Exposure to water-pipe smoking, whether flavored or unflavored, has been shown to instigate inflammation and oxidative stress in BALB/c mice. This consequently results in alterations in the expression of inflammatory markers and antioxidant genes. This study aimed to scrutinize the impact of Epigallocatechin gallate (EGCG)—a key active component of green tea—on inflammation and oxidative stress in BALB/c mice exposed to water-pipe smoke. The experimental setup included a control group, a flavored water-pipe smoke (FWP) group, an unflavored water-pipe smoke (UFWP) group, and EGCG-treated flavored and unflavored groups (FWP + EGCG and UFWP + EGCG). Expression levels of IL-6, IL1B, TNF-α, CAT, GPXI, MT−I, MT−II, SOD−I, SOD−II, and SOD-III were evaluated in lung, liver, and kidney tissues. Histopathological changes were also assessed. The findings revealed that the EGCG-treated groups manifested a significant decline in the expression of inflammatory markers and antioxidant genes compared to the FWP and UFWP groups. This insinuates that EGCG holds the capacity to alleviate the damaging effects of water-pipe smoke-induced inflammation and oxidative stress. Moreover, enhancements in histopathological features were observed in the EGCG-treated groups, signifying a protective effect against tissue damage induced by water-pipe smoking. These results underscore the potential of EGCG as a protective agent against the adverse effects of water-pipe smoking. By curbing inflammation and oxidative stress, EGCG may aid in the prevention or mitigation of smoking-associated diseases. Water-pipe smoking, also known as hookah or shisha, has experienced a surge in popularity globally, predominantly among the youth . The onc2O2) and molecular oxygen (O2) [2O2 can be cell-damaging [2O2 into water and molecular oxygen, countering oxidative damage [2O2 reduction is managed by glutathione peroxidase 1 (GPX1) [Cellular defense against free radicals primarily hinges on a set of antioxidant enzymes, like superoxide dismutases (SODs), metallothioneins (MTs), catalase (CAT), and glutathione peroxidases (Gpxs). These enzymes act as a protective shield, preventing cellular damage from reactive oxygen species (ROS) ,6. Out ogen (O2) . Excessidamaging . To neute damage . Without1 (GPX1) . Additio1 (GPX1) . Notably1 (GPX1) .Smoking, a primary contributor to many ailments, induces inflammatory reactions largely by releasing specific proteins or cytokines like TNFα, IL6, and IL1β . ResearcNatural compounds, especially flavonoids, are renowned for their protective attributes against drug-induced adverse effects. Green tea (Camellia sinensis) has been extensively praised for its health-promoting properties, attributed to its abundant antioxidants and anti-inflammatory agents ,17. GreeConsistent scientific evidence underscores EGCG’s protective potential against oxidative damage, especially in essential organs like the heart, brain, and kidneys . The proThis research endeavors to amalgamate molecular analysis with histopathological assessments, delineating the contrasting effects of flavored versus unflavored water-pipe smoking. By examining the influence of Epigallocatechin gallate (EGCG), the prominent active component in green tea, our study investigates its efficacy in moderating inflammation and oxidative stress. We meticulously measured the expression of inflammatory markers and antioxidant genes in the lung, liver, and kidney tissues of BALB/c mice subjected to water-pipe smoke. Our methodology incorporates a strategic experimental design with control and EGCG-treated cohorts, each exposed to both flavored and unflavored smoke variants.The innovation of this study is anchored in its comparative analysis, which scrutinizes the protective efficacy of EGCG against the toxicological impacts of different types of water-pipe smoke. Through an intricate evaluation of gene expression and histopathological changes, we seek to unveil the nuanced protective role of EGCG. This investigation stands to contribute substantially to the field, as it may confirm EGCG’s potential as a mitigative agent against the detrimental effects of water-pipe smoking. The implications are profound; our findings aim to inform and enhance preventive and therapeutic strategies, thereby fostering advancements in public health policies targeted at water-pipe smoking-related complications.From the provided data, it is evident that the gene expression levels for various inflammatory markers  were differentially impacted by the respective treatments.IL-6 levels significantly increased in the flavored water-pipe smoke (FWP) group (3.64 ± 0.12) and the unflavored water-pipe smoke (UFWP) group (3.11 ± 0.18) compared to the control group (1.07 ± 0.09). However, this significant increase is noticeably diminished upon EGCG treatment, as seen in the significantly decreased levels in the FWP + EGCG group (2.08 ± 0.30) and the UFWP + EGCG group (1.36 ± 0.05) .A similar pattern is observed for IL1B levels, with a significant increase in the FWP group (4.28 ± 0.09) and UFWP group (3.27 ± 0.19) relative to the control group (1.07 ± 0.12). Yet again, this significant escalation is mitigated with EGCG treatment, leading to significantly decreased levels in the FWP + EGCG (2.82 ± 0.08) and UFWP + EGCG (1.97 ± 0.24) groups .For TNF-α, the control group demonstrates a level of 1.01 ± 0.02. This level significantly increases in the FWP (6.01 ± 0.28) and UFWP (5.44 ± 0.15) groups. Consistent with the other markers, EGCG treatment results in a significant decrease in inflammation, as reflected in the reduced levels in the FWP + EGCG (4.36 ± 0.05) and UFWP + EGCG (3.58 ± 0.13) groups .In conclusion, the data indicate a potent anti-inflammatory effect of EGCG in BALB/c mice exposed to both flavored and unflavored water-pipe smoke. This is substantiated by the significantly decreased expression of the inflammatory markers IL-6, IL1B, and TNF-α . The EGCThe group treated with EGCG alone showed no marked variation in the expression of Antioxidant Genes CAT, GPXI, MT−I, MT−II, SOD−I, SOD−II, and SOD-III relative to the control group.The expression of the Catalase (CAT) gene significantly increases in groups exposed to flavored water-pipe smoke  and unflavored water-pipe smoke  compared to the control group (1.07 ± 0.12). However, EGCG treatment significantly decreases these elevated levels in both the FWP + EGCG (3.70 ± 0.14) and UFWP + EGCG (2.22 ± 0.17) groups .A similar pattern is evident for Glutathione Peroxidase XI (GPXI). The FWP (6.00 ± 0.14) and UFWP (6.91 ± 0.15) groups showed significant increases compared to the control group (1.02 ± 0.04). However, this significant rise is moderated upon EGCG treatment in the FWP + EGCG (4.17 ± 0.10) and UFWP + EGCG (4.42 ± 0.17) groups .Metallothionein-I (MT−I) gene expression follows the same trend, with significant increases in the FWP (6.40 ± 0.14) and UFWP (5.25 ± 0.21) groups compared to the control group (1.04 ± 0.09). EGCG treatment significantly lowers this spike in the FWP + EGCG (2.80 ± 0.14) and UFWP + EGCG (3.90 ± 0.13) groups .For the Metallothionein-II (MT−II) gene, expression significantly increased in the FWP (3.90 ± 0.14) and UFWP (2.53 ± 0.11) groups compared to the control group (1.07 ± 0.09). Notably, EGCG treatment significantly lowers this spike in the FWP + EGCG (1.09 ± 0.16 and UFWP + EGCG (−1.27 ± 0.10) groups .Superoxide Dismutase I (SOD−I), essential for reactive oxygen species detoxification, experiences a significant escalation in the FWP (6.70 ± 0.28) and UFWP (8.33 ± 0.17) groups compared to the control group (1.07 ± 0.12). EGCG treatment significantly decreases this rise in the FWP + EGCG (5.32 ± 0.11) and UFWP + EGCG (3.75 ± 0.07) groups .In contrast, Superoxide Dismutase II (SOD−II) reveals a milder but still significant increase in the FWP (2.00 ± 0.14) and UFWP (2.50 ± 0.14) groups. Interestingly, EGCG treatment significantly lowers this spike in the FWP + EGCG (1.23 ± 0.04) and UFWP + EGCG (−0.48 ± 0.04) groups.Lastly, for Superoxide Dismutase III (SOD−III), EGCG treatment significantly decreases the enhanced expression induced by smoke, demonstrated by the significantly lower levels in the FWP + EGCG (4.35 ± 0.14) and UFWP + EGCG (3.05 ± 0.21) groups. This decline underscores the potency of EGCG in counteracting the oxidative stress caused by both flavored (8.91 ± 0.15) and unflavored (6.76 ± 0.08) water-pipe smoke .The group treated with EGCG alone showed no marked variation in the expression of Antioxidant Genes CAT, GPXI, MT−I, MT−II, SOD−I, SOD−II, and SOD−III relative to the control group.For the Catalase (CAT) gene, the groups exposed to FWP (4.22 ± 0.17) and UFWP (3.70 ± 0.14) significantly increased in expression compared to the control group (1.07 ± 0.12). However, the elevated levels were significantly mitigated with EGCG treatment in both FWP + EGCG (1.86 ± 0.10) and UFWP + EGCG (1.57 ± 0.09) groups .A similar trend was observed for the Glutathione Peroxidase XI (GPXI) gene. Expression significantly increased in the FWP (2.94 ± 0.06) and UFWP (3.86 ± 0.14) groups compared to the control group (1.02 ± 0.04). Upon EGCG treatment, these heightened levels were significantly reduced in the FWP + EGCG (1.64 ± 0.05) and UFWP + EGCG (2.17 ± 0.10) groups .For the Metallothionein-I (MT−I) gene, expression significantly increased in the FWP (2.49 ± 0.12) and UFWP (1.83 ± 0.06) groups compared to the control group (1.04 ± 0.09). Intriguingly, the EGCG-treated groups, FWP + EGCG (−1.08 ± 0.11) and UFWP + EGCG (−1.61 ± 0.08) showed expression levels significantly lower than the control group, suggesting that EGCG might suppress this gene’s expression upon smoke exposure .The Metallothionein-II (MT−II) gene followed a similar pattern. Expression significantly increased in the FWP (3.22 ± 0.17) and UFWP (5.48 ± 0.11) groups compared to the control group (1.07 ± 0.09). With EGCG treatment, the levels were reduced but remained higher than the control in the FWP + EGCG (2.16 ± 0.08) group