR (24 h). Cultured chondrocytes were pretreated with 10 mM NAC, 10 μM GW1929, or 10 μM DPI for 1 h and then treated with 10 ng/ml IL-1β; western blot was used to detect the expression of PPARγ, NOX2, p38, p-p38, ERK1/2, p-ERK1/2, JNK1/2, and p-JNK1/2 (24 h).Cultured chondrocytes were pretreated with 10 mM NAC, 10 μl binding buffer into which 5 μl of FITC Annexin V and 5 μl propidium iodide (PI) were added for 15 min at 25°C in the dark. After incubation, additional 400 μl binding buffer was added, and chondrocytes were analyzed with a FACScan flow cytometer . Results were quantified and assembled by the FlowJo software .To quantify the percentage of chondrocytes undergoing apoptosis, the FITC Annexin V Apoptosis Detection Kit was used as described previously . BrieflyThe ROS level in chondrocytes was measured by using a commercialized kit according to the manufacturer's optimized instructions. Briefly, the number of chondrocytes to be tested was counted using a cell count board. Then, cells were suspended in diluted DCFH-DA (10 mM) in PBS and incubated at 37°C for 20 min. After washing twice with PBS, ROS production in these cells was measured by using a FACScan flow cytometer .2 in cell supernatant was measured by ELISA according to the manufacturer's protocol. Optical density was detected by the microplate reader at 450 nm .The PGEμg) according to the manufacturer's protocol. COX-2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified using SYBR® Select Master Mix in a Bio-Rad iQ5 real-time PCR system. The specific primer sequences  are presented in Total RNA was extracted from treated samples using TRIzol. The High-Capacity cDNA Reverse Transcription kit was used to reverse transcribe total RNA  or lucigenin (20 μM), to initiate the reaction, followed by immediate measurement of chemiluminescence using a luminometer .Cells were grown in six-well culture plates and incubated with endosulfan for the indicated time intervals. Cells were gently scraped and centrifuged for 10 min at 4°C. The cell pellet was resuspended in 35 ml of ice-cold DMEM medium per vial and then kept on ice. 5 Caspase-9 and caspase-3 activities were measured by colorimetric assay kits according to the manufacturer's instructions. Briefly, cells were collected and lysed using the lysis buffer provided. The caspase-9 and caspase-3 activity colorimetric assays are based on the hydrolysis of the peptide substrate acetyl, resulting in the release of p-nitroaniline moiety, which has a high absorbance at 405 nm that was detected by a microplate reader .μg of protein was separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked for 1 h at room temperature in tris buffered saline with Tween-20 with 5% nonfat milk, the PVDF membrane was then incubated with rabbit anti-rat PPARγ antibody, rabbit anti-rat NOX2 antibody, rabbit anti-rat p38 antibody, rabbit anti-rat p-p38 antibody, rabbit anti-rat ERK1/2 antibody, rabbit anti-rat p-ERK1/2 antibody, rabbit anti-rat JNK1/2 antibody, rabbit anti-rat p-JNK1/2 antibody, and rabbit anti-rat GAPDH antibody, respectively  overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were detected with the enhanced chemiluminescence assay kit. The GAPDH signal was used as an internal loading control, and relative expression levels were quantified by Quantity One software .Chondrocytes were harvested and lysed in RIPA buffer for total protein extraction. The protein concentration of each sample was determined by the BCA protein assay kit. After that, 10 P < 0.05 was considered to be statistically significant.Data shown in our study were represented as means ± SD from three independent experiments. One-way ANOVA was conducted for comparison between multiple groups, and The primary cultured cells were identified as chondrocytes by immunofluorescence staining of collagen II and SOX9 .β continuous treatments . The results showed that the viability of chondrocytes decreased by 0.12% (P > 0.05), 0.35% (P > 0.05), 0.52% (P > 0.05), 1.33% (P > 0.05), 2.87% (P > 0.05), 4.14% (P > 0.05), and 6.04% (P > 0.05) after 1 ng/ml IL-1β treatment at seven time points compared with the vehicle group, respectively. After 10 ng/ml IL-1β treatment, the viability of chondrocytes decreased by 0.58% (P > 0.05), 1.79% (P > 0.05), 2.48% (P > 0.05), 3.18% (P > 0.05), 9.72% (P < 0.05), 22.31% (P < 0.001), and 6.04% (P < 0.001) at seven time points compared with the vehicle group, respectively. After 30 ng/ml IL-1β treatment, the viability of chondrocytes decreased by 1.74% (P > 0.05), 3.18% (P > 0.05), 4.90% (P > 0.05), 6.24% (P > 0.05), 15.86% (P < 0.01), 35.62% (P < 0.001), and 70.74% (P < 0.001) at seven time points compared with the vehicle group, respectively , 10 μM DPI (NOX2 inhibitor), 10 μM SB203580 (p38MAPK inhibitor), 10 μM PD98059 (ERK inhibitor), or10 μM SP600125 (JNK inhibitor), respectively, for 1 h and then treated with 10 ng/ml IL-1β. Cell proliferation was detected at 12 h, 24 h, and 48 h. Compared with the vehicle group, the viability of chondrocytes decreased by 7.97% , 2.22% , 3.51% , 4.41% , 5.17% , 7.32% , and 4.11%  at 12 h, respectively. There was a significant difference between the IL-1β group and the NAC group (P < 0.01). There was no significant difference between the IL-1β group and agonist or inhibitor groups (P > 0.05). Compared with the vehicle group, the viability of chondrocytes decreased by 21.34% , 6.20% , 7.83% , 9.86% , 11.89% , 14.64% , and 10.22%  at 24 h, respectively. There were significant differences between the IL-1β group and NAC or agonist or inhibitor groups. Compared with the vehicle group, the viability of chondrocytes decreased by 45.75% , 14.85% , 17.90% , 22.09% , 27.07% , 33.15% , and 22.07%  at 48 h, respectively. There were significant differences between the IL-1β group and the NAC or agonist or inhibitor groups  after different concentrations of IL-1ectively . Since tr groups .β, the apoptotic rate of chondrocytes increased by about 5 times (P < 0.001) compared with the vehicle group. When pretreated with NAC or agonist or inhibitors, the apoptotic rate increased by 1.31 times , 1.74 times , 2.27 times , 2.76 times , 3.36 times , or 2 times , respectively, compared with the vehicle group. Among all these chemicals, NAC showed the best antiapoptotic effect, decreased by 61.46% (P < 0.001), compared with the IL-1β group. Meanwhile, the other compounds all possessed different degrees of antiapoptosis, which presented as 54.20% , 45.44% , 37.30% , 27.18% , and 50.10%  compared with the IL-1β group, respectively. Compared with the NAC group, the apoptotic rate increased by 18.83% , 41.55% , 62.67% , 88.92% , or 29.45% , respectively , which indicated enhanced intracellular oxidative stress. When pretreated with the chemicals, the fluorescence intensity of intracellular ROS increased by 9% , 40.18% , 14.01% , 67.28% , 96.26% , or 57.94% , respectively, compared with the vehicle group, but decreased by 65.93% , 53.12% , 61.87% , 44.06% , 34.37% , or 47.18% , respectively, compared with the IL-1β group. NAC showed the best antioxidative effect according to our data. Compared with the NAC group, the fluorescence intensity of intracellular ROS increased by 37.61% , 11.92% , 64.22% , 92.66% , or 55.04% , respectively . When pretreated with NAC or agonist or inhibitors, PGE2 content in cell supernatant increased by 75.72% , 16.18% , 38.15% , 70.80% , 43.35% , or 93.64% , respectively, compared with the vehicle group, but decreased by 43.42% , 116% , 82.42% , 47.54% , 75.80% , or 30.14% , respectively, compared with the IL-1β group. Compared with the NAC group, PGE2 content in cell supernatant decreased by 51.24% , 27.19% , 2.87% , or 22.58%  or increased by 10.19% , respectively (PGEectively .P < 0.01) in chondrocytes treated with IL-1β, compared with the vehicle group. When pretreated with NAC or agonist or inhibitors, the mRNA expression of COX-2 increased by 69% , 38% , 7% , 53% , 103% , or 37% , respectively, compared with the vehicle group, but decreased by 43.19% , 75.36% , 126% , 58.16% , 19.21% , or 76.64% , respectively, compared with the IL-1β group. Compared with the NAC group, the mRNA expression of COX-2 decreased by 22.46% , 57.94% , 10.45% , or 23.35%  or increased by 20.11% , respectively . When pretreated with other chemicals, the levels of NOX increased by 4.9 times , 4.1 times , 4.9 times , 5.1 times , or 5.2 times , respectively, but decreased by 6%  compared with the vehicle group. The NOX activities decreased by 11% , 25% , 83% , 11% , 7% , or 6% , respectively, compared with the IL-1β group. Compared with the NAC group, the levels of NOX decreased by 16%  or 81%  but increased by 1% , 5% , or 6% , respectively . When pretreated with NAC or agonist or inhibitors, the caspase-9 activities increased by 2.4 times , 1.6 times , 2.5 times , 4.7 times , 5.1 times , or 3.7 times , respectively, compared with the vehicle group. The levels of caspase-9 decreased by 62% , 74% , 60% , 25% , 12% , or 40% , respectively, compared with the IL-1β group. Compared with the NAC group, the caspase-9 activities decreased by 31%  but increased by 2% , 94% , 120% , or 54% , respectively . When pretreated with NAC or agonist or inhibitors, the caspase-3 activities increased by 2.5 times , 2.4 times , 2.4 times , 4.6 times , 3.2 times , or 3.1 times , respectively, compared with the vehicle group. The levels of caspase-3 decreased by 58% , 59% , 56% , 19% , 45% , or 46% , respectively, compared with the IL-1β group. Compared with the NAC group, the caspase-3 activities decreased by 3%  but increased by 4% , 92% , 29% , or 30% , respectively . Pretreated with GW1929, PPARγ was activated, and there was no significant difference between the vehicle group and the GW1929 group (P > 0.05). The effect of GW1929 was better than that of the NAC group . Pretreated with DPI, PPARγ activity was lower than that of the vehicle group , but higher than that of the IL-1β group , and there was no significant difference between the DPI group and the NAC group (P > 0.05) (After treatment with IL-1 > 0.05) .β, the activation of NOX2 increased by 200% compared with the vehicle group (P < 0.001). There was no significant difference between the IL-1β group and the NAC group (P > 0.05). Pretreated with GW1929, NOX2 activity increased by 140% compared with the vehicle group (P < 0.01), but decreased by 20% compared with the IL-1β group (P < 0.01). The effect of GW1929 was weaker than that of the NAC group . Pretreated with DPI, NOX2 activity returned to normal and there was no significant difference between the vehicle group and the DPI group (P > 0.05) (After treatment with IL-1 > 0.05) .β, p38 activity increased by 13% (P < 0.05), while p-p38 activity increased by 300% (P < 0.001), compared with the vehicle group. Pretreated with GW1929, the activation of p38 decreased by 26% compared with the vehicle group (P < 0.05) and decreased by 34% compared with the IL-1β group (P < 0.01), while p-p38 activity increased by 80% compared with the vehicle group (P < 0.05) but decreased by 56% compared with the IL-1β group (P < 0.001). Both p38 and p-p38 activities were weaker than that of the NAC group (P < 0.01). Pretreated with DPI, the activation of p38 decreased by 38% compared with the IL-1β group (P < 0.05) and 49.42% compared with the NAC group (P < 0.01). There was no significant difference between the vehicle group and the DPI group (P > 0.05), while p-p38 activity increased by 200% compared with the vehicle group (P < 0.01) but decreased by 26% compared with the IL-1β group (P < 0.01). There was no significant difference between the NAC group and the DPI group (P > 0.05) , but p-ERK1/2 activity increased by 270% compared with the vehicle group (P < 0.001). Pretreated with GW1929, the activation of ERK1/2 increased by 28% compared with the vehicle group (P < 0.01) and increased by 20% compared with the IL-1β group (P < 0.01). ERK1/2 activity was stronger than that of the NAC group (P < 0.05), while p-ERK1/2 activity increased by 150% compared with the vehicle group (P < 0.001) but decreased by 30% compared with the IL-1β group (P < 0.01). The activation of p-ERK1/2 was weaker than that of the NAC group (P < 0.05). Pretreated with DPI, ERK1/2 activity increased by 57% compared with the vehicle group (P < 0.01) and 50% compared with the IL-1β group (P < 0.01). ERK1/2 activity was stronger than that of the NAC group (P < 0.01), while p-ERK1/2 activity increased by 68% compared with the vehicle group (P < 0.01) but decreased by 50% compared with the IL-1β group (P < 0.001). The activation of p-ERK1/2 was weaker than that of the NAC group (P < 0.01) , but p-JNK1/2 activity increased by 300% compared with the vehicle group (P < 0.001). Pretreated with GW1929, there was no significant difference in JNK1/2 activity between the vehicle group and the GW1929 group (P > 0.05) or the IL-1β group and the GW1929 group (P > 0.05) or the NAC group and the GW1929 group (P > 0.05), while p-ERK1/2 activity increased by 260% compared with the vehicle group (P < 0.01), but there was no significant difference in p-JNK1/2 activity between the IL-1β group and the GW1929 group (P > 0.05) or the NAC group and the GW1929 group (P > 0.05). Pretreated with DPI, there was no significant difference in JNK1/2 activity between the vehicle group and the DPI group (P > 0.05) or the IL-1β group and the DPI group (P > 0.05) or the NAC group and the DPI group (P > 0.05), while p-ERK1/2 activity increased by 240% compared with the vehicle group (P < 0.01), but there was no significant difference in p-JNK1/2 activity between the IL-1β group and the DPI group (P > 0.05) or the NAC group and the DPI group (P > 0.05) (Figures After treatment with IL-1 Figures .OA is characterized by gradual degeneration of articular cartilage, new bone formation, and synovial hyperplasia, which may eventually lead to pain, joint dysfunction, and disability , 18. CliThe NOX family is mainly through the activation of ROS to complete the physiological and pathological functions. NOX of phagocytes has no activity in resting cells. ROS is activated when stimulated by pathogenic microorganisms, inflammatory mediators, and external factors, which are related to host defense. Different from phagocytic NOX, nonphagocytic NOX maintains a certain activity under physiological conditions and produces intracellular and extracellular ROS . ROS proROS is a kind of substance formed in aerobic metabolism and aerobic environment. It contains oxygen in molecular composition and has higher chemical activity than oxygen itself. The increase of ROS can cause serious oxidative damage to the main components of cells, such as DNA, protein, and lipid , 25. Amo2 in the GW1929 group, DPI group, and PD98059 group was lower than that in the NAC group, indicating that the GW1929 group, DPI group, or PD98059 group was stronger than the NAC group in anti-inflammatory effect. Although there was no significant difference between the SB203580 group and the NAC group or the SP600125 group and the NAC group, the concentration of PGE2 of the SB203580 group and the SP600125 group was lower than that of the IL-1β group (γ, NOX2, and MAPK signaling pathways play a regulatory role in the production of PGE2. In downregulation of COX-2, the effect of the PD98059 group was weaker than that of the NAC group. There was no significant difference between the SB203580 group and the NAC group. But the expression of COX-2 in the SB203580 group and the PD98059 group was lower than that in the IL-1β group. The SP600125 group, the GW1929 group, and the DPI group had better effect on the downregulation of COX-2 than that of the NAC group (γ and NOX2 had direct regulation on COX-2 as well as P38MAPK and JNK.ROS can directly attack mitochondria and cause oxidative damage. Mitochondrial damage in OA chondrocytes can increase the sensitivity of chondrocytes to inflammatory stimulation , 30. Dur1β group . These rAC group . It is sROS is involved in signal transduction and is closely related to cell development and fate. ROS is the basis of the physiological function of the body. It regulates many kinds of signal transduction, protein structure, transcription factors, and genes through direct reaction and regulates their functions . As a siMAPKs are the key components of intracellular signaling pathways, which are related to cell proliferation, differentiation, apoptosis, cytokine response, and MMP expression , 32. Stu2, and iNOS. In addition, SB203580 can inhibit the process of chondrocyte hypertrophy, reduce the synthesis of collagen X, and delay the aging process of chondrocytes [MAPK signaling pathways play a key role in the signal transduction of OA cartilage injury, which receives extracellular stimulation to transfer into the nucleus, regulate transcription factors and their downstream genes, and finally react at the cellular level. Inhibition of MAPK signaling pathways can reduce lipid peroxidation of inflammatory cells, regulate apoptosis-related genes, and eventually lead to inhibition of cell proliferation and apoptosis, thus protecting articular cartilage . P38MAPKdrocytes .α/β/γ, which are widely distributed in the human body. PPARγ is the most deeply studied. Early studies mainly focused on the lipid metabolism regulation of PPARγ, but recent studies have shown that PPARγ also plays a role in the regulation of inflammatory response, matrix decomposition, and synthesis [κB, and other inflammatory response genes [γ is present in all major cells of human joints, including chondrocytes. Recent studies have found that PPARγ expression in OA patients or animal models is significantly reduced [γ in chondrocytes will lead to structural and functional changes, such as decreased synthesis and secretion of the matrix of chondrocytes required, increased synthesis and secretion of matrix metalloproteinases, and increased release of inflammatory factors and cytokines. This effect is consistent with the changes of chondrocytes during OA [γ was involved in the pathogenesis of OA. IL-1β induced oxidative stress, increasing chondrocyte apoptosis rates and decreasing PPARγ protein activity, which consisted of the pathophysiology of OA. GW1929, as an agonist, can directly increase PPARγ activity and thus inhibit oxidative stress. DPI can reduce ROS production by inhibiting NOX2 activity, which also inhibits oxidative stress (γ was not the nuclear transcription factor that directly activated NOX2 expression but may regulate NOX2 indirectly by activating other downstream proteins. The detailed mechanism underlying needs to be further confirmed. PPARγ activation could downregulate the p38MAPK signaling pathway and inhibit NOX2. PPARγ had no direct effect on ERK1/2, but NOX2 could regulate the phosphorylation of ERK1/2. Both PPARγ and NOX2 had no effect on JNK.PPAR has three isomeric forms, PPARynthesis , 41. It se genes , 42. PPA reduced . Downreguring OA . Based oe stress . We infeβ can induce the expression of COX-2 and PGE2 in chondrocytes through the NOX2/ROS/p38MAPK signaling pathway, and PPARγ expression is downregulated during this period. Activation of PPARγ can significantly inhibit the expression of COX-2 and PGE2 in chondrocytes induced by IL-1β and resist the injury of chondrocytes induced by IL-1β, so as to alleviate the pathogenesis of OA.IL-1


Vascular smooth muscle cell senescence plays a pivotal role in the pathogenesis of atherosclerosis. Anagliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor for the treatment of hyperglycemia. Recent progress indicates that DPP-4 inhibitors show a wide range of cardiovascular benefits. We hypothesize that Anagliptin plays a role in vascular smooth muscle cell senescence and this may imply its modulation of atherosclerosis. Here, the beneficial effect of Anagliptin against interleukin 1β (IL-1β)-induced cell senescence in vascular smooth muscle cells was studied to learn the promising therapeutic capacity of Anagliptin on atherosclerosis. Firstly, we found that Anagliptin treatment ameliorated the elevated secretions of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and macrophage chemoattractant protein-1 (MCP-1). Secondly, our findings indicate that exposure to IL-1β reduced telomerase activity from 26.7 IU/L to 15.8 IU/L, which was increased to 20.3 and 24.6 IU/L by 2.5 and 5 μM Anagliptin, respectively. In contrast, IL-1β stimulation increased senescence- associated β-galactosidase  staining to 3.1- fold compared to the control group, it was then reduced to 2.3- and 1.6- fold by Anagliptin dose-dependently. Thirdly, Anagliptin dramatically reversed the upregulated p16, p21, and downregulated sirtuin1 (SIRT1) in IL-1β-treated vascular smooth muscle cells. Lastly, the protective effect of Anagliptin against cellular senescence in vascular smooth muscle cells was abolished by silencing of SIRT1. In conclusion, Anagliptin protects vascular smooth muscle cells from cytokine-induced senescence, and the action of Anagliptin in vascular smooth muscle cells requires SIRT1 expression.  