and significantly decreased in the UFWP + EGCG (1.29 ± 0.08) group .For Superoxide Dismutase I (SOD−I), the expression level significantly increased in the FWP (2.79 ± 0.11) and UFWP (1.90 ± 0.13) groups compared to the control group (1.07 ± 0.12). Surprisingly, the EGCG-treated groups, FWP + EGCG (−2.10 ± 0.14) and UFWP + EGCG (−1.39 ± 0.06) exhibited levels significantly lower than the control group .The Superoxide Dismutase II (SOD−II) gene exhibited a minor but still significant increase in the FWP (1.26 ± 0.08) and UFWP (2.10 ± 0.16) groups compared to the control group (1.01 ± 0.02). Surprisingly, the EGCG-treated groups, FWP + EGCG (−1.10 ± 0.14) and UFWP + EGCG (−0.73 ± 0.04) exhibited levels significantly lower than the control group .Lastly, for Superoxide Dismutase III (SOD−III), expression was significantly higher in the FWP (3.25 ± 0.21) and UFWP (2.88 ± 0.11) groups than in the control group (1.03 ± 0.04). After EGCG treatment, the expression significantly decreased but remained above control levels in FWP + EGCG (1.27 ± 0.04) and UFWP + EGCG (0.56 ± 0.03) groups .The group treated with EGCG alone showed no marked variation in the expression of Antioxidant Genes CAT, GPXI, MT−I, MT−II, SOD−I, SOD−II, and SOD-III relative to the control group.The expression levels of the Catalase (CAT) gene in lung tissue significantly increased in the groups exposed to flavored (23.53 ± 0.04) and unflavored (18.66 ± 0.13) water-pipe smoke compared to the control group (1.07 ± 0.12). Notably, EGCG treatment significantly reduced these elevated levels in both the FWP + EGCG (13.21 ± 0.15) and UFWP + EGCG (9.11 ± 0.30) groups .A similar trend is observed in Glutathione Peroxidase XI (GPXI) expression, with significantly heightened levels in the FWP (18.50 ± 0.06) and UFWP (16.33 ± 0.18) groups relative to the control group (1.02 ± 0.04). This significant increase is notably attenuated by EGCG treatment, as seen in the FWP + EGCG (11.71 ± 0.20) and UFWP + EGCG (6.06 ± 0.36) groups .The Metallothionein-I (MT−I) gene follows this pattern, showing significantly elevated expression in the FWP (9.21 ± 0.30) and UFWP (11.17 ± 0.24) groups compared to the control group (1.04 ± 0.09). This significant increase is notably mitigated by EGCG treatment, with expression in the FWP + EGCG (3.32 ± 0.17) and UFWP + EGCG (1.84 ± 0.08) groups dropping considerably .Metallothionein-II (MT−II) expression also significantly increased in the FWP (12.91 ± 0.08) and UFWP (8.62 ± 0.08) groups from the control group level of 1.07 ± 0.09. The impact of EGCG treatment is significant, markedly reducing expression to 4.72 ± 0.18 (FWP + EGCG) and 2.88 ± 0.11 (UFWP + EGCG) .For Superoxide Dismutase I (SOD−I), we observe a significant upswing in expression levels in the FWP (8.36 ± 0.16) and UFWP (6.24 ± 0.16) groups relative to the control group (1.07 ± 0.12). Once again, EGCG treatment significantly mitigates this increase, lowering levels to 3.01 ± 0.16 (FWP + EGCG) and 1.86 ± 0.08 (UFWP + EGCG) .Superoxide Dismutase II (SOD−II) demonstrates a similar significant increase in the FWP (12.72 ± 0.12) and UFWP (13.82 ± 0.17) groups from a control level of 1.01 ± 0.02. With EGCG treatment, these expression levels are significantly reduced, as evident in the FWP + EGCG (6.12 ± 0.31) and UFWP + EGCG (4.79 ± 0.12) groups .Lastly, Superoxide Dismutase III (SOD−III) experiences a moderate but still significant increase in expression levels in the FWP (4.80 ± 0.01) and UFWP (3.67 ± 0.16) groups from the control level of 1.03 ± 0.04. Following EGCG treatment, these levels significantly decreased to 2.38 ± 0.11 (FWP + EGCG) and 1.32 ± 0.16 (UFWP + EGCG) .Exposure to both flavored and unflavored water-pipe smoke instigated observable inflammatory alterations in the liver, lung, and kidney tissues of BALB/c mice. These modifications included interstitial inflammation characterized by lymphocyte and plasma cell infiltration in the lungs C,D, portA distinct variation in the degree of inflammation was observed between the flavored and unflavored water-pipe smoke exposure groups. Specifically, the flavored water-pipe smoke exposure resulted in more pronounced inflammation in the liver, lung, and kidney tissues D when coThe present investigation substantiates the evolving scientific literature underscoring the therapeutic potency of Epigallocatechin gallate (EGCG) in mitigating both the oxidative stress and inflammation triggered by inhaling both flavored and unflavored water-pipe smoke. This examination employed BALB/c mouse models to emulate the biological interactions of humans with such smoke. This claim draws on the foundational research that illustrates the powerful anti-inflammatory and antioxidant properties of EGCG, thus spotlighting its potential utility in managing smoke-induced diseases ,27.An extensive review of the existing literature indicates diverse no-observed adverse-effect levels (NOAELs) for EGCG in both rats and mice. Specifically, two 90-day oral toxicity studies of green tea extract (GTE) identified NOAELs of 500 mg/kg ,29. FurtAlthough these results suggest a reasonable safety margin for EGCG in animal models, it is acknowledged that the NOAELs and the biological effects can be influenced by multiple factors. These include extraction techniques, source plant material, and accompanying polyphenols. These complexities emphasize the challenges in direct extrapolation to human contexts ,35,36.To further contextualize our study, the chosen dose of 50 mg/kg/day has been used in previous research to explore the effects of EGCG on different diseases in mice ,36,37,38Through rigorous evaluation during this study, a pronounced surge in the expression of inflammatory markers, specifically Interleukin-6 (IL-6), Interleukin 1 beta (IL1B), and Tumor Necrosis Factor-alpha (TNF-α), was observed. The pathological role of these markers, pivotal in sparking numerous inflammatory diseases, has been thoroughly established in contemporary medical research . InteresContrarily, upon introducing EGCG to the experimental setup, a remarkable decrease in the expression of these inflammatory markers was noted, thereby validating the existing literature highlighting the capability of polyphenolic compounds like EGCG to efficiently suppress inflammatory responses ,41.Our investigation further delved into the impact of EGCG on the expression of antioxidant genes within the kidney, liver, and lung tissues of mice subjected to both flavored and unflavored water-pipe smoke. In line with previous research, a substantial increase in the expression of antioxidant genes, including Catalase (CAT), Glutathione Peroxidase XI (GPXI), Metallothionein-I (MT−I), Metallothionein-II (MT−II), Superoxide Dismutase I (SOD−I), Superoxide Dismutase II (SOD−II), and Superoxide Dismutase III (SOD−III), was observed. This trend suggests that smoke inhalation triggers oxidative stress, which in turn leads to an upregulated antioxidant gene expression ,42.Interestingly, the administration of EGCG was associated with a discernable decrease in the expression of these antioxidant genes across all examined tissues, hinting at a potential reduction in oxidative stress. This observation aligns with a plethora of research emphasizing the antioxidant capabilities of EGCG ,17,19.Flavored and unflavored water-pipe smoking was observed to upregulate the expression levels of inflammatory and antioxidant genes—including IL-6, IL1B, TNF-α, CAT, GPXI, MT−I, MT−II, SOD−I, SOD−II, and SOD−III—in the lung, liver, and kidney tissues of the animal model. This upregulation, indicative of the body’s defensive response to oxidative stress caused by exposure to harmful substances in water-pipe smoke, emphasizes the body’s efforts to counteract potential cellular damage through a surge in antioxidant and inflammatory genes. However, when treated with EGCG, a prominent polyphenol in green tea recognized for its robust antioxidant capabilities, there was a pronounced decrease in these gene expressions. This suggests that EGCG may reduce the body’s urgency to amplify its antioxidants when combating oxidative stress. Such findings align well with the study by Al-Awaida et al., [Moreover, it is crucial to consider the inherent biochemical properties of EGCG as an electron donor and a proficient scavenger of reactive oxygen species (ROS) like superoxide, peroxyl, hydroxyl, and peroxynitrite radicals. These properties lend further credence to EGCG’s prospective usefulness as an antioxidant therapeutic in conditions characterized by oxidative stress and inflammation. The radical scavenging qualities of EGCG are instrumental to its antioxidant capacity and seem to be significantly implicated in the protective effects observed in our study .This distinct characteristic of EGCG could also play a critical role in tempering the inflammatory response. By scavenging reactive oxygen species, EGCG could potentially stunt the propagation of the inflammatory cascade incited by oxidative stress, hence mitigating the overexpression of pro-inflammatory markers . These aIn aggregate, our findings provide substantial support for the therapeutic promise of EGCG in the context of smoke-associated diseases. Nonetheless, it is crucial to emphasize that EGCG is not a panacea for the adverse effects of water-pipe smoking. The primary public health goal should remain focused on reducing or quitting tobacco use. Future research endeavors should aim to further unravel the underlying mechanisms of EGCG’s protective effects, determine optimal dosing regimens, and investigate the long-term impacts of its consumption. Such concerted efforts could lay the groundwork for more effective strategies to offset the adverse health effects associated with water-pipe smoke exposure.In this investigation, we also scrutinized the protective efficacy of EGCG against the injurious effects of water-pipe smoke exposure in lung, kidney, and liver tissues. Our findings are in harmony with preceding research into EGCG’s