As the eriments . VSMC seeriments . Severaleriments . P21 is eriments . The traeriments . As an ieriments . Recentleriments . In addieriments . TherefoAnagliptin  is a nov2 at 37°C. The treatment reagent IL-1β was purchased from R&D Systems (#201-LB), and Anagliptin was obtained from Sigma-Aldrich (#4943). To test the cytotoxicity of Anagliptin, hVSMCs were treated with a series of Anagliptin ranging from 0.1 to 50 μM. For all other experiments, cells were challenged with IL-1β (10 ng/mL) [This study was performed in accordance with the principles of the World Medical Association Declaration of Helsinki for medical research using human-derived cells. Experimental protocols were approved by the Ethical Committee of Yan’an University. Human vascular smooth muscle cells (hVSMCs) were obtained from Guandao Biological Engineering  and cultured in a Dulbecco’s Modified Eagle Medium (DMEM) supplied with 10% fetal bovine serum (FBS) in 5% CO0 ng/mL)  or AnaglSystems #1-LB, andCells were plated in a 96-well plate (5000 cells per well). After necessary treatment, CCK-8 solution was added to the cells and incubated for 3 hours at 37°C. Lastly, optical density (OD) was determined using 450 nM.+, diaphorase, 0.004% BSA, 0.15% sucrose, and INT. After incubation for 30 minutes, OD at 490 nm was measured after eliminating the reaction.The cytotoxicity was further confirmed by detecting the release of LDH using a commercial kit . Briefly, the cells were incubated with 1% Triton X-100 for 45 minutes, followed incubation of the samples in the dark in the cultural system containing lactate, NAD−ΔΔCt method. The following primers were used:In brief, an RNA isolation kit  was applied to isolate total RNAs from hVSMCs, followed by being transcribed into cDNA according to the instruction of the qRT-PCR mRNA Detection Kit . Subsequently, the Lightcycler 480 Detection System  was used to perform real-time PCR, with GAPDH taken as the internal control for normalization. The circle times were recorded for the calculation of target genes according to the 2MCP-1 ; IL-6 ; TNF-α ; p16 , p21 , SIRT1 , GAPDH .The total proteins were isolated from the treated hVSMCs using the lysis buffer, and quantified using the bicinchoninic acid (BCA) kit . Subsequently, the proteins were run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and further transferred to the polyvinylidene fluoride (PVDF) membrane . After 3 washes with PBST buffer, the membrane was incubated with the primary antibody against p16, p21, SIRT1, and β-actin overnight in a cold room and incubated with HRP-conjugated secondary antibody  at room temperature for 1.5 hours. Lastly, the membranes were exposed to an ECL detection system .The concentrations of TNF-α, IL-6, and MCP-1 in the supernatant of treated hVSMCs were detected using the ELISA commercial kits . The following Quantikine ELISA kits were purchased: IL-6 (#D6050), TNF-α (#PDTA00D), and MCP-1(#DCP00). TNF-α, IL-6, and MCP-1 were measured as perthe detailed procedure described previously .The CHAPS buffer was used to lyse the treated hVSMCs, followed by centrifugation at 15,000 × g for 30 minutes and quantifying the concentration of total proteins using the BCA kit . The TeloTAGGG Telomerase PCR ELISA Plus Kit  was used to measure the telomerase activity. The TRAP-PCR reaction system contained 0.5 mg of each sample and 10 pM of each primer, followed by quantification using RT-PCR assay.2 in fresh SA-β-gal staining solution. Lastly, the percentage of SA-β-gal positive blue cells was counted after being photographed.The Senescence Detection kit  was used. Briefly, cells were washed with PBS buffer and fixed using 2% paraformaldehyde for 5 min, followed by washing and incubated at 37°C in the absence of COAnalysis of Variance (ANOVA) with Tukey’s Multiple Comparison post-hoc test was used to compare the difference among groups. P < 0.05 was considered statistically significant.We aimed to investigate whether Anagliptin possesses a protective effect against IL-1β- induced insults in VMSCs. We demonstrated that Anagliptin prevented cellular senescence by reducing SA-β-gal but increasing telomerase activity in IL-1β- challenged VMSCs. Mechanistically, we found that this effect was mediated by SIRT1.23 and our test results. To test the cytotoxicity, cells were stimulated with Anagliptin at the concentrations of 0.1, 0.25, 0.5, 2.5, 5, 25, and 50 μM for 24 hours. No obvious difference was observed in the cell viability when Anagliptin was lower than 5 μM  . Xia 3333 reportThe senescence of VSMCs accompanied by the gained senescence-associated secretory phenotype (SASP) could lead to vascular inflammation, hence the loss of vascular function . Our datin vivo experiments are warranted to validate the findings.A recent study shows that selective elimination of senescent cells by silencing key regulators in mice can improve age-related chronic diseases . It is oTo sum up, our data revealed that Anagliptin prevented IL-1β-induced cellular senescence in vascular smooth muscle cells by up-regulating SIRT1. Thus, we concluded that the DPP-4 inhibitor Anagliptin has a protective role in vascular smooth muscle cells.


GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, HMBS, B2M, RPS15, and ACTB) in peripheral blood mononuclear cells (PBMCs) of cattle populations reared at cold arid high-altitude hypoxia and hot arid normoxia environments. Four different statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder were used to assess the stability of these genes. A total of 30 blood samples were collected: six adult heifers each of Ladakhi (LAC) and Holstein Frisian crosses (HFX) and 4 Jersey (JYC) cows from cold arid high-altitude hypoxia environments (group I) and five adult heifers each of Sahiwal (SAC), Karan Fries (KFC), and Holstein Friesian (HFC) cows from hot arid normoxia environments (group II). Combined analysis of group I and group II resulted in identification of a panel of RGs like RPS9, RPS15, and GAPDH that could act as a useful resource to unravel the accurate transcriptional profile of PBMCs from diverse cattle populations adapted to distinct altitudes.To estimate gene expression in a reliable manner, quantitative real-time polymerase chain reaction data require normalisation using a panel of stably expressed reference genes (RGs). To date, information on an appropriate panel of RGs in cattle populations reared at cold arid high-altitude hypoxia and hot arid tropical normoxia environments is not available. Therefore, the present study was carried out to identify a panel of stably expressed RGs from 10 candidate genes ( Bos indicus lineage adapted to various agro-climatic zones from high land to hot tropical regions. Leh-Ladakh, also known as a “COLD DESERT” is a part of the western Himalayan agro-climatic and high-altitude temperate sub-agro-climatic zone in India. Ladakh is situated at an altitude of 3,500–5,500 m with difficult terrain and harsh climate conditions such as extreme temperature (−40°C in winter and 35°C in summer), low humidity (25–40%), low precipitation (80–300 mm), and low oxygen level (nearly 60–70% of the oxygen concentration at sea level). In spite of harsh weather, this region is blessed with several unique livestock populations such as yak, cattle, horses, sheep, goat, donkey, and double-hump camel. Over thousands of years of the evolutionary process, these animals have developed the special ability to survive in cold and hypoxia environments prevalent in Ladakh. Amongst all the livestock species, the native cattle of Ladakh known as “Ladakhi cattle” are the major livestock species that play an important role in the agriculture economy of the region. The cattle from the Trans-Himalayan region of Ladakh are short in stature and well-adapted to the high-altitude environment. Based on morphometric data on 275 animals and genetic characterization using microsatellite markers (unpublished data), this cattle population was observed to be highly distinct from native cattle breeds adapted to other agroclimatic zones of India. Recently, our group was successful in delineating the distinct transcriptome signatures of peripheral blood mononuclear cells (PBMCs) of high-altitude-adapted Ladakhi cattle and tropically adapted Sahiwal cattle , Jersey cattle (exotic), and Frieswal cattle {Sahiwal x Holstein Frisian cross}, and three cattle populations from the hot dry and semi-arid condition of Haryana state, viz., Sahiwal cattle (native), Holstein Frisians cattle (exotic), and Karan Fries cattle {Tharparkar x Holstein Frisian}. Ladakhi cattle (LAC) are the native breed of the Bos indicus lineage and are naturally adapted to the high-altitude region of Ladakh. Jersey cattle (JYC) are of the Bos taurus lineage and originated from temperate regions, while Frieswal cattle (HFX) are a cross-bred cattle population. Both Jersey and Frieswal cattle are non-native to Leh-Ladakh and have been reared in limited organized farms in the region since the last 2 decades. Amongst the cattle populations selected from the hot semi-arid region, Sahiwal cattle (SAC) are a very popular native cattle breed of Bos indicus lineage and are known for their adaptation potential to hot dry and tropical conditions. Holstein Frisian cattle (HFC) are of Bos taurus lineage and are non-native to India. However, Holstein Frisian cattle have been widely used in India in various cross-breeding programmes to enhance milk production of local breeds. Karan Fries cattle (KFC) are a popular cross-breed that were developed several decades ago by crossing Tharparkar cattle (native) with Holstein Frisian. Therefore the study has included two populations of native cattle: one from high-altitude  and the other from hot arid tropical regions ; and two populations of cross-breeds and two populations of exotic cattle from two extreme altitudes. The geographical coordinates of the sampling site representing the hot arid climate were latitude– 29° 3′ 56.7828″ N, and longitude 76° 2′ 25.7892″ E. The geographical coordinates of the sampling site from the high-altitude region of Ladakh were latitude– 34° 9′ 9.3168″ N, and longitude 77° 34′ 37.3764″ E. About 7–8 ml of whole blood samples was collected aseptically from the external jugular vein of the animals in sterile EDTA-coated vacutainer tubes.The blood sampling of animals was performed in accordance with the relevant guidelines and regulations as approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-National Bureau of Animal Genetics Resources (NBAGR), Karnal. The study has included three cattle populations from the cold arid high-altitude region of Leh-Ladakh, In total, 30 blood samples were collected from adult heifers; five each of Sahiwal (SAC), Karan Fries (KFC), and Holstein Friesian (HFC) cows from the hot arid normoxia environment and 5 heifers each of Ladakhi (LAC), Holstein Frisian crosses (HFX), and Jersey (JYC) cows from the cold arid high-altitude hypoxia environment. The entire workflow of the qPCR experiment is depicted in 2+ and Mg2+ free; Hyclone, Utah) and was gently over laid on a Histopaque-1077  followed by centrifugation at 4000 RPM for 30 min at RT. After removing the buffy coat in a separate 15 ml tube, cells were treated with 2 ml of chilled RBC lysis buffer for 5 min at RT and washed twice with 1 × PBS . Finally, the cells were suspended in 1.0 ml of ice cold Trizol reagent , homogenized, and stored at −80°C. Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions . The extracted RNA was further purified using RNeasy mini kit columns . To remove the traces of genomic DNA, rnase free DNase enzyme was used according to the manufacturer’s instructions . RNA concentration and purity were checked using a NanoDrop ND-1000 spectrophotometer  and Experion Bio-analyzer . The OD260/OD280 absorption ratio for different samples varied from 1.92 to 2.10. The RNA integrity number (RIN) values for all purified RNA samples were in the satisfactory range (6.5–8.5). The RNA integrity of each sample was also confirmed by visualizing 28S and 18S ribosomal bands on 1.5% agarose gel. The cDNA was synthesized using a Revertaid First strand cDNA synthesis kit  as described in our previous studies  using the density gradient centrifugation method as described by  studies . BrieflyGAPDH), ribosomal protein L-4 (RPL4), eukaryotic elongation factor 1 alpha (EEF1A1), ribosomal protein S9 (RPS9), hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ubiquitin expressed transcript (UXT), hydroxyl methylbilane (HMBS), beta 2-microglobulin (B2M), ribosomal protein S15 (RPS15), and beta actin (ACTB). The primers specific for these 10 RGs were available in the laboratory and have been utilized successfully in several of our previous studies , 0.4 μL each of 10 μM forward and reverse primers, and 0.2 μL of DNase/RNase free water. Each qRT-PCR reaction was performed in duplicate to check the quality by assessing intra-assay variation. The amplification was carried out in a 96-well block using a LightCycler 480-II real-time PCR instrument  with the following conditions; 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C (denaturation), and 1 min at 60°C . In order to evaluate the quality of qPCR reactions in terms of nonspecific amplification and primer-dimer formation, a dissociation curve for each gene was obtained by increasing the temperature from 60°C to 95°C. A six-point relative standard curve was prepared for each gene by using five-fold serial dilutions of pooled cDNA samples in duplicate. The amplification specificity for each primer was checked by the presence of a single band of expected size on 2.5% agarose gel , and alsviz., geNorm  are considered to have higher expression stability , crossing point standard deviation [{SD (±CP)} <1], and coefficient of variance (CV [%CP]). In BestKeeper analysis, genes with low SD (<1), low CV, and high coefficient of correlation (r) are generally considered stably expressed and vice versa.The qPCR data recorded for each gene were subsequently analysed to evaluate the expression stability. Four statistical approaches, , geNorm , NormFin, geNorm , BestKee, geNorm , and the, geNorm  were usetability . In additability . This anhttps://www.heartcure.com.au/reffinder/) was also employed to estimate the overall ranking of the 10 RGs by assigning an appropriate weight to each gene  in PBMCs of cattle types from cold arid high-altitude hypoxia and hot arid normoxia environments. The specificity of each primer pair was ascertained by the presence of the single specific amplicon of expected size on 2.5% agarose gel  to 27.86 (HMBS). On the basis of their average Ct scores, the 10 RGs were classified into group-I (abundantly expressed), group-II (moderately expressed), and group-III (least expressed). Group-1 included EEF1A1, B2M, RPS15, and RPL4 genes with a high expression level and average Cq scores of 17.66, 18.18, 19.35, and 19.68, respectively. Group-II included RPS9, GAPDH, and ACTB that displayed an intermediate expression level with average Cq scores of 20.33, 21.69, and 22.27, respectively. Group-III included UXT, HPRT1, and HMBS with the least expression and average Cq scores of 24.76, 26.18, and 27.86, respectively (EEF1A1 exhibited the lowest coefficient of variations (lowest variability across samples). On the other hand, the HMBS gene showed the highest coefficient of variation. Based on this parameter, EEF1A1, B2M, RPS15, and RPL4 RGs were most stable while UXT, HPRT1, and HMBS genes were the least stable across the combined dataset  are summarized in ectively . Conside dataset .EEF1A1 showed the highest maximum and HMBS showed the least expression level. The average Cq values of individual RGs in cold arid hypoxia and hot arid normoxia groups are summarized in Similar to the combined dataset, in cold arid as well as hot arid normoxia groups, RPS9 and RPS15 showed the highest expression stability (M = 0.464), followed by RPL4 (M = 0.527) and GAPDH (M = 0.539), whereas HPRT1 was the least stable gene (M = 1.228). On the basis of average expression stability measure, RGs were arranged from most stable  to the least stable : RPS9 = RPS15 > RPL4> GAPDH > HMBS > EEF1A1> B2M > UXT > ACTB > HPRT1  and normoxia hot arid group  was based on the geNorm algorithm. The expression stability of individual RGs was evaluated first by combining RT-qPCR data for all 30 PBMCs samples (cold arid hypoxia and hot arid normoxia groups). All the RGs in combined group analysis showed stable expression with a stability index within the acceptable range . In the combined analysis,  > HPRT1 . Since t > HPRT1 . It is gand RPL4 .RPL4 = EEF1A1 >RPS9> HMBS > RPS15 > B2M > GAPDH > HPRT11 > UXT > ACTB (RPL4 and EEF1A1 showed the highest expression stability (M = 0.255) followed by RPS9 (M = 0.285) and HMBS (M = 0.312), whereas ACTB was least stable (M = 0.940). The pair-wise variation analysis for different combinations of RGs was well below the threshold value of 0.15. The results obtained for V2/3 (0.092), V3/4 (0.075), V4/5 (0.097), and V5/6 (0.077) combinations indicated that RPL4 and EEF1A1 would provide the most accurate normalisation  and hot arid normoxia group . The M values for all the 10 RGs in the hypoxia cold arid group were <1.5. The ranking of RGs in order of expression stability within this group were: T > ACTB ; Table 3lisation . The expHPRT1 = RPS9> RPS15 > GAPDH > RPL4> EEF1A1> HMBS > ACTB > B2M > UXT (HPRT1 (M = 0.229) and RPS9 (M = 0.229) showed the highest stability followed by RSP15 (M = 0.305) whereas UXT was least stable (M = 0.819). The results for V2/3 (0.109), indicated that RPS9 and HPRT1 would provide reliable normalisation in PBMCs of the hot arid normoxia group , the ranking of RGs in order of expression stability were: 2M > UXT ; Table 3ia group . The resssed RGs .RPL4, RPS9, and RPS15 were found to be most stable with stability values of 0.282, 0.289, and 0.292 respectively. On the other hand, UXT, ACTB, and HPRT1 were least stable with stability values of 0.605, 0.737, and 1.288, respectively  analysis covering both the conditions and all samples (cold arid hypoxia and hot arid normoxia), ectively . The graof 0.808 . The graRPS15, RPS9, and HPRT1 as the three most stable RGs with stability values of 0.107, 0.134, and 0.136, respectively , HMBS (CV ± SD = 1.97 ± 0.55), RPS15 (CV ± SD = 2.85 ± 0.55), RPS9 (CV ± SD = 2.85 ± 0.58), RPL4 (CV ± SD = 3.15 ± 0.62), B2M (CV ± SD = 3.46 ± 0.63), and EEF1A1 (CV ± SD = 4.35 ± 0.77). Only three genes, UXT (CV ± SD = 4.18 ± 1.03), ACTB (CV ± SD = 5.81 ± 1.29), and HPRT1 (CV ± SD = 7.93 ± 2.07) showed SD > 1 and hence were considered unacceptable as RGs. Additionally, the BestKeeper software was used to measure the inter-gene relation amongst the 10 RGs in the combined dataset by calculating Pearson correlation coefficient (r) values. Several RG pairs GAPDH/RPL4 (r = 0.861) RPS9/RPS15 (r = 0.849), RPS9/HMBS (r = 0.847), RPL4/RPS9 (r = 0.834), and HMBS/RPS5 (r = 0.832) showed high correlation coefficients  calculated for each gene was used to estimate the correlation value between BI and each RG. As shown in RPS9 (r = 0.901), RPL4 (r = 0.894), RPS15 (r = 0.890), and GAPDH (r = 0.849). The high correlation values strongly suggested their suitability as reliable RGs in the experimental set up of the study.The descriptive statistics for BestKeeper analysis of the combined dataset are shown in ficients . The RG RPS15 (CV ± SD = 1.44 ± 0.28), HMBS (CV ± SD = 1.14 ± 0.32), GAPDH (CV ± SD = 1.46 ± 0.32), EEF1A1 (CV±SD = 2.18 ± 0.38), RPL4 (CV ± SD = 2.04 ± 0.41), RPS9 (CV ± SD = 2.15 ± 0.44), B2M (CV ± SD = 2.85 ± 0.52), and HPRT1 (CV ± SD = 2.12 ± 0.60) (ACTB (CV ± SD = 5.85 ± 1.34) and UXT (CV ± SD = 4.62 ± 1.17) and were unstable. The inter-gene relation showed very high correlation coefficient values (r) for EEF1A1/RPS9 (r = 0.93), EEF1A1/RPL4 (r = 0.90), and RPL4/RPS9 (r = 0.86) (r) of individual RGs with the BestKeeper index resulted in high values for EEF1A1 (r = 0.865), RPS9 (r = 0.858), and RPL4 (r = 0.846). These results strongly suggest that EEF1A1, RPS9, and RPL4 with low CV, low SD, and high coefficient of correlation to the BestKeeper index should be the ideal panel for the cold arid high-altitude hypoxia group.The BestKeeper analysis for the cold arid hypoxia group and hot arid normoxia group was also conducted separately. In the cold arid hypoxia group, the following 8 RGs showed lower a coefficient of variation (CV) and standard deviation (SD):  ± 0.60) . The rem = 0.86) . FurtherGAPDH (CV ± SD = 3.44 ± 0.74), B2M (CV ± SD = 4.07 ± 0.74), HMBS (CV ± SD = 3.47 ± 0.96), RPS 9 (CV ± SD = 2.85 ± 0.79), UXT (CV ± SD = 3.28 ± 0.80), HPRT1 (CV ± SD = 3.35 ± 0.81), RPS15 (4.30 ± 0.83), EEF1A1 (CV ± SD = 5.09 ± 0.91), and RPL4 (CV ± SD = 4.75 ± 0.93) showed low SD and CV values. Whereas ACTB (CV ± SD = 54.82 ± 1.04) showed SD > 1 and hence was considered unstable.The BestKeeper analysis for the hot arid normoxia group also resulted in several RGs with SD < 1 . The folr) for GAPDH/RPL4 (r = 0.930), EEF1A1/RPL4 (r = 0.951), RPS9/HPRT11 (r = 0.981), RPS15/HMBS (r = 0.923), HMBS/ACTB (r = 0.908), HPRT11/RPL4 (r = 874), HPRT11/EEF1A1 (r = 861), HMBS/RPS9 (r = 0.874), and RPS9/RPS15 (r = 0.965) (RPS9 (r = 0.978), RPS15 (0.971), HPRT1 (r = 0.962), RPL4 (0.905), and GAPDH (r = 0.904) (The inter-gene relation showed a strong correlation coefficient (= 0.965) . The hig= 0.904) . The higRPS9 (1.41), RPS15 (1.86), and GAPDH (2.83) as the three most stable RGs, followed by RPL4 (3.41), HMBS (3.98), EEF1A1 (6.48), and B2M (6.48), whereas UXT (8.00), ACTB (9.00), and HPRT1 (10.00) ranked as the three least stable RGs (EEF1A1 (1.41), RPL4 (2.11), and RPS15 (3.16) as the most stable RGs; while HPRT11 (8.00), UXT (9.00), and ACTB (10.00) as the most unstable RGs. In the hot arid normoxia group, the analysis displayed RPS9 (1.32), HPRT1 (2.71), and GAPDH (2.83) as most stable while EEF1A1 (6.96), UXT (8.41), and ACTB (8.46) as unstable RGs.Additionally, the RefFinder algorithm was used to evaluate the comprehensive ranking of individual RGs in combined, cold arid hypoxia, and hot arid normoxia group datasets . In the able RGs . In the EPAS1 and two target genes associated with heat stress response such as HSP70 and HSP27 were generated in PBMC samples of high-altitude-adapted and tropically adapted cattle populations. As shown in RPS19, RPS15, and GAPDH) normalised the target gene data more accurately. We expected higher expression of high-altitude-associated genes in PBMCs of high-altitude cattle populations. On the other hand, genes related to heat stress response such as HSPs should have higher expression in PBMCs of cattle populations from the tropical region. As shown in alpha and EPAS1 was higher in high-altitude (HA)-adapted cattle while genes like HSP70 and HSP27 were more expressed in low-altitude (LA) cattle breeds. Further, the relative expression, standard deviation (SD), and standard error (SE) of the four target genes also supported the high quality of the panel of reference genes used  resulted in an unexpected pattern of expression. As shown in On the other hand, normalisation of same target genes with least stable genes  is being employed as a most preferred tool to validate gene expression data. Even though qRT-PCR is the most sensitive technique, it suffers from several analytical variations like differences in the amount of starting material, RNA extraction, and efficiency of the reverse transcription process. To a great extent the effect of these non-biological variables can be nullified by normalising gene expression data by a panel of stable reference genes (RGs). As suggested in the “Minimum information for publication of Quantitative Real-time PCR Experiments” (MIQE) guideline , accuracnditions .RPS9, RPS15, and GAPDH were marked as the most stable RGs for combined data. In the cold arid hypoxia group; RPL4, EEF1A1, and RPS15 were the most stable RGs, while in the hot arid normoxia cattle group, RPS9, HPRT1, and GAPDH were identified as the most stable RGs.The major challenge in any biological experiment involving different tissues, cell types, disease state, and physiological and or developmental stages is the knowledge about appropriate RGs whose expression remains constant without any observable variations across samples . It has RPS9, RPS15, and, to a certain extent, RPL4 genes were identified as stable RGs in the three groups . Both RPS9 and RPS15 genes are part of the ribosomal component of the small 40S subunit while RPL4 is a 60S ribosomal protein L4. In the past, these ribosomal genes were also identified as ideal reference genes in many studies involving the mammary gland of dairy cows (T. castaneum) during a fungal infection (GAPDH, the other stable RG in our study, has also been identified as an appropriate reference gene in several other studies (GAPDH, EEF1A1 and HPRT1 were the other genes that ranked amongst the first three most stable RGs in cold arid hypoxia and hot arid normoxia groups, respectively. EEF1A1 regulates the enzymatic delivery of aminoacyl tRNAs to the ribosome while HPRT11 catalyses the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate, and the regulates generation of purine nucleotides through the purine salvage pathway. To the best of our knowledge, this is the first study to assess the expression stability of 10 candidate RGs in cattle populations adapted to distinct altitudes.In the present analysis, iry cows , mammaryiry cows , differeiry cows , heat-stiry cows , and PBMiry cows . It is kiry cows  and requnfection . Similarnfection . In our  studies . Along wRPL4, EEF1A1, and RPS15 RGs were the most stable, while in the hot arid normoxia group, RPS9, HPRT1, and GAPDH were identified as the most stable RGs. Further, the combined analysis resulted in identification of a panel of RPS9, RPS15, and GAPDH RGs that could act as a useful resource to unravel the accurate transcriptional profile of peripheral blood mononuclear cells of cattle populations adapted to tropical and high-altitude conditions.This study has identified an altitude-specific panel of RGs in PBMCs of cattle populations from hot arid normoxia and cold arid high-altitude hypoxic environments. In the cold arid hypoxia group,


Bifidobacterium animalis subsp. lactis A6 (BAA6) had protective effects against diet-induced obesity. However, whether BAA6 enhances FAO to ameliorate the development of obesity has not been explored. After being fed with high-fat diet (HFD) for 9 weeks, male C57BL/6J mice were fed HFD or BAA6 for 8 weeks. In vitro study was carried out using 3T3-L1 adipocytes to determine the effect of BAA6 culture supernatant (BAA6-CM). Here, we showed that administration of BAA6 to mice fed with HFD decreased body weight gain (by 5.03 g) and significantly up-regulated FAO in epididymal adipose tissues. In parallel, FAO in 3T3-L1 cells was increased after BAA6-CM treatment. Acetate was identified as a constituent of BAA6-CM that showed a similar effect to BAA6-CM. Furthermore, acetate treatment activated the GPR43-PPARα signaling, thereby promoting FAO in 3T3-L1 cells. The levels of acetate were also elevated in serum and feces (by 1.92- and 2.27-fold) of HFD-fed mice following BAA6 administration. The expression levels of GPR43 and PPARα were increased by 55.45% and 69.84% after BAA6 supplement in the epididymal fat of mice. Together, these data reveal that BAA6 promotes FAO of adipose tissues through the GPR43-PPARα signaling, mainly by increasing acetate levels, leading to alleviating the development of obesity.Fatty acid β-oxidation (FAO) is confirmed to be impaired in obesity, especially in adipose tissues. We previously proved that  Obesity is a common health problem for individuals today, which could increase the risk of metabolic diseases . It is cAkkermansia muciniphila (AKK) could increase FAO to ameliorate obesity [Lactobacillus genus, to a high-fat diet (HFD)-fed mice promotes FAO in fat tissues, thereby protecting against obesity [Lactobacillus genus have been shown to be able to reduce fat percentage in obese people [FAO is recognized as an essential factor in obesity ,6. It ha obesity , which h obesity . Several obesity ,19. Furte people . NotablyG protein-coupled receptors (GPRs) play pivotal roles in cell signaling to maintain energy homeostasis ,22. TheyBifidobacterium animalis subsp. lactis A6 (BAA6) is a probiotic which can stay active under gastric juice environment [ironment . Additioironment . HoweverBAA6 (CGMCC No. 9273) was obtained from the feces of a centenarian . BAA6 was incubated in none oxygen conditions, as described in previous study .AKK (JCM 30893) was provided by Japan Collection of Microorganisms RIKEN BioResource Research Center  and incubated in none oxygen condition, as presented previously .9 colony-forming units (CFU)/mL, were centrifugated at 3000× g at 4 °C for 15 min. BAA6-CM was obtained from the cell-free supernatant. Then, the collected BAA6 cells were washed twice and reconstituted by physiological saline. Plate count was used to measure bacterial concentration. The dead BAA6 was prepared at 80 °C for 30 min.BAA6 culture supernatant (BAA6-CM) was obtained as with the previous method . Dead BAn = 6) and HFD (n = 22) for 9 weeks. Composition of diets was shown in n = 6): (1) HFD group; (2) HFD + BAA6 group, gavage of BAA6 at 109 CFU/kg per day for another 8 weeks; (3) HFD + AKK group (positive group), gavage of AKK at 109 CFU/kg per day for another 8 weeks. Mice were orally administrated with 0.9% saline solution in ND and HFD groups. Body weight and food intake were measured every week. When the experiment ended, mice were fasted for 12 h and then anesthetized by CO2. Blood and fat tissues were collected. The fat tissues and serum were preserved at −80 °C for further detection. The experimental processes were approved by the Animal Experimentation Committee of China Agricultural University (AW18080202-1).Male C57BL/6J mice  were provided by Beijing HFK Bioscience Co. Ltd. . They were raised under a stable temperature (23 ± 1 °C) and 12 h light/dark cycle for 1 week. Mice were randomly assigned into two groups: normal diet  with Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum  at 37 °C and incubated to confluence. Subsequently, cells were differentiated, employing DMEM with FBS (10%), insulin (2 µg/mL), 3-isobutyl-1-methylxanthine (0.5 mmol/L), and dexamethasone (0.25 µmol/L) for 3 days. They continued incubation with insulin for another 5 days to fully differentiate into adipocytes [The 3T3-L1 preadipocytes were obtained from American Type Culture Collection . Cells were grown in six-well plates (2 × 10ipocytes .4 cells/well). Subsequently, cells were differentiated into adipocytes. They were treated with BAA6-CM  for 1–5 days and dead BAA6  for 1–4 days, respectively. After treatment, CCK8 reagent was added and incubated for 1 h at 37 °C. Microplate reader  was used to assess cell viability (at 450 nm) [The 3T3-L1 cell viability was detected by cell counting kit-8 assay  according to the manufacturer’s instructions. In brief, 3T3-L1 preadipocytes were grown in 96-well plates (1 × 10 450 nm) .Total cholesterol (TC), TG, and high-density lipoprotein cholesterol (HDL-C) were analyzed by No. 3 Hospital of Beijing University .Body fat was assessed by Body Composition Analysis . Oxygen consumption and respiratory exchange ratio (RER) were measured through mouse Comprehensive Laboratory Animal Monitoring System metabolic cages .TG concentration was detected through commercial TG assay kit  as reported previously .Hematoxylin and eosin assay, in epididymal adipose tissues, was performed as with the previous method . Oil Redβ oxidation assessment was estimated through a Seahorse Bioscience XF96 Analyzer , as described previously . Brieflyg was used to centrifuge the mixture at 4 °C for 10 min. The supernatant was used for further analysis. Agilent 7890B instrument, coupled to a 7000D Triple Quadrupole gas chromatography mass spectrometry system with a DB-FFAP column , was utilized. The carrier gas was helium, and the flow rate was 1.2 mL/min. The oven temperature was held at 90 °C for 1 min, risen to 100 °C at a rate of 25 °C/min, risen to 150 °C at a rate of 20 °C/min, held on 0.6 min, risen to 200 °C at a rate of 25 °C/min, and kept for 0.5 min after running for 3 min. The quantitation of acetate in BAA6-CM, serum, and feces was counted using constructing calibration curves.Acetate was detected by GC-MS/MS. Wuhan MetWare Biotechnology was in charge of extracting the sample and quantifying metabolites, as previously described . Briefly2+) was measured as previously reported [5 cells/well). Then, cells were treated with DMEM, including 5 μmol/L Fura-2/AM  for 45 min at 37 °C. Then, the 3T3-L1 cells were incubated with phosphate-buffered saline containing BSA (0.2%) for 5 min. The ratio of fluorescence intensities was assessed by changing excitation wavelengths of