antioxidant and anti-inflammatory properties and provide compelling evidence of its potential to alleviate the morphological alterations instigated by water-pipe smoke exposure ,17,44.Mice exposed to clean air in our control group displayed normal tissue morphology in the lung, kidney, and liver, reinforcing previous research findings . HoweverNotably, smoke-exposed mice that received EGCG treatment demonstrated protected tissue morphology. These observations further endorse prior research positing that the polyphenolic compounds in EGCG confer potent antioxidant and anti-inflammatory effects capable of reducing cellular damage from toxins ,47.Despite these encouraging findings, it is important to underscore that EGCG should not be considered a universal remedy for the deleterious effects of water-pipe smoking. Further research is needed to fully understand how EGCG exerts these protective effects, along with studies on the optimal dosage, duration, and potential long-term impacts of EGCG consumption .Public health initiatives should continue to highlight the detrimental health effects of water-pipe smoking and the importance of reducing exposure. However, our findings underscore the potential of EGCG as a complementary protective measure against the harmful effects of exposure to such substances.The study utilized 48 male BALB/c mice, aged 9–12 weeks and weighing 25–30 g.Control Group: Subjected to ambient air without any external treatment.Flavored Smoke Group: Exposed to flavored water-pipe smoke.Unflavored Smoke Group: Exposed to unflavored water-pipe smoke.Flavored Smoke + EGCG Group: Exposed to flavored smoke and administered Epigallocatechin gallate  at a dosage of 50 mg/kg ,37,38.Unflavored Smoke + EGCG Group: Exposed to unflavored smoke and administered EGCG at a dosage of 50 mg/kg.Ambient Air + EGCG Group: Subjected to ambient air and given EGCG at a dosage of 50 mg/kg.We divided the mice into six distinct groups, each comprising eight mice. For 90 days, each group underwent unique daily treatments as follows:For groups receiving EGCG, the compound was administered orally via gavage precisely one hour before daily smoke exposure, in accordance with the protocol referenced in .Mice were accommodated in a controlled environment with a temperature of 22 ± 1 °C, relative humidity between 65–70%, and a 12-h light/dark cycle . For samA digital smoke delivery system was utilized, featuring an 8 mm thick Plexiglas inhalation chamber measuring 30 cm × 22.5 cm × 10.5 cm. The chamber capacity accommodated eight BALB/c mice simultaneously . The devIn the first 30 s, mice were exposed to either flavored or unflavored tobacco smoke.In the subsequent 30 s, fresh air was introduced into the chamber to purge any remaining smoke.The final 30 s allowed the mice a period for respiration, during which they inhaled the introduced fresh air.The 90-s smoking procedure was divided into three distinct phases:This entire 90-s cycle was repeated 20 times back-to-back during each daily session, which was conducted seven days a week. Consequently, the total exposure time amounted to 30 min per group, each day. Following this regimen, tissues  were collected for genetic analysis and histopathological examination.For this, 50 mg of the frozen tissue was mixed with 2 mL of lysis buffer using the Promega RNA extraction Mini Kit .Total RNA was extracted from tissues using Promega RNA extraction Mini Kit reagent , accordiOne microgram of the RNA samples was reversely transcribed into cDNA using reverse transcription with Improm-II Reverse Transcriptase Promega , following the manufacturer’s protocol and instructions.The CFX96™ Thermal Cycler Real-Time System  was utilized using SYBR Green Master Mix Promega . Each 20 µL reaction mixture contained 10 μL of SYBR Green Master Mix, 0.5 μL of forward primer (5 μM), 0.5 μL of reverse primers (5 μM), 1 μL of cDNA template, and 8 μL of RNase-free water.^-ΔΔCt method [Expression levels of specific genes in different tissue types were evaluated, along with the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase . The reactions were performed in triplicate for consistency . The relt method . SpecifiAfter the 90-day protocol, tissue samples from the lungs, liver, and kidneys were fixed in 10% buffered formaldehyde for a week.Tissue sections of 5 μm were stained with hematoxylin and eosin (H&E). A Leica microscope with a 16-bit camera documented the samples .All statistical computations were executed using GraphPad Prism version 8.0 .The data derived from the study were assessed for normal distribution across all experimental groups, thereby allowing for the use of parametric tests in data comparison.Data were amassed from three distinct trials, each comprising five replicates. Results are presented as mean ± standard deviation (SD).For multiple group comparisons, a one-way analysis of variance (ANOVA) was conducted. Tukey’s post hoc test was employed following ANOVA for multiple comparisons. Student’s t-test was utilized for pairwise comparisons.p-value less than 0.05 was deemed indicative of statistical significance.A Our study provides strong evidence demonstrating the mitigating effects of Epigallocatechin gallate (EGCG), a prominent compound found in green tea, on the inflammatory and oxidative stress responses caused by both flavored and unflavored water-pipe smoke exposure in BALB/c mice. Histopathological assessments further substantiated EGCG’s beneficial impact, revealing its capacity to protect against smoke-induced tissue damage in the liver, lungs, and kidneys. However, the exact molecular mechanisms by which EGCG exerts these protective effects remain unclear and warrant further investigation. Employing techniques such as RNA-sequencing and proteomics in future studies may provide more comprehensive insights into these pathways.


The refractoriness of tumor cells to apoptosis represents the main mechanism of resistance to chemotherapy. Smac/DIABLO mimetics proved to be effective in overcoming cancer-acquired resistance to apoptosis as a consequence of overexpression of the anti-apoptotic proteins XIAP, cIAP1, and cIAP2. In this work, we describe a dual-targeting peptide capable of selectively activating apoptosis in cancer cells. The complex consists of a fluorescent periodic mesoporous organosilica nanoparticle that carries the short sequences of Smac/DIABLO bound to the αvβ3–integrin ligand. The dual-targeting peptide @PMO shows significantly higher toxicity in αvβ3-positive HeLa cells with respect to αvβ3-negative Ht29 cells. @PMO exhibited synergistic effects in combination with oxaliplatin in a panel of αvβ3-positive cancer cells, while its toxicity is overcome by XIAP overexpression or integrin β3 silencing. The successful uptake of the molecule by αvβ3-positive cells makes @PMO promising for the re-sensitization to apoptosis of many cancer types. Tumor cells can escape apoptosis, which is the main mechanism of resistance to chemotherapy . Apoptosin vivo  by downregulating their expression when apoptotic stimuli are present; b) by upregulating natural proapoptotic proteins; c) by directly inhibiting the action of IAPs on caspases . All of in vivo .The target therapy approach allows overcoming the need for selectivity toward cancer cells by conventional chemotherapeutic drugs. Conjugation with peptide ligands capable of recognizing receptors overexpressed on tumor cells ensures selectivity and improves cellular uptake . In thisUse of nanoparticles (NPs) as nanocarriers is an emerging approach. The potential of nanocarriers is that they improve drug efficacy, solubility, and stability, thereby reducing drug toxicity . Nanopar3Si-R-Si-(OEt)3, where the organic group -R- is localized within the channel walls (Periodic mesoporous organosilicas (PMOs) are versatile nanoparticles. They were first synthesized in 1999 by Ozin, Stein, and Inagaki. PMOs are synthesized by sol–gel self-assembly processes of silsesquioxane of the type (EtO)el walls . They exel walls ; and intel walls .In this work, we connected the cyclic RGD with the short sequence Alanine-Valine-Proline-Isoleucine (AVPI) of Smac/DIABLO, which is responsible for the cytotoxic activity, via a lysine bridge and coupled it to nanoparticles. The system showed high uptake, attributed to the presence of the fibronectin competitor that binds the integrin αvβ3, allowing it to selectively interact and pass through the cell membrane of cancer cells . At the The chemical reagents and the solvents were purchased from Sigma Aldrich  and used without further purification. The reactions were carried out in an unmodified domestic microwave oven .N,N′-dicyclohexylcarbodiimide (DCC) (3.3 mmol) were added as coupling reagents. The mixture was stirred for 3 h. The resin was washed three times with the following solvents: DCM (5 mL), MeOH (5 mL), and DMF (5 mL). The peptide bond formation was evaluated using the Kaiser test. The Fmoc protective group was removed by a solution of piperidine (20%) in DMF (5 mL) for 45 min at room temperature. After filtering and washing with DCM, MeOH, and DMF, the complete Fmoc deprotection was evaluated using the Kaiser test. The steps of coupling and deprotection were repeated for each amino acid.The 2-chlorotrityl chloride resin (Cl-Trt-resin) preloaded with amino acid (1.1 mmol) was swollen in DCM (5 mL). The 9-fluorenylmethoxycarbonyl (Fmoc) amino acid (3.3 mmol) was dissolved in DMF (5 mL), and 1-hydroxybenzotriazole (HOBt) (3.3 mmol) and SS22 was treated with a solution of trifluoroacetic acid (TFA) and triisopropylsilane/water/phenol (TIPS/H2O/PhOH (9:0.5:0.5 and v/v 20 mL)) and left to be stirred for 2.5 h at room temperature. After that, the suspension was filtered and washed with ether (Et2O)/TFA (95:5 and v/v 10 mL), Et2O, and methanol. The combined filtrates were evaporated under vacuum. The residual oil was treated with ice-cold ether to yield the product SS24  as a white precipitate. The solid was recovered by centrifugation. Purities were determined