between 340 and 380 nm, with emission at 510 nm. The concentration was calculated as in the previous study [The content of intracellular calcium  was used to transfect cells by Lipofectamine 2000  , accordip < 0.05.All values were shown as mean ± standard deviation (SD). SPSS 21.0  was used to analyze data. The Shapiro–Wilk test was used to assess Gaussian distribution. Equal variance was analyzed by Levene’s test. Statistical comparison was measured by Tukey’s post-test after ANOVA analysis. Differences were considered significant when To investigate the effect of BAA6 in alleviating the development of obesity in mice, the levels of body weight, food intake, fat mass, and serum lipid profiles of mice were measured. There was no remarkable difference in food intake of all mice . In HFD 2) reduced in HFD group in both light and dark phases, while it was increased by BAA6 and AKK treatments , which had positive effects on the host . Acetateacterium . Similaracterium . Dietaryacterium . Furtheracterium . As expeEvidence has pointed out that the PPARα pathway has a primary action in the regulation of FAO . Virtual2+/CAMKKβ pathway [2+ and p-CAMKKβ were increased after treatment with the GPR43 agonist [2+ (by 45.85% and 33.76%) and p-CAMKKβ (by 17.61- and 4.60- fold) in vitro. Besides, inhibition of GPR43 signaling abrogated expression of p-AMPK protein [2+, p-CAMKKβ, p-AMPK, and PPARα. Moreover, acetate and BAA6-CM-induced enhancement of FAO were also destroyed by GPR43 knockdown. These findings indicated that BAA6-generated acetate could activate GPR43 signaling to promote FAO.GPRs played an important role in cell signaling, especially in energy homeostasis, and regulated various adipocyte functions, such as FAO ,23. GPR4 pathway . Several agonist . In line protein . To furtIn conclusion, our study revealed that BAA6 supplementation could increase FAO in adipose tissues and alleviate the development of obesity. Acetate, an important metabolite of BAA6, was a pivotal factor to promote FAO through the GPR43-PPARα signaling. This study indicates that BAA6 may be a potential therapy to ameliorate diet-induced obesity. However, there are some limitations in our study. First, different age and sex of the mice need to be considered. In the future, clinical trials could be conducted to confirm the beneficial effect of BAA6 in obesity and obesity-related disorders.


Then, sheets were submitted to TiO2 photocatalysis at UV radiation at 254 nm (TiO2/UV) with a pH value of 4.5 ± 0.2 and a TiO2 concentration of 1 gL−1. We achieved a complementary effect on the transformation of LDPE films. With the first treatment, ablation was generated, which increased hydrophilicity. With the second treatment, the cavities appeared. The changes in the LDPE sheets’ hydrophobicity were measured using the static contact angle (SCA) technique. The photocatalytic degradation curve at 400 h revealed that the DC-LPP photocatalysis sequential process decreased SCA by 82°. This was achieved by the incorporation of polar groups, which increased hydrophilicity, roughness, and rigidity by 12 and 38%, respectively. These sequential processes could be employed for LDPE and other material biodegradation pretreatment.Low-density polyethylene (LDPE) sheets (3.0 ± 0.1 cm) received sequential treatment, first by the action of direct-current low-pressure plasma (DC-LPP) with a 100% oxygen partial pressure, 3.0 × 10 On an everyday basis, one of the most employed polymers worldwide is low-density polyethylene (LDPE) with industrial, commercial, and domestic uses. Low-density polyethylene transformation and/or biotransformation is difficult and slow because it is a high molecular weight polymer with hydrophobic carbonated chains and low nitrogen content, thus generating a major environmental problem affecting the use of water, resources, soil, and air. During LDPE incineration and its subsequent disposal in sanitary landfills, gaseous emissions, such as COx and volatile organic compounds (VOCs) are generated ,2. There2) [2, plasma action results in double bond formation in LDPE’s polymeric chain, with groups such as vinyl and vinyldenes (R1R2C=CH2) with hydrogen release (H2) or with the participation of hydrogen in a second reaction to form water (H2O) [2 release, where its formation occurs with the participation of methyl groups (CH3-) present in LDPE [3-O-OH) on the material’s surface, due to the ease of breaking O-O bonds that later form free radicals, such as carboxy (C-O•), carboxyl (COO•) [3•), or alkoxy (CH3-O•) [2 atoms from the polymeric chain, various polar groups are formed on the LDPE surface, such as (hydroxyl (OH-), carbonyl (C=O), and carboxyl (C=OOH)) [DC-LLP modifies LDPE surface (ablation) by randomly inserting onto the plastic’s surface carboxyl polar groups (-COOH) and hydroxyl groups (-OH) ,7 that g2) ,7,15 or 2) ,11,16. I(CH3-O•)  on the s(CH3-O•) . Last, d(C=OOH)) ,20,21. T(C=OOH)) .2, are a physical/chemical alternative to modify or transform different types of plastic [2 begins when the oxide semiconductor is electromagnetically irradiated (λ < 400 nm) and a photoelectron excitation takes place, where photon energy (hv) is absorbed by the electron in the TiO2 valence band (VB), which causes energy transitions between the VB and the conduction band (CB) to ensue. This dynamic generates vacancies or holes in the spaces previously occupied by VB electrons, producing an electron–hole pair (e−/h+) [−/h+) can produce a series of oxidation–reduction reactions, where photogenerated e- in CB can form O2•−, and the photogenerated h+ in VB can form OH•. Likewise, the photogenerated holes initiate oxidative stages where adsorbed organic molecules on the material’s surface can be oxidized to become CO2 and water. Relating to plastic photocatalytic oxidation, various authors propose reactive oxygen species (ROS) randomly oxidize LDPE carbon chains, in the amorphous phase as well as the crystal phase [On the other hand, advanced oxidation processes, such as photocatalysis with TiO plastic ,23,24,25 (e−/h+) . Electroal phase ,25,26.2/UV photocatalysis as technologies to transform LDPE sheets.Both DC-LLP and photocatalysis modify the LDPE’s chemical structure and mechanical properties, favoring its degradation, yet a combined action between both will notably increase the material transformation. Currently, this combination has not been studied. Therefore, the objective of this study was to select reaction conditions and/or operating conditions to perform plasma discharge and TiO2 LDPE sheets (pristine LDPE) results before being submitted to experimentation appear in Clean 3 cm2 plasma was used for LDPE pretreatment for consecutive biodeterioration with P. ostreatus. In this work, it was demonstrated that plasma discharge favored the material’s hydrophobicity and subsequent colonization and biodeterioration by the fungus [2, was used for plasma discharge since various authors have described its use for LDPE surface modification [In previous work by Gómez-Méndez et al. , O2 plase fungus . In the fication ,31,32.2 discharge, 41% to 100% Ar plasma discharge, and 44% to the (1:1) mix of gases discharge  met with conditions 1 and 2 previously described in the methodology . Out of ischarge .p = 0.0126)  of (26 ± 5)° and (30 ± 5)°, respectively . For 100% O2 (p = 0.4275) ° treatment followed by the 3.0 × 10−2 treatment mbar, 600 V with (21 ± 3)° (2 (1:1) (p = 0.0001), ° followed by 2.6 × 10−2 mbar, 700 V with an SCA of (30 ± 2)° (p = 0.0139) ° followed by 100% O2 treatment, 3.0 × 10−2 mbar, 600 V (21 ± 3)°. However, no significant differences were observed between them (p = 0.9981). Therefore, the lowest voltage treatment was used (For discharge of 100% Ar ( 0.0126) , the two 0.4275) , the gre21 ± 3)° B. The mi0.0001),  showed t30 ± 2)° C. The co 0.0139)  showed twas used D.2 discharge is that Ar results in chain cleavage and bond rupture on LDPE’s surface, generating Alkyl radicals (C•) by splitting of C-H and C-C bonds. These radicals prefer to interact with adjacent polymer chains rather than with polar groups, forming crosslinks [2 discharge is very reactive and efficient in incorporating the surface polar groups, such as carbonyl (C=O), carboxyl (C=O-OH), and hydroxyls (-OH), which are responsible for decreasing SCA due to hydrophobicity changes [2 discharge. In addition, once the discharge ends, C• on the surface can react with atmospheric O2 [The main difference between Ar And Oosslinks ,34, whil changes ,36. Thusheric O2 , and wheheric O2 ,373•) [2) or ozone (O3), allowing the formation of alkoxy (C-O•). Furthermore, LDPE-carbonated radicals can react with O2 radicals from the environment and form peroxide (C-O-O•) and hydroperoxide (H-O-O•) radicals [Abou Rich et al.  describe3•) . These rradicals .2 plasma discharge at different voltages and time of discharge, SCA decrease was greater in comparison with only O2 plasma treatment. Likewise, they described how the time/voltage ratio required to decrease SCA was lower for O2 in comparison with Ar discharge [2 plasma forms oxidized structures crosslinked with LDPE’s surface, suggesting a more rapid transformation due to the incorporation of high-density functional groups [2 discharge with lower voltages (600 and 700 V) in comparison with Guruvenket et al. [Guruvenket et al.  observedischarge . Ar–O2 pl groups . In the t et al. , as was t et al. .2 plasma discharge treatment is illustrated in 2 plasma treatment. On the other hand, Abou Rich et al. [Generally, LDPE suffers non-permanent physical and chemical modifications on its surface during and after plasma discharge . Plasma-h et al.  describeHydrophobic recovery can occur through different mechanisms: (a) the reorganization of the polymer’s surface when low and high molecular weight molecules, not modified by the discharge, diffuse from the material’s core to the outer layers ; (b) dif2 plasma for 6 min and control are in 2 plasma discharge under the conditions was effective in increasing the material’s hydrophilicity. Hydrophobic recovery after treatment appears in The static contact angle for LDPE treated with O2 3.0 × 10−2 mbar and 600 V) SCA (21 ± 3)°, final weight (4.2 ± 0.1) mg, roughness (10 ± 3) nm, Young´s modulus (41 ± 6) MPa, and yield strength (11 ± 2) MPa. In comparison, pristine LDPE-treated material presented a 76% SCA decrease (p = 0.0139) with a 100% increase in roughness, whereas Young´s modulus and yield strength increased by 21% and 12%, respectively, but not significantly in comparison with pristine LDPE sheets (p = 0.05234).The following results were obtained from LDPE sheets submitted to the best treatment: (100% O−1 (CH2 asymmetric stretching), 1460 cm−1 (CH2 band bending), and 729 cm−1 (CH2 rocking deformation). It is important to highlight that a decrease in these signals appeared after treatments , vinylenes (R1R2C=CH2), and trans vinylenes (-CH2=CH2-), whose absorption bands appear between 890 and 960 cm−1  spectra for pristine LDPE sheets (black line) and post-plasma treatment with 100% argon (red line), argon–oxygen gas mix (brown line), and 100% oxygen (blue line) appear in eatments B–D. For ectively E. Figure process . Rajanda process  describe of mass . The sam of mass  specifie of mass . Shikova of mass  describe960 cm−1 E. In theectively . These r2 (100%) exhibited much more change (2 treatment was (10 ± 3) nm (p = 0.0196). As determined by AFM after discharge, LDPE’s surface roughness changes doubled in comparison with pristine LDPE. Thus, this demonstrates the existence of processes leading to the material’s ablation, which removed the surface layers of the material [2 gas exposure.LDPE’s morphological surface changes after plasma treatment were evidenced by SEM, whereas atomic force microscopy (AFM) revealed roughness results. Pristine LDPE surface without treatment is illustrated in e change D due to 2 0% exhibimaterial . In this2 treatment , demonstrating O2 plasma discharge was more effective in generating ablation processes on LDPE’s surface in comparison with Ar discharge.As shown by scanning electron microscopy (SEM) images, the greatest change was observed for Oreatment D, followt  (8.90 wt%) and oxygen (O2) (89.62 wt%)  D. On the.62 wt%) C, shows  wt%) . Therefore, the main factors and interactions among factors were evaluated. For SCA, the R2 was 0.8223, while for the final weight it was 0.8795. For the viscoelastic properties of Young’s modulus and the yield strength, the R2 were 0.9800 and 0.8100, respectively. There was a high correlation among predicted and observed values with precision values greater than 4.0, confirming results were not the result of experimental noise , with contributions of 45% and 40%, respectively. Additionally, standardized effects with their respective signs were +43.6, −6.5, −6.1, and +0.5; indicating the aforementioned factors could be employed at their low level. pH and TiO2 concentration were also influential factors ; regarding LDPE sheet weight loss, the highest contribution percentage was obtained with pH (60%) (pH and TiOpH (60%) .p < 0.0001). For Young’s modulus, pH and TiO2 concentration and their interaction were significant , followed by AB interaction (p = 0.0007), and TiO2 concentration , with contribution percentages of 70, 20, and 30%, for factor A, B, and AB interaction, respectively. Again, it was observed that a decrease in yield strength was favored by low levels of each factor and even their interaction was employed , which were significant (centages . These remployed .p = 0.0032), final weight (p = 0.0001), Young’s modulus (p < 0.0001), and yield strength (p = 0.0095), were observed (−1 TiO2 and pH 4.5) to perform photocatalytic transformation curves because the lowest contact angle and lowest weight at 300 h of UV light exposure were observed.Mean comparison among treatments demonstrated significant differences for SCA , enabling their oxidation [2 composite. These phenomena permitted more and better contact between the photo-exciter catalyzer and LDPE sheets, bringing about higher oxidation and photoinduced wear by the action of free radicals, and hence, photocatalytic degradation of the material. In the present work, the initial pH was 4.5 ± 0.2 and ended at 7.9 ± 0.2, an increase that could be attributed to (-OH) ion increase in the solution [2 was a negative charge, thus separating from the material due to charge similarity. However, as observed in SEM images, cavity generation could have facilitated TiO2 particle capture, continuing with the photocatalytic degradation, which suggests that the photo-generated e− are transferred to adsorbed O2, whose reduction allows the superoxide anion radical production (O2•−), which in turn favors hydroxyl radical formation (•OH) as dominant species [The treatment that presented the best results in terms of weight and SCA decrease was T1 pH 4.5 ± 0.2 and 1 gL−1 TiO2 . pH is axidation . Therefosolution . After e species , and thi−1 (at 0 h), to (6.73 ± 0.77) mg L−1 at 400 h, which could have been associated with fragmentation and hydrolysis of certain carbonated fractions of the polymer. Furthermore, the final weight decreased to 0.2 mg  and final weight data. Total carbon concentration increased from (4.07 ± 1.04) mg Lo 0.2 mg C.2 was a critical factor since high concentrations can minimize radiation reaching the material’s surface. Too high concentrations can decrease the effect UV can have on the system and photocatalytic degradation velocity, generating in the solution a “screen”-like effect [TiOe effect .2 to form aldehydes and carboxylic acids [2 concentrations in T1 treatment  in a solid phase, and not the other way around, as it is described for most reports in the literature [2 adhered to a surface that can form films, which diminish the photocatalytic activity since the photocatalytic activity is less in a state of suspension [2 particles were in an acid solution. According to Wang et al. [2 solubility [This work innovated in performing a photocatalysis with TiOterature ,47. Thisterature , TiO2 adspension . For treg et al. , acid pHlubility . AlthougAs in this work, others researchers have published that a mixture of the anatase–rutile phases, , present−1 (at 0 h), to (6.73 ± 0.77) mg L−1 at 400 h.While the process of photocatalysis took place, a decrease in SCA was observed, with a final value of (16 ± 3)°, which after 400 h of treatment represented an 82% reduction in comparison with pristine LDPE. For Young’s modulus, an increase was observed after the first 100 h, which gradually decreased to end in (47 ± 5) Mpa, representing a 38% increase A. Yield For SCA photolysis control, values were (61 ± 9)°, a value greater than treatments, a final weight of (4.2 ± 0.3) mg, like T4, and Young’s modulus of (48 ± 4) MPa, and yield strength of (4 ± 1) MPa, comparable with T2. Collectively, these data indicate photolysis by itself is not sufficient to generate a hydrophilic event on LDPE sheets and facilitate their degradation, yet in combination with plasma discharge, they can significantly increase the transformation effect.2 plasma-treated (red line), and sequential plasma followed by photocatalysis treatment  appear in −1, CH2 asymmetric stretching; 2851 cm−1, CH2 symmetric stretching; 1471 cm−1, CH2 bending and deformation; and 719 cm−1, CH2 rocking and deformation, were also present in spectra with plasma treatment (P + F), although with less intensity  (−1 and 1000 cm−1 (−1 (−1 and between 1600–1800 cm−1) (−1 (FTIR spectra for pristine LDPE (black line), Ontensity B–D). Sigrmation) E. Moreov000 cm−1 G were recm−1  F, which m−1)  (LDPE SEM images obtained from the 2−1 TiO2) B,E resul−1 TiO2) B’ and 11−1 TiO2) E’ for th−1 TiO2)  B present−1 TiO2) E, displac matrix , due to  authors ,58,59.When comparing LDPE’s physical properties after the sequential plasma-photocatalysis processes with pristine material, it was observed that treatment generated a complementary effect that maintained hydrophilicity, attaining an 81.6% decrease for SCA. Another important change observed was a 38% increase in Young’s modulus, demonstrating these processes increased the material’s rigidity by restricting atom movement by the incorporation of polar groups, by crosslinking and bond cleavage in LDPE amorphous region, evidenced as cavities on the surface, which increase the crystalline zone proportion .LDPE sheets were purchased from a local market in Bogotá, D.C., Colombia. They were prepared for experiments as described .2, and a mix of Ar–O2 at a 1:1 ratio), voltage (V), and plasma chamber pressure (mbar). Selection of operating conditions and their effect on LDPE are detailed in Within a high vacuum chamber (18 cm × 18 cm) with glass walls and stainless-steel lids and flanges, two flat electrodes, anode, and cathode, were placed with parallel cylinders separated by varying “s” distances to generate a low-temperature self-sustained DC plasma. Argon and oxygen reactive gas mix were rarefied at different concentrations. . Plasma 2, 13 for Ar, and 14 for the Ar–O2 mix (p < 0.05). The analysis was performed by ANOVA employing SAS® software . Following, a new statistical analysis was performed for each block using the same tests previously selected . With the obtained results, subsequent experimental conditions were established [Once treatments that met these conditions were identified, operation settings that would decrease static contact angle (SCA) in LDPE sheets were selected as follows: 5treatments for Or–O2 mix . Differeablished ,43,60.2 plasma discharge for 30, 60, 120, and 240 s of exposure. As control, pristine LDPE sheets were used and results were expressed as final SCA in degrees ± standard deviation.Employing the glow discharge treatment that generated the lowest SCA, a new assay was performed in the wet chamber  to deterAdditionally, following Mortazavi and Nosonovsky , LDPE sh2 Aldrich™) mixture of rutile and anatase (99.5% purity) was employed. To observe morphological characteristics and semi-quantitative composition, scanning electron microscopy images were captured, coupled with energy dispersion spectroscopy (SEM/EDS). A Jeol™  JSM 6490LV scanning electron microscope with 30kV, SEI signal, and 5000–1000× magnification was used. Observations were performed by depositing 0.5 g TiO2 on a glass substrate [2 crystal structure, X-ray diffraction was performed (XRD) using an X-Ray diffractometer Siemens D-5000  equipment operated with a Cu Kα = 1.5418 Å anode [For photocatalysis, titanium dioxide  (4.7 L min−1). Positioned within the reactor and in parallel with the UV lamp, inert clamps were installed to hold previously O2 plasma-irradiated LDPE sheets (14 sheets of 3 cm2) [For the photocatalysis experiments, a 2.5 L photoreactor  with a work effective volume (WEV) of 1.2 L was employed. A 15 W UV lamp (Phillips™) with UV-C (280–100 nm) light emission capacity located within the photo-reactor and protected with a quartz glass jacket was employed. Within the reactor’s lower part, three ports for air injection were located and the air was injected at 10 ftf 3 cm2)   level of 4.5 ± 0.2 and a (+1) level of 9.0 ± 0.2. Factor B was TiO2 in gL−1, with a (−1) level of 1.0 and a (+1) level of 10 gL−1 (TiO2 was added within the reactor and homogenized by air injection with UV lamps off).Condition selection for LDPE sheet photocatalytic transformation was performed through a 22 1 gL−1), T2: +1 − 1 , T3: −1 + 1 , and T4: +1 + 1 , performed in triplicate during 300 h of photocatalysis. After each treatment, LDPE sheets were removed to analyze response variables: SCA, final sheet weight, Young’s modulus, and yield strength [®  and SAS®  [The design generated four codified treatments as follows: T1: −1 − 1  .2 factorial design treatment was employed to perform photocatalytic degradation curves during 400 h; every 100 h, three LDPE sheets were removed to analyze changes associated with final weight (mg), SCA (°), Young’s modulus (Mpa), and yield strength (Mpa). Additionally, SEM and Fourier-transform infrared spectroscopy (FTIR) studies were performed. As controls, LDPE sheets exposed only to photolysis and plasma discharge were employed. Determinations for controls were performed at the beginning and the end of the process. Moreover, to determine total organic carbon (TOC) [2 were sampled, separating the chemical compound by sedimentation. Results obtained at 400 h of treatment were analyzed by ANOVA to determine differences among treatments. Additionally, multiple variable correlations with a confidence interval of 95% were performed using SAS® software  [The selected 2on (TOC) , pH aquestitute) .To obtain LDPE’s surface microscopic information (atomic and/or molecular) in terms of its macroscopic properties, a correlation between static contact angle and tension or surface adhesion was established (hydrophilicity or wettability) ,65 supp.−1 testing speed, 250% maximum strain and 5 N maximum stress.LDPE sheet Young´s modulus  and yielScanning electron microscopy (SEM) ,68 (JeolN is surface height data number and Atomic force microscopy (AFM) ,64 [. All images were formatted to 1280 × 860 pixels and their noise was reduced by a Gaussian filter aided by a Euclidean curve shortening flow process to enhance edges [Computational SEM image processing techniques were used to measure surface cavities produced by sequential treatment  on the material’s surface, making the material hydrophilic, and a 94.89% increase in roughness, due to ablation and an increase in rigidity because of carbonated chain relative movement decrease from crosslinking and oxide reduction events. These oxidations facilitated the photocatalytic activity of a TiO2 solution , generating cavities on LDPE’s surface or expanding the ablation initiated by the plasma discharge, yet retaining high hydrophilicity and material’s rigidity, hence demonstrating a complementary effect of both processes. To the best of our knowledge, this is a pioneering work employing LDPE sheets in a sequential O2 plasma and TiO2/UV photocatalysis in solution, maintaining low SCA. Our data suggest sequential treatment with physicochemical processes in a complementary manner should be evaluated on plastic transformation.LDPE sheets treated with 100% O


R2 = 0.29). LM II (β = 0.39), anxiety (β = −0.29), and education (β = 0.21) were significant predictors of “understanding of alternative treatments” (adjusted R2 = 0.30). Anxiety (β = −0.36) and age (β = −0.22) were significant predictors of “appreciation” (adjusted R2 = 0.18). Age (β = −0.31) and anxiety (β = −0.28) were significant predictors of explained variance in “reasoning” (adjusted R2 = 0.17). Patients with anxiety had lower scores on all five MacCAT-T subscales: “understanding,” without 3.8 [SD = 1.2] vs. with 2.6 [SD = 1.1]; “understanding of alternative treatments,” without 2.9 [SD = 2.2] vs. with 1.3 [SD = 1.8]; “appreciation,” without 2.9 [SD = 1.1] vs. with 1.9 [SD = 1.2]; “reasoning,” without 4.0 [SD = 2.0] vs. with 2.7 [SD = 1.7]; and “expressing a choice,” without 1.9 [SD = 0.4] vs. with 1.5 [SD = 0.6]. Considering the effects of BPSD, cognitive function, and age/education when assessing consent capacity in persons with AD is important. Reducing anxiety may contribute to improved capacity in persons with AD.This study aimed to clarify how behavioral and psychological symptoms of dementia (BPSD) and cognitive function affect the decision-making capacity of persons with Alzheimer's disease (AD) in a real informed consent situation about anti-dementia drug prescriptions. The participants were 76 patients with AD. We used the MacArthur Competence Assessment Tool to assess the capacity for consent to treatment (MacCAT-T). We simultaneously used the Mini-Mental State Examination, Executive Interview, Executive Clock Drawing Task, Logical Memory I of the Wechsler Memory Scale-Revised (LM I), LM II, and Neuropsychiatric Inventory (NPI) to assess cognitive function and psychiatric symptoms. We calculated the correlations between the MacCAT-T scores and the demographic, neuropsychological, and psychiatric variables. Once the univariable correlations were determined, we performed simple linear regression analyses to examine if the regression equations were significant. In the final analyses, we incorporated significant variables into stepwise multiple linear regression analyses to determine the most significant predictors of mental capacity. Age (β = −0.34), anxiety (β = −0.27), and LM I (β = 0.26) were significant predictors of “understanding” (adjusted  At present, as the number of people with dementia encountered in clinical practice rapidly increases, providing treatment to cognitively impaired older patients is becoming problematic. Japan is the most aged country worldwide  as compared to healthy individuals  are also important factors that affect the capacity to consent to treatment  who were older than 65 years were recruited from outpatients at memory clinics from three hospitals in Japan: the Kyoto Prefectural University of Medicine, the North Medical Center Kyoto Prefectural University of Medicine, and the Ujioubaku Hospital. The sample size was calculated using G* Power 3.1  was administered as a caregiver-based clinical instrument that evaluates neuropsychiatric symptoms of dementia  is an observation scale for assessing six domains: memory, orientation, judgment, community affairs, home and hobbies, and personal care. It has the following scoring scale: CDR 0, healthy; CDR 0.5, questionable dementia; CDR 1, mild dementia; CDR 2, moderate dementia; and CDR3, severe dementia  based on the interview recordings. Then, a study session was conducted in which the implementation and evaluation criteria based on the scoring results was discussed until the standard was determined. The next seven participants were interviewed by one of the five psychologists (including YK and KO). Each participant was scored separately based on the recordings. We discussed the implementation and evaluation criteria again and prepared a scoring manual.P < 0.05 was considered significant. To understand the clinical significance of the MacCAT-T scores, Spearman correlation coefficients between the MacCAT-T and the demographic variables, CDR, neuropsychological, and psychiatric variables were calculated. Once the univariable correlations were determined, we performed simple linear regression analyses to examine if the regression equations were significant. In the final analyses, we incorporated significant variables into a stepwise multiple linear regression analyses to determine the most significant predictors of mental capacity. In addition, for NPI items with significant univariate correlations, participants were divided into two groups based on the presence or absence of neuropsychiatric symptoms for each NPI subscale. Then, we compared the scores on each scale of the MacCAT-T scores between the two groups using the Mann–Whitney U-test.Data were analyzed using SPSS 26.0 J for Windows . SD = 2.9) years. The classification of anti-dementia drugs was 55 donepezil (72.37%), 11 galantamine (14.47%), 5 rivastigmine (6.58%), and 5 memantine (6.58%). Demographic and neuropsychological data for each CDR group are shown in Seventy-six participants (22 men and 54 women) participated in the study. The mean education history of the participants was 10.6 , which is almost perfect, indicating that most people with AD could state a clear treatment choice. In contrast, “understanding: benefits and risks ,” “understanding of alternative treatments ,” “reasoning: comparison ,” and “reasoning: generate consequences ” did not meet half of the full score determined for each item.MacArthur Competence Tool for Treatment performances for all participants and for each CDR group are shown in Correlations between MacCAT-T scores and demographic and cognitive variables and NPI symptoms are presented in Simple linear regression analyses indicated that age, education, CDR, MMSE-J, J-EXIT25, J-CLOX1, LM I, and anxiety contributed significantly to “understanding.” Age, education, MMSE-J, J-CLOX1, LM I, and LM II, and anxiety contributed significantly to the “understanding of alternative treatments.” Age and anxiety contributed significantly to “Appreciation” and “Reasoning.” Multiple linear regression analyses using these variables indicated that age, anxiety, and LM I explained the variance in “understanding.” LM II, anxiety, and education explained the variance in “understanding of alternative treatments.” Anxiety and age explained the variance in “appreciation.” Age and anxiety explained the variance in “reasoning.” “Expressing a choice” was excluded from the regression analysis. These results are shown in SD = 1.2] vs. with 2.6 [SD = 1.1], U = 216.0, p = 0.001; “understanding of alternative treatments,” without 2.9 [SD = 2.2] vs. with 1.3 [SD = 1.8], U = 261.5, p = 0.005; “appreciation,” without 2.9 [SD = 1.1] vs. with 1.9 [SD = 1.2], U = 248.0, p = 0.002; “reasoning,” without 4.0 [SD = 2.0] vs. with 2.7 [SD = 1.7], U = 306.5, p = 0.025; and “expressing a choice,” without 1.9 [SD = 0.4] vs. with 1.5 [SD = 0.6], U = 333.5, p = 0.007 . Individuals with anxiety had lower scores on all five MacCAT-T subscales: “understanding,” without 3.8 [ = 0.007 .Our results revealed that “understanding of alternative treatments,” “understanding: benefits and risks,” “reasoning: comparison,” and “reasoning: generate consequences” scores were less than half of that required for each item. Thus, these items were particularly difficult for individuals with AD. The result that participants with dementia have impaired “understanding” and “reasoning” is consistent with a previous study . The patients/participants provided their written informed consent to participate in this study.YK designed the study, collected and analyzed the data, and wrote the paper. JN designed the study, collected the data, and wrote the paper. TM designed the study, collected the data, and assisted with the writing of the paper. KN and KO collected the data and assisted with the writing of the paper. YE, KI, HK, SN, and MM designed the study and assisted with the writing of the paper. TAF supervised the statistical design of the study and assisted with the writing of the paper. All authors revised and approved the final manuscript and are accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.