to be >95% by analytical RP HPLC and elemental analysis . The identity of the compounds was confirmed by 1H NMR, 2D NMR, and ESI MS analyses.The half peptidyl resin 1H NMR  δ: 0.86 , 0.88 , 0.90 , 1.20 , 1.35–1.40 , 1.45–1.53 , 1.70–1.74 , 2.71–2.76 , 3.30–3.33 , 3.76 , 4.11 , 4.27–4.30 , 4.40–4.50 , 7.34 , 7.42 , 7.51 , 7.86 , 7.90–8.10 , 8.14 . ES-MS m/z [M+2H]2+/2 and [M+H]+found: 403.7 and 806.2, calculated: 806.4 calculated for C42H59N7O9. Elementary analysis for C42H59N7O9 calculated: C 62.51, H 7.49, N 12.15, found: C 62.32, H 7.62, N 12.02.Fmoc Ala-Val-Pro-Ile-Gly-Lys(Boc)-OH and Arg(Pbf)-Gly-Asp(OtBu)-phe-Lys(Cbz)OH(SS23 and SS16): the peptidyl resin was treated with a solution of trifluoroethanol/acetic acid/dichlomethane (TFE/CH3COOH/DCM (1:1:4 and v/v 20 mL)) and allowed to stir for 90 min at room temperature. After that, the suspension was filtered and washed with Et2O, DCM, and methanol. The solvents were collected and evaporated under vacuum. The residual oil was treated with ice-cold ether to yield products SS23  and SS16  as white precipitates. The solids were recovered by centrifugation.Peptide cleavage 1H NMR FmocAla-Val-Pro-Gly-Lys(Boc)OH (SS23)  δ: 0.86 , 0.88  0.90 , 1.20 , 1.35–1.40 , 1.43 , 1.45–1.53 , 1.70–1.74 , 2.71–2.76 , 3.30–3.33 , 3.76 , 4.11 , 4.27–4.30 , 4.40–4.50 , 5.60–5.65 , 7.34 , 7.42 , 7.51 , 7.86 , 7.90–8.10 , 8.14 . ES-MS m/z found: 805.5 [M-100] and 928.5 [M+Na], calculated: 906.5 calculated for C47H67N7O11. Elementary analysis for C47H67N7O11 calculated: C 62.30, H 7.45, N 10.82, found: C 62.49, H 7.32, N 10.94.SS16 (0.6 mmol) was solubilized in DMF (5 mL) and added drop by drop using a syringe pump to a mixture formed by diphenyl phosphoryl azide (DPPA) (1.8 mmol) and NaHCO3 (12 mmol) in DMF (10 mL) for 12 h at room temperature. After the reaction time, the solvent was removed under high vacuum. The solid was dissolved in 30 mL of ethyl acetate and washed with a solution of HCl 1 M (5 mL), a saturated solution of NaHCO3 (5 mL), and brine (5 mL). The solvent was removed under vacuum, and the crude product was purified by column chromatography ) to yield the final product SS21 . ES-MS m/z found for C52H71N9O12S 1046.2 [M+H]+ calculated: 1046.5.The linear peptide SS21 was solubilized in iPrOH in a flask, and HCOONH4 (4 equiv.) and Pd/C 10% (5% in weight with respect to the peptide) were added. The solution was irradiated in a microwave at 600 W for three cycles of 30 s each. The reaction was monitored by TLC under the disappearance of the starting material.Peptide SS23 (0.14 mmol) was dissolved in DCM/DMF (3:1), HOBt (0.16 mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) (0.16 mmol) were added to it. After 10 min, the cyclic peptide SS25 (0.15 mmol) and N,N-diisopropylethylamine (DIPEA) were added (0.30 mmol) and stirred at room temperature. After 3 h, the solvent was removed under vacuum. The residual oil was solubilized with 30 mL of ethyl acetate and washed with HCl 1 M (5 mL) and a saturated solution of NaHCO3 (5 mL). The solvent was evaporated under vacuum, and the crude product was purified by column chromatography ) to yield the final product SS30  as colorless oil. ES-MS m/z found for C91H130N16O20S 1698.9 [M-100] and 1821.9 [M+Na]+calculated: 1799.9.The Fmoc linear peptide SS25 (0.50 mmol) and SS30 (0.087 mmol) were dissolved in a mixture formed by TFA, triisopropylsilane, and water (90:5:5 v/v/v and 20 mL). The final solution was stirred for 1 h. After removing the solvent, the products were triturated in ether and recovered by centrifugation to yield the final products SS27 and SS33 as TFA salt and used without further purification. Purities were determined to be >95% by analytical RP-HPLC and elemental analysis . The identity of the compounds was confirmed by 1H NMR, 2D gCOSY, and ESI MS analyses.Peptides 1H NMR cyclic[RGDfK]  δ: 0.70–0.90 , 1.42–1.65 , 1.70–1.72 , 2.38 , 2.67–2.70 , 2.79–2.81 , 2.83–2.85 , 3.07–3.10 , 3.25 , 3.94–3.96 , 4.04 , 4.13–4.18 , 4.43–4.45 m, 1H, PheHα), 4.63–4.65 , 7.15–7.26 , 7.34 , 7.58 , 7.63 , 7.67–7.70 , and 8.06–8.10 . 280 mg, 0.46 mmol, 92%. ES-MS m/z found 604.5 [M+1], calculated: 604.3 calculated for C27H41N9O7. Elementary analysis for C27H41N9O7 calculated: C 53.72, H 6.85, and N 20.88, found: C 53.89, H 6.74, and N 21.10.1H NMR  δ: 0.75–0.87 , 0.88–0.95 , 1.12 , 1.33–1.39 , 1.46–1.53 , 1.54–1.64 , 1.90–2.02 , 2.66–2.70 , 2.81–2.91 , 2.92–3.03 , 3.24–3.30 , 3.43–3.46 , 3.53–3.62 , 3.64–3.75 , 4.00–4.05 , 4.07–4.23 , 4.30–4.40 , 4.41–4.47 , 6.71–6.76 , 6.93–7.03 , 7.39–7.54 , 7.15–7.27 , 7.81–7.92 , 7.96 , and 8.09–8.17 . ES-MS m/z and found: 696.9 [M+2H]2+/2 and 1414.75 [M+Na]+, calculated: 1392.75 calculated for C69H98N16O15. Elementary analysis for C69H98N16O15 calculated: C 59.51, H 7.17, and N 16.09, found: C 59.32, H 7.05, and N 16.26.2O/ethanol (90 mL and 33 mL) and a 32 wt% ammonia (1.2 mL) solution. The reaction mixture was stirred at room temperature for 1 h before the addition of 1,2-bis(trimethoxysilyl)ethane (BTME) (0.005 mol). The aforementioned reaction mixture was continuously stirred for an additional time of 48 h at room temperature. The CTAB mesoporous template was removed by stirring the sample in ethanol (50 mL) with a 32 wt% aqueous solution of HCl (1.5 gr) at 50°C for 6 h. The resulting solid was recovered by centrifugation , washed with ethanol three times, and dried at 60°C in vacuum (Hexadecyltrimethylammonium bromide (CTAB) (1.33 mol) was dissolved in Hn vacuum .IR-780 (10 mg) was dissolved in dimethylsulfoxide (DMSO) (5 mL) and sonicated to complete dissolution of the dye. Then, the mixture was added drop by drop to 10 mL of water containing PMO (100 mg). The solution was stirred, in the dark, overnight and at room temperature. After this, the PMO was washed thrice with water, and the nanoparticles were recovered by centrifugation and left to dry at room temperature .IR-780PMO-OH, we coated them with 3-isocyanatopropyltrymethoxy silane (ICPTES) and then with peptides. For ICPTES coating, IR-780PMO-OH (100 mg) was suspended in toluene (5 mL) and triethylamine  and sonicated for 15 min. After that, ICPTES was added (1.6 mL). The reaction mixture was stirred overnight at room temperature. The nanoparticles were recovered by centrifugation and washed thrice with toluene.To functionalize the Peptides (20 mg) were dissolved in DMSO (1 mL) and TEA (120 μL) was added. The reaction mixtures were left sonicated for 10 min and then added to the PMO (20 mg). After stirring overnight at room temperature, the nanoparticles were washed twice with water and ethanol and recovered by centrifugation. PMO was left to dry under vacuum at 60°C.Fmoc-peptides@PMO were dissolved in a solution of 20% piperidine in DMSO. The reaction mixtures were sonicated for 10 min and stirred for 40 min. After that, the nanoparticles were washed thrice with ethanol and recovered by centrifugation. The step of deprotection was repeated for 25 min. The PMOs were recovered by centrifugation, then washed thrice with ethanol, and dried at room temperature.2O/CH3CN/0.1% TFA to 2:8 H2O/CH3CN/0.1% TFA in 20 min, flow rate 0.5 mL min−1. The exact mass was determined using a QTOF apparatus. The products were purified by flash chromatography, which was performed with silica gel (particle size: 40–60 µm) obtained from Merck. The purity was determined to be ≥ 95% by analytical RP HPLC, exact mass, and elemental analysis. Elemental analysis was performed with an EACE 1110 CHNS/O analyzer . NMR spectra were recorded by Bruker at 400 MHz in 8:2 DMSO–d6/H2O (water presaturation) and 13C NMR spectra at 100 MHz. Two-dimensional methods  were used to support and confirm the assignment. The chemical shift δ is reported in ppm relative to the residual proton signal of the solvent: DMSO-d6 2.50 ppm (1H), 39.52 ppm (13C).Analytical RP HPLC was carried out on a C18 column ; DAD 210/246 nm, mobile phase from 9:1 HThe nanoparticles were analyzed through dynamic light scattering (DLS) measurements performed with a Zetasizer Nano ZS , He–Ne laser 633 nm, Max 4 mW, using polystyrene cuvettes . PMOs were dispersed in distilled water and sonicated for 20 min. The measurements were conducted at 20°C. Scanning electron microscopy, specifically the JSM-7610 FPLUS model by JEOL in Udine, Italy, was used to acquire high-resolution images of PMO nanoparticles. All images were collected using different magnifications.ex 240 nm and λem 282 nm) (ex 397 nm and λem 280 nm)   and Fmoc 280 nm)  and plot-1 using a Cary 630 spectrometer by Agilent. Background and sample spectra were acquired before each acquisition at a spectral resolution of 1 cm-1, with 128 scans per measurement.IR spectra were measured within the wavenumber range of 4,000 to 650 cm−1), and streptomycin (100 μg ml−1) . MCF10A was cultured in Ham’s F12: Dulbecco’s modified Eagle’s medium (DMEM) 1:1 medium  supplemented with 5% horse serum (Gibco), penicillin (100 U/ml), streptomycin (100 μg/ml), L-glutamine (2 mM) (Lonza), insulin (0.01 mg/ml), hydrocortisone (500 ng/ml), cholera toxin (100 ng/ml) , and epithelial growth factor (20 ng/ml) . Oxaliplatin was purchased from Tocris  and dissolved in DMSO .BJ, HeLa S3, SW480, and Ht-29 were purchased from ATCC , and HCT-116 was purchased from Ubigene . MCF10A and 293 Ampho-Phoenix cells were previously described and characterized . Cancer For the resazurin-to-resorufin reduction assays, cells were grown in a 96-well plate for 120 min at 37 °C with resazurin solution (0.15 mg/ml) . The fluorescence of resorufin was quantified on a PerkinElmer EnSpire 2,300 Multilabel Reader (ex 550 nm/em 590 nm). For the Apo-ONE assay, 25,000 cells were seeded in a 96-well white polystyrene microplate . After 24 h, cells were treated for 36 h with oxaliplatin and oxaliplatin+SS35. Cells were then lysed and subjected to caspase-3/7 (DEVDase) activity assays by using the Apo-ONE Homogeneous Caspase-3/7 Assay  according to the manufacturer’s instructions. After 30 min of incubation, fluorescence was recorded in the linear phase of the kinetics with a Synergy H1 plate reader . For the trypan blue viability assay, cells were harvested, pelleted at 1500 rcf, and incubated for 5 min with a PBS/0.4% Trypan Blue solution (Gibco). At least 300 cells were counted for each condition.SS35 (0.1 μg/ml), or both, for 48 h. Then, living cells were harvested and counted. A total of 1000 living cells were seeded in 60-mm adhesion plates , cultured for 14 days, and stained for 20 min with methylene blue (5 g/L of 50% (v/v) ethanol/water, 1 mL/plate). Colonies were counted with clone.JARA total of 25,000 HeLa S-3 cells were seeded in a 96-well plate . After 24 h, they were treated with 20 µM oxaliplatin or SS7, SS35, SS28, and SS36 for 3 h; cells were then harvested and resuspended in phosphate-buffered saline (PBS). Single-cell suspensions were prepared as previously described , membranes were incubated overnight with the following primary antibodies: anti-caspase 3 , anti-HA , anti-actin , and anti-histone H3 . HRP-conjugated secondary antibodies were obtained from Cell Signaling Technology , and blots were developed with Super Signal West Dura . For fluorescence-based detection, AF660 or AF760 secondary antibodies were used , and images were acquired at Odyssey M Imaging System .pWZL-Hygro Hemagglutinine-XIAP (HA-XIAP) was obtained by subcloning through a restriction-ligation-based approach the ORF of XIAP (BamHI/EcoRI) from pEBB-XIAP (Addgene Plasmid #11558) in pWZL-Hygro HA. Transfections of 293 Ampho-Phoenix cells were carried out with polyethylenimine  using a 2:1 ratio of PEI (µl): DNA (µg). A pWZL-Hygro empty plasmid was used as a control. Retroviral infection of HeLa S-3 cells was performed with an M.O.I of 0.1–0.3 at 32°C by using Ampho cells as packaging cells. Two consecutive infections were performed, the second 48 h after the first. Cells were selected with 250 μg/ml hygromycin B . Polyclonal cultures were then obtained and started to be used 2 weeks after the end of selection. For Integrin Subunit Beta 3 (ITGB3) silencing, 300,000 HeLa cells were seeded in 35-mm plates and transfected with 40 ng endoribonuclease-prepared short interfering RNAs (esiRNAs)  and 5.5 µl Lipofectamine 3,000 .HeLa cells were lysed using TRIzol . Totally 1.0 μg of total RNA was DNAse I-treated  and retro-transcribed by using 100 units of M-MLV reverse transcriptase  in the presence of 1.6 μM oligo (dT) and 4 μM random hexamers . Real-time quantitative reverse transcriptions (qRT-PCRs) were performed using SYBR Green technology (KAPA Biosystems). Data were analyzed by the comparative threshold cycle (delta delta Ct ΔΔCt) using HPRT and ACTB as normalizers. The primers used for ITGB3 amplification in qRT-PCR are FW: GTGACCTGAAGGAGAATCTGC and RV: CCGGAGTGCAATCCTCTGG.p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Unless otherwise indicated, all the data in the figures were represented as arithmetic mean ± the standard deviation from at least three independent experiments.For experimental data, Student’s t-test was employed. The Mann–Whitney test was applied when normality could not be assumed. For comparisons between more than two samples, the ANOVA test was applied coupled with Kruskal–Wallis and Dunn’s multiple comparison tests. Excel and GraphPad Prism were used for statistical analysis. We denoted significance as *SS23, SS24, and SS16, we used a Cl-Trt resin in the presence of HOBt/DCC under the previously optimized conditions . The cyclization of SS16 was performed under high-dilution conditions in the presence of DPPA/NaHCO3/DMF, yielding c [Arg (pbf)-Gly-Asp(OtBu)-phe-Lys (Cbz)]  and characterized by reversed-phase HPLC and 1H NMR. The purities were determined to be >95% by analytical RP-HPLC .To prepare the linear peptides nditions . The steavengers . For pepde chain . The peps (Cbz)] . After hs (Cbz)]  and treauct SS33 . The pep2O. The reaction was stirred for 12 h in the dark, then washed with water, and recovered by centrifugation. To functionalize PMO with peptides, the outer shell of the nanoparticle was reacted previously with ICPTES and TEA, with a procedure optimized in our laboratory  measured has a diameter of 495 nm with a PDI of 0.12 . After c-1 were assigned to Si-C. The presence of a broad absorption band at 3384.42 cm-1 was assigned to Si-OH. The presence of a urea bond is confirmed by stretching vibrations at 1647.48 and at 1546.84 cm-1 in SS28, at 1651.21 and at 1558.02 cm-1 in SS35, and at 1654.93 and at 1558.02 cm-1 in SS36 spectrums  in ethanol (2 mL) were prepared, and the absorption spectra were measured. The absorbance values of peptides linked to PMOs were adjusted to account for potential interference caused by PMOs, which can scatter or absorb light in the visible and UV ranges, leading to inaccuracies in absorbance measurements of chromophores. The absorbance spectrum of PMO was subtracted from the absorbance spectrum of the sample containing the peptide@PMO. In the region of absorption of the chromophore, we observed a linear and well-defined relationship between the absorbance of PMO and wavelength. To generate the PMO absorbance spectrum under the chromophore’s absorption band, we performed linear regression on the absorbance values outside the chromophore’s band. This allowed us to establish a baseline for the absorption of the PMO. The amount of peptide functionalized on the PMO was determined using the formula obtained from the calibration curves. The absorbance is at λmax = 257 nm for Phe and λmax = 267 nm for Fmoc-Ala. The quantification values are reported in Once calibration curves were established, solutions of SS28, SS35, and SS36), two cancer cell lines were selected: Ht-29, a human colorectal adenocarcinoma cell line expressing low levels of the heterodimeric αvβ3 integrin, and HeLa, a human cervical epithelioid carcinoma cell line expressing high amounts of αvβ3  was equivalent between Ht-29 and HeLa cells. A more efficient uptake by HeLa cells was observed for SS28  and SS35  .To prove the successful uptake of the three molecules  . This evSS28 and SS35 between Ht-29 and HeLa cells was indeed due to differential expression levels of αvβ3, we silenced ITGB3 expression in HeLa cells for 48 h and monitored the uptake of SS28, SS35, and SS36 for 3 hours after administration of the compounds. In HeLa cells successfully deprived of the integrin β3  the engagement of αvβ3 decreases HeLa fitness and b) the lower dose is sufficient to block αvβ3 and achieve efficacy.After establishing the differential uptake of the described compounds in Ht-29 and HeLa cells, we performed standard biological assays to evaluate the induction of apoptosis. First, Ht-29 and HeLa cells were treated with increasing doses  of n assays . HeLa ceto Ht-29 , which ito Ht-29 . Both HtLa cells . InteresSS28) and the chimeric peptide RGD-AVPI   . Althougde SS35) . To indi at 37°C .SS28 and SS35, as these normal cell lines were characterized by the highest mRNA levels of ITGAV on the basis of what emerges from the analysis of the transcriptome available on GENT2 . To prove this, we treated HeLa cells with 10 µM OxPt as a single agent or in combination with 0.6 μg/ml SS35. As shown in SS35 increases the anti-cancer activity of OxPt, and this effect correlates with increased activity of caspases 3 and 7  cell lines, Ht-29, HCT-116, and SW480, that were previously described for having increasing expression levels of αvβ3: Ht-29 << HCT-116 < SW480  the functionalization on the nanoparticles. To prepare the peptides, we used solid-phase peptide synthesis, as reported in SS23 and SS16, the cleavage was carried out with TFE and acetic acid to maintain the protective group  and cyclic RGD (SS25) were coupled in standard conditions and then deprotected to yield the final compound SS33  and Phenylalanine (λex 240 nm and λem 282 nm) to establish the calibration curves (To quantify the amount of peptide loading on the PMOs, we used Fmoc Alanine (λn curves . We founn curves .SS28 and SS35 by αvβ3-positive cancer cells. This is consistent with what was previously reported (The functionalization of the nanoparticles with RGD peptides to engage αvβ3 integrins proved to be successful in increasing the uptake of reported . αvβ3 tareported . Howeverreported  and the reported .SS35 triggers apoptosis in HeLa cells and in all the αvβ3-positive colorectal cancer cells tested. When used in combination with oxaliplatin, SS35 treatment potentiates the induction of cell death, achieving almost a complete response. Oxaliplatin was selected for the combined treatment as platinum-based compounds are widely used in neoadjuvant and adjuvant therapies for advanced cervical (SS35 are not informative about the specific mechanism of cell death triggered. In particular, further experiments are required to clarify whether SS35 would be effective in sensitizing caspase-8 mutated cells to OxPt.cervical  and colocervical . Moreovecervical  and cervcervical  to extricervical , necroptcervical , and immcervical . Mechanicervical . As oxalcervical , the addSS35 molecule would also perturb integrin engagement, maybe increasing the toxicity of the AVPI peptide. Specific in vivo experiments are required to evaluate this possibility, as the engagement of αvβ3 is expected to be transient and reversible. In particular, additional in vivo experiments will allow us to evaluate the efficacy of SS35 in remodeling tumor matrix interactions, as well as its pharmacodynamics and its ability to modulate the tumor microenvironment. However, despite the limitations of the cellular models used, our data demonstrate that RGD-coated nanoparticles provide an efficient way to target tumor cells overexpressing αvβ3 integrin with SMAC mimetics.As it has been reported that inhibition of αvβ3 ligation to the matrix induces anoikis in different cancer cells  and blocSS35 shows the synergic effect when used with oxaliplatin, which is interesting for the development of new cancer therapies.In conclusion, we demonstrated the efficacy of DTP@PMO as a mimetic of Smac/DIABLO.


Mitochondria play a key role in the cerebral ischemia-reperfusion injury. Although the extracellular signal-regulated kinase 1/2 inhibitor PD98059 (PD) is a selective and reversible flavonoid that can protect the mitochondria in a rat model of cardiac arrest/cardiopulmonary resuscitation, its role requires further confirmation. In this study, we investigated whether PD could maintain mitochondrial homeostasis and decrease reactive oxygen species (ROS) production in neuroblastoma (SH-SY5Y) cells exposed to oxygen–glucose deprivation/reperfusion (OGD/R). PD improved the mitochondrial morphology and function, reversed the increase in ROS production and cell apoptosis, and reduced total-superoxide dismutase and Mn-superoxide dismutase activities induced by OGD/R. PD decreases ROS production and improves mitochondrial morphology and function, protecting SH-SY5Y cells against OGD/R-induced injury. Brain i2a cells . Based o22.12.Human neuroblastoma SH-SY5Y cells were obtained from the China Center for Type Culture Collection . They were verified to be of the correct lineage and uncontaminated by other cell types or organisms. The cells were cultured in a mixed substrate, comprising a 1:1 mixture of Minimal Essential Medium (MEM) and F12  and 10% fetal bovine serum (Gibco BRL) at 37°C under 5% COSH-SY5Y cells were stochastically separated into five groups as follows: normal control group (control), DMSO (solvent for PD) control group (DMSO), OGD/R group, PD low-dose group , and PD high-dose group .2.22 to maintain the oxygen concentration around <0.1% at 37°C for 5 min. Following different periods of OGD , ischemic–hypoxic cells were re-incubated in MEM/F12 medium at 37°C under 5% CO2 for 24 h.The OGD/R model was established as previously described . For the2.35 cells/mL and incubated for 24 h. Six wells were used for each treatment and control group. After OGD/R, a mixture of 90 μL of DMEM and 10 μL of CCK‐8 was added to each well. The absorbance of the samples at 450 nm was measured following incubation at 37°C for 2.5 h in the dark.Cell viability was determined using a CCK-8 cell counting kit  following the manufacturer’s instructions. The cells were seeded in 96-well plates at 2 × 102.4Intracellular ROS levels were determined using the 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescence probe . Following treatment, the cells were collected and incubated with 0.1% DCFH-DA in DMEM at 37°C for 20 min. The cells were then washed three times with serum-free DMEM. DCFH fluorescence was recorded in three randomly selected fields. The experiment was repeated three times. The intensity of DCFH fluorescence was assessed using ImageJ Pro Plus 6.0 (Media Cybernetics).2.56 cells/well and incubated for 24 h. The cells were then lysed in ice-cold RIPA buffer for 30 min. After centrifugation at 12,000×g for 15 min at 4°C, the supernatant was collected. Protein levels were assessed using a BCA protein assay kit (Beyotime Biotechnology). The Cu/Zn-SOD level in the supernatant was determined according to the manufacturer’s instructions. The absorbance of the samples at 550 nm was measured using an ultraviolet spectrophotometer .SOD activity was determined using a SOD assay kit  according to the manufacturer’s protocol. The cells were seeded in six-well plates at 1 × 102.6MMP was examined using a fluorescent probe kit (Beyotime Biotechnology). Briefly, the cells were cultured in six-well plates with JC-1 dye working solution for 20 min at 37°C and then washed twice with JC-1 staining solution. The medium was added to each well. Red (JC-1 aggregates) and green (JC-1 monomer) fluorescence were recorded in three randomly selected fields. Fluorescence intensity was estimated using Image J Pro Plus 6.0.2.74 for 1 h. The samples were dehydrated with 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 90% ethanol:90% acetone (1:1), 90% acetone, and 100% acetone three times for 10 min per step, and then incubated at 25°C for 1 h. The samples were then embedded in epoxy resin, placed at 35°C for 15 h, then at 45°C for 12 h, and finally at 60°C for 24 h. The cell masses were cut into ultrathin slices and stained. The mitochondrial structure was observed using transmission electron microscopy , and three different fields were captured.The cells were treated with 4% glutaraldehyde at 4°C for 1 h, rinsed twice with pre-cooled phosphate-buffered saline (PBS), and then treated with 1% OsO2.8Apoptosis was evaluated using a Hoechst 33258 staining kit (Beyotime Biotechnology). The cells were harvested, washed, mixed with 4% (v/v) paraformaldehyde at 25°C for 30 min, further washed, and stained with 500 μL of Hoechst 33258 for 5 min. Fluorescence microscopy  was used to observe nuclear morphology.2.96 cells/well. After treatment, the cells were harvested and rinsed twice with PBS. The cells were stained with a 1:1 mixture of V-FITC and PI for 15 min under dark conditions. Thereafter, 400 μL of Annexin V binding buffer was added to each well. Finally, apoptosis was measured using flow cytometry after 1 h.Apoptosis was measured using flow cytometry with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Early apoptosis of cells was detected by binding Annexin V to phosphatidylserine exposed laterally. PI is a nucleic acid dye that cannot pass through normal cells with intact cell membranes and early apoptotic cells but can pass through the cell membranes of middle and late apoptotic cells and necrotic cells to stain the nucleus. The kit (Beyotime Biotechnology) uses Annexin V combined with PI to distinguish cells at different stages of apoptosis. Flow cytometric analyses were performed using a BD FACSCalibur Flow Cytometer . Briefly, the cells were seeded in six-well plates at 1 × 102.10g for 15 min at 4°C, the supernatant was collected. Protein expression was measured using a BCA protein assay kit. Identical amounts of protein were loaded into a sodium dodecyl-sulfate-polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% bovine serum albumin for 1 h and then incubated with primary antibodies against phosphor-extracellular-regulated protein kinase 1/2 , cleaved-caspase 3 (1:500), caspase 3 (1:500), Bax , Bcl-2 (1:500), Drp-1 (1:500), Mfn1 , Opa1 , and Cyto C (1:500) at 4°C overnight. All the primary antibodies were purchased from Cell Signaling Technology. Thereafter, the membrane was incubated with an IRDye800CW-coupled secondary antibody at 25°C for 2 h. Finally, the membranes were visualized using a western blotting detection system with the Li-Cor Odyssey Scanner imaging densitometer , and the assessed bands were quantitated using the ImageJ Pro Plus 6.0. software.The cells were lysed in cold RIPA buffer with a 1% protease inhibitor cocktail and phenylmethylsulfonyl fluoride for 30 min. After centrifugation at 13,000×2.11p < 0.05 were considered significant.Statistical analyses were performed using SPSS software 22.0. Values are presented as mean value ± standard deviation. The Kolmogorov–Smirnov test was used to test for normality and equal variance. All data were processed using a one-way analysis of variance. Results with 33.1Neuroblastoma SH-SY5Y cells were subjected to OGD for 1, 2, and 3 h, followed by reoxygenation for 24 h. Inverted phase-contrast microscopy was used to determine cellular morphology. With prolonged OGD, the number of adherent cells decreased, whereas the number of floating cells increased . This suCell viability was assessed using a CCK-8 kit, according to the manufacturer’s instructions. Compared with that of the control group (100% ± 0.33%), cell viability decreased with prolonged OGD, as follows: 81% ± 0.17% at 1 h, 56% ± 0.25% at 2 h, 35% ± 0.14% at 3 h, and 26% ± 0.10% at 4 h. Considering an optimal cellular density of 50–60% for drug evaluation, cells were subjected to OGD of 2 h in the subsequent experiments .3.2p < 0.01). However, treatment with PD reversed these changes (p < 0.01 vs the control group) but decreased in the PD-H group (p < 0.05 vs the OGD/R group)  (101.30 ± 11.48), dimethyl sulfoxide (DMSO) control (DMSO) (137.43 ± 10.89), OGD/R (378.01 ± 45.10), PD low dose (PD-L) (248.69 ± 37.75), and PD high dose (PD-H) (201.99 ± 10.78). OGD/R increased the ROS levels compared with the control ( changes . This inR group) .3.3p < 0.01). The Mn-SOD activity increased in both PD-L and PD-H groups than in the OGD/R group , indicating lower MMP levels. A significant enhancement was observed in the MMP in the PD-H group compared with that in the OGD/R group (p < 0.05)  .3.6p < 0.01). Opa1 expression decreased considerably in the PD-L and PD-H groups compared with that in the OGD/R group (p < 0.01)  . Compare < 0.01) . Compare < 0.01) .3.7No significant differences in nuclear morphology were observed in the DMSO group compared with that in the control group; however, in the OGD/R group, the staining pattern was dispersed and deep, indicating that the nuclei were disrupted. Compared with OGD/R, PD-L and PD-H significantly improved nuclear morphology .p < 0.05 and p < 0.01, respectively)  .3.8p < 0.05). PD-H lowered the cleaved-caspase-3 level compared with OGD/R (p < 0.01), and both PD-L and PD-H increased the Bcl-2/Bax level compared with OGD/R   .4in vitro, we established an OGD/R model and found that PD alleviated the ROS production following OGD/R injury, mitochondrial injury, and apoptosis in SH-SY5Y cells.This study proved that the ERK inhibitor PD reduced ROS production, maintained mitochondrial morphology and function, and alleviated the mitochondria-dependent apoptosis in SH-SY5Y cells caused by OGD/R. Brain injury following CA/CPR remains a major concern worldwide. The findings of our previous study indicated that PD plays a brain-protection role in a rat model of CA/CPR . To expl2, resulting in the overproduction of ROS [Oxidative stress is an important pathological factor that increases with the changes in mitochondrial function during CIRI . Oxygen n of ROS ,19. Inacn of ROS .Mitochondrial function plays a vital role in cellular fate. Mitochondria-dependent apoptosis is common during ischemia-reperfusion ,22. MMP Mitochondria are highly dynamic, exhibiting fusion and fission ,27. MitoAlthough an emerging effect was observed in the total expression of Opa1, Mfn1, and Drp1, the number of western blot repeats was low, which is a limitation of the current study. Thus, dynamic variations in Opa1, Mfn1, and Drp1 levels require observation from multiple time points; however, this report only observed the expression of these proteins during OGD of 2 h.5Collectively, our results indicate that the ERK inhibitor PD protected SH-SY5Y cells from OGD/R damage by reducing ROS production, maintaining mitochondrial morphology and function, and alleviating mitochondria-dependent apoptosis. The present findings, together with those of our previous studies in CA/CPR rats, imply that the ERK inhibitor and mitochondrial may be the potential therapeutic target in CIRI.


The accurate stratification of head and neck squamous cell carcinoma (HNSCC) patients based on prognostic differences, using robust biomarkers or signatures, is crucial for guiding clinical interventions. Our study aimed to develop a predictive signature for head and neck squamous cell carcinoma outcomes based on T-cell receptor-related genes (TCRRGs). Using The Cancer Genome Atlas HNSCC dataset, GSE41613, and GSE65858, we identified two HNSCC clusters based on TCRRG expression. These clusters showed differences in overall survival (OS) and immune infiltration. A robust TCRRG-based prognostic signature comprising MAP2K7, MAPK3, MAPK9, ORAI1, PSMA1, UBB, and ZAP70 was subsequently constructed and validated across multiple HNSCC cohorts. A nomogram model was then constructed for personalized HNSCC treatment guidance. Functional analyses indicated notable changes in biological functions and pathways between high- and low-risk groups, with the high-risk group exhibiting a suppressive immune environment. Utilizing this TCRRG-based signature, we may precisely forecast HNSCC outcomes, offering enhanced therapeutic strategies.Backgroud: The stratification of head and neck squamous cell carcinoma (HNSCC) patients based on prognostic differences is critical for therapeutic guidance. This study was designed to construct a predictive signature derived from T-cell receptor-related genes (TCRRGs) to forecast the clinical outcomes in HNSCC. Methods: We sourced gene expression profiles from The Cancer Genome Atlas (TCGA) HNSCC dataset, GSE41613, and GSE65858 datasets. Utilizing consensus clustering analysis, we identified two distinct HNSCC clusters according to TCRRG expression. A TCRRG-based signature was subsequently developed and validated across diverse independent HNSCC cohorts. Moreover, we established a nomogram model based on TCRRGs. We further explored differences in immune landscapes between high- and low-risk groups. Results: The TCGA HNSCC dataset was stratified into two clusters, displaying marked variations in both overall survival (OS) and immune cell infiltration. Furthermore, we developed a robust prognostic signature based on TCRRG utilizing the TCGA HNSCC train cohort, and its prognostic efficacy was validated in the TCGA HNSCC test cohort, GSE41613, and GSE65858. Importantly, the high-risk group was characterized by a suppressive immune microenvironment, in contrast to the low-risk group. Our study successfully developed a robust TCRRG-based signature that accurately predicts clinical outcomes in HNSCC, offering valuable strategies for improved treatments. Head and neck squamous cell carcinoma (HNSCC) is a pervasive and aggressive cancer type originating from the epithelial layers of the oral cavity, larynx, and pharynx . The occTumor immunotherapy has emerged as a promising therapeutic approach for patients with recurrent or metastatic HNSCC . Immune In the present study, we established a TCRRG-based risk prediction model employing The Cancer Genome Atlas (TCGA) HNSCC train cohort, with its robustness successfully corroborated in the TCGA HNSCC test cohort, GSE41613, and GSE65858. Additionally, a predictive nomogram model using the TCRRG risk signature was developed to accurately forecast the OS of HNSCC patients. Notably, our study unveiled a substantial rise in immune cell infiltration and improved immune functions within the low-risk group in contrast to the high-risk group. These observations shed light on the potential mechanisms contributing to the favorable clinical outcomes identified in the low-risk group.n = 97) and GSE65858 (n = 270) were based on the GPL570 (Affymetrix Human Genome U133 Plus 2.0 Array) and the GPL10558 , respectively. All patients in the TCGA HNSCC cohort underwent surgery, and a subset of these patients also received postoperative radiotherapy. Within the GSE41613 cohort, 43 patients were treated with a multi-modality, whereas 53 patients underwent a combination of treatment modalities. In the GSE65858 cohort, 78 patients received a single therapeutic approach, 189 patients were treated with multiple modalities, and 3 patients received palliative care, though specific treatment details were not provided. The TCR-related genes were obtained from the ImmPort Portal  and GSEA databases (GSEA software version 4.3.2), including REACTOME_TCR_SIGNALING, BIOCARTA_TCR_PATHWAY, PID_TCR_PATHWAY, and KEGG_T_CELL_RECEPTOR_SIGNALING_PATHAWY.RNA-seq transcriptome data and associated clinicopathological information for HNSCC patients were acquired from the TCGA database. The clinical samples originated from diverse head and neck regions, including the oral cavity, tongue, floor of the mouth, tonsil, gum, palate, lip, larynx, oropharynx, pharynx, and hypopharynx. The study included a total of 497 HNSCC cases. Additionally, microarray data and relevant clinical information were obtained from the Gene Expression Omnibus databases using the accession numbers GSE41613 and GSE65858. The microarray data for GSE41613  regression method. A unique TCRRG-based prognostic signature was derived via multivariate Cox proportional hazards regression analysis. A risk score for each patient was computed as the sum of each gene’s score, derived by multiplying the expression of each gene by its coefficient. The TCGA HNSCC train cohort was divided into high-risk and low-risk groups based on the median value of risk scores. The Kaplan–Meier method and log-rank test were utilized to calculate the difference in OS between these groups. Moreover, a comparison of OS, stratified by various clinicopathological parameters between these groups, was conducted. A receiver operating characteristic (ROC) curve was generated to assess the prediction accuracy of the prognostic model. The utility of the risk score as an independent prognostic factor was analyzed using univariate and multivariate Cox regression analyses. Finally, the robustness of the TCRRG-based prognostic signature was validated in independent HNSCC cohorts, which included the HNSCC test cohort, GSE41613, and GSE65858.A nomogram model, incorporating risk scores and clinicopathological parameters such as age, gender, tumor grade, and TNM stage, was established. Calibration curves were employed to assess the accuracy of predicting the 1-year, 2-year, and 3-year OS rates in HNSCC.A volcano plot was utilized to depict differentially expressed genes (DEGs) between high- and low-risk groups. Following this, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to pinpoint enriched biological processes and pathways. These analyses were carried out using the “clusterProfiler” R package (R software version 4.2.3).In order to calculate the stromal score, immune score, and tumor purity in high-risk and low-risk groups, the ESTIMATE R package (R software version 4.2.3) was employed. The ggplot2 R package (R software version 4.2.3) was used to illustrate these differences. The CIBERSORT method was performed to determine the disparities in infiltration abundance of 22 immune cell types within high-risk and low-risk groups. Violin plots, derived from the ggplot2 R package (R software version 4.2.3), graphically displayed these variations in immune infiltrating cell types among distinct subgroups. Furthermore, the immune function in the low-risk and high-risk groups was estimated via single sample Gene Set Enrichment Analysis (ssGSEA). The most pronounced positive and negative correlations between risk scores and immune cell types were visualized with a scatter plot.The mutation profiles of HNSCC samples were sourced from the TCGA Data Portal, and the ‘maftools’ package was used to contrast the mutational profiles between high-risk and low-risk groups.http://tide.dfci.harvard.edu, accessed on 8 December 2022) was utilized to evaluate the potential immune therapy response in the TCGA HNSCC cohort. By following procedures provided by the TIDE platform, median centering of the expression matrix for both low-risk and high-risk groups was performed and submitted for analysis. This yielded important metrics such as cancer-associated fibroblast (CAF) score, T-cell exclusion score, and T-cell dysfunction score, all of which were compared between the two risk groups. The Tumor Immune Dysfunction and Exclusion (TIDE) web platform  was used for all statistical analyses. To evaluate the difference in OS between specified groups, we applied Kaplan–Meier curves and performed the log-rank test. Additionally, we utilized the two-sided t-test to compare the differences between the two groups. A CSF2, INPP5D, MAP2K1, MAP2K7, MAPK3, MAPK9, ORAI1, PIK3R3, PSMA1, PSMA7, PSMD10, PSMD2, PSMD7, SKP1, UBB, UBE2D2, and ZAP70 . In contrast, INPP5D, MAP2K7, MAPK3, ORAI1, PIK3R3, and ZAP70 were deemed as protective genes (HR < 1). For the construction of the prognostic signature, seven TCRRGs—MAP2K7, MAPK3, MAPK9, ORAI1, PSMA1, UBB, and ZAP70—were selected, and their coefficients were obtained from the LASSO algorithm (MAP2K7 expression + (−0.484) × MAPK3 expression + (0.58118) × MAPK9 expression + (−0.38196) × ORAI1 expression + (0.477007) × PSMA1 expression + (0.48365) × UBB expression + (−0.46462) × ZAP70 expression. On the basis of the median risk score value, HNSCC patients were divided into high-risk and low-risk groups following the computation of risk scores (p = 1.353 × 10−6) compared to those in the low-risk group (p = 1.621 × 10−4) (p = 4.029 × 10−2) (p = 1.712 × 10−3) (p = 1.5 × 10−2) or >60 (p = 8.3 × 10−3), for male patients (p = 1.3 × 10−3), for patients at stages III–IV (p = 3 × 10−4) or T3–T4 (p = 1.1 × 10−4), and for patients with (p = 4.3 × 10−2) or without lymph node metastasis (LNM) (p = 5.3 × 10−3). Conversely, female patients (p = 7.6 × 10−2), those at stages I–II (p = 1.1 × 10−1), or at T1–T2 (p = 1.6 × 10−1) did not demonstrate significant differences in OS between high- and low-risk groups (p = 6.5 × 10−4), both male (p = 3.2 × 10−2) and female patients (p = 1.4 × 10−2), those at stages III–IV (p = 8.4 × 10−4) or T3–T4 (p = 4.7 × 10−3), and those with LNM (p = 2.8 × 10−3). However, no significant difference in OS was observed for patients aged ≤60 (p = 2.6 × 10−1), those at stages I–II (p = 4.6 × 10−1) or T1–2 (p = 1.4 × 10−1), and those without LNM (p = 1.5 × 10−1) (p = 4.3 × 10−2), those at stages I–II (p = 1 × 10−2) or T1–T2 (p = 3.1 × 10−2), and those without LNM (p = 2.6 × 10−2). No significant difference in OS was identified for patients aged ≤60 (p = 1.1 × 10−1) or >60 (p = 1.8 × 10−1), female patients (p = 6.6 × 10−1), those at stages III–IV (p = 1.9 × 10−1) or T3-4 (p = 2.8 × 10−1), and those with LNM (p = 3 × 10−1) (p = 3.8 × 10−2) or >60 (p = 1.7 × 10−2), male patients (p = 2.8 × 10−4), and those at stage III–IV (p = 2.4 × 10−4). There was no significant difference in OS for female patients (p = 5.6 × 10−1) or those at stage I–II (p = 8.2 × 10−1) between the high- and low-risk groups (p < 1 × 10−3). In contrast, for HPV-positive patients, there was no significant difference in OS between the high- and low-risk groups (p < 1 × 10−3)  , GSE6585 × 10−2) , and GSE × 10−3) . Followik groups . For the × 10−1) . In the  × 10−1) . In the k groups . Moreovek groups . We also × 10−3) .To further scrutinize the predictive accuracy of the TCRRG-based risk signature, ROC curves were generated for OS prediction, and the corresponding areas under the curve (AUCs) were computed. Additionally, the predictive effectiveness of the risk signature was compared against various clinicopathological parameters, such as age, gender, grade, and stage. In the TCGA HNSCC train cohort, as demonstrated in p < 1 × 10−3, HR = 1.504, 95% CI = 1.345–1.682) and OS  as an independent prognostic indicator  and risk score  were significantly correlated with survival  and risk score  as independent prognostic determinants for HNSCC  and served as independent prognostic predictors  for HNSCC patients  HNSCC cases had somatic mutations, with TTN, TP53, and MUC16 showing the highest frequency of alterations  had somatic mutations in the low-risk group. The most frequent alterations were erations . Moreoveerations .We then identified the DEGs between high- and low-risk groups in the TCGA HNSCC cohort, using a threshold of |log2FC| > 1 and FDR < 0.05 A. Our GO+ cells, activated memory CD4+ T-cells, and activated dendritic cells. In contrast, the high-risk group showed a higher proportion of resting memory CD4+ T-cells, M0 macrophages, and resting dendritic cells  [MAPK3 integrates cytoplasmic signals to induce transcriptional changes in the context of differentiation, proliferation, and survival . It has mulation . Followimulation . At thiss (APCs) ,37. Conss (APCs) ,39. Mores (APCs) .The cytoplasmic tyrosine kinase ZAP70 is one of the additional signaling molecules that the TCR relies on to transduce signals. It can associate with the TCR complex and is required to initiate the canonical biochemical signal pathways downstream of the TCR . NovotnáDespite the promising results, our study has several limitations that need to be addressed. Although the validity of the TCRRG-based signature has been confirmed across multiple independent HNSCC cohorts, further validation through large-scale independent studies remains essential for fully establishing the signature’s reliability and wide applicability. Additionally, this analysis did not include key clinical parameters such as therapeutic resistance and molecular features. The available TCGA HNSCC dataset, as well as the GSE41613 and GSE65858 datasets, provided limited or no information regarding these essential clinical and molecular attributes. Moreover, our case series lacks the immunohistochemical evaluation, and in particular, the Combined Positive Score (CPS). Combining CPS and our TCRRG-based signature might be important for the diagnosis and therapeutic guidance of patients with HNSCC . PatholoIn summary, we effectively constructed and verified a reliable prognostic signature utilizing TCRRG that exhibits a substantial correlation with the unfavorable clinical outcome among patients with HNSCC. This signature may function as a significant molecular biomarker for tracking the development of HNSCC and may provide critical insights into the selection of suitable therapeutic approaches.


Nardostachys jatamansi DC, is well-known for its promising therapeutic applications, such as being used as a drug on anti-inflammatory, antidepressant, cardioprotective, anti-neuroinflammatory, anti-arrhythmic, anti-periodontitis, etc. However, its stability under varying environmental conditions and its degradation products remain unclear. In this study, four main degradation products, including two previously undescribed compounds [2–deoxokanshone M (64.23%) and 2–deoxokanshone L (1.10%)] and two known compounds [desoxo-narchinol A (2.17%) and isonardosinone (3.44%)], were firstly afforded from the refluxed products of nardosinone in boiling water; their structures were identified using an analysis of the extensive NMR and X–ray diffraction data and the simulation and comparison of electronic circular dichroism spectra. Compared with nardosinone, 2–deoxokanshone M exhibited potent vasodilatory activity without any of the significant anti-neuroinflammatory activity that nardosinone contains. Secondly, UPLC–PDA and UHPLC–DAD/Q–TOF MS analyses on the degradation patterns of nardosinone revealed that nardosinone degraded more easily under high temperatures and in simulated gastric fluid compared with the simulated intestinal fluid. A plausible degradation pathway of nardosinone was finally proposed using nardosinonediol as the initial intermediate and involved multiple chemical reactions, including peroxy ring-opening, keto–enol tautomerization, oxidation, isopropyl cleavage, and pinacol rearrangement. Our findings may supply certain guidance and scientific evidence for the quality control and reasonable application of nardosinone-related products.Nardosinone, a predominant bioactive product from  Nardostachys jatamansi DC., which has been extensively used for centuries in Himalayan countries to treat neurological and cardiovascular diseases  with yields of 64.23%, 1.10%, 2.17%, and 3.44%, respectively. Column chromatography , preparative thin-layer chromatography (TLC), and recrystallization methods were combined for the chemical investigation of the refluxed products of nardosinone in boiling water ; this le1) was afforded as a colorless crystal or white amorphous powder and could be easily dissolved in methanol instead of dichloromethane. Its molecular formula of C12H16O2 was established based on the analysis of its HRESI–MS (high-resolution electrospray ionization mass spectrometry)  and NMR (nuclear magnetic resonance) data (νmax: 3331 cm−1) and conjugated double bonds (νmax: 1584 cm−1 and 1546 cm−1) in compound 1. The 1H and 13C NMR and HSQC (heteronuclear single-quantum correlation) data  and 0.87 ; δC 19.4 and 15.4], three methylenes , two olefinic methines , one aliphatic methine [δH 1.62  and δC 38.5], and four quaternary carbons . The NMR data indicated that compound 1 was a C12 norsesquiterpenoid. Comprehensive analysis of 1H–1H COSY (1H–1H correlation spectroscopy), HSQC, and HMBC (heteronuclear multiple bonding correlation) spectra of compound 1 resulted in the full assignment of its 1H and 13C NMR data, as shown in 1H–1H COSY spectrum suggested the presence of one spin coupling system (H–1 to H–4 and H3–11), as shown in 2–2 and C–1, C–3, C–4, C–9, and C–10; between H–4 and C–3, C–5, C–6, C–11, and C–12; between H–6 and C–5, C–7, C–8, C–10, and C–12; and between H–8 and C–6, C–7, C–9, and C–10 established the chemical scaffold of 4–hydroxy-8,8a-dimethyl-6,7,8,8a-tetrahydronaphthalen-2(1H)-one ring for compound 1  spectra with those calculated for 4R,5S–1 and 4S,5R–1 using the TDDFT  method , two methylenes , two olefinic methines , one aliphatic methine [δH 2.68  and δC 32.1], and five quaternary carbons , suggesting that compound 3 may be a C15 sesquiterpenoid. Comprehensive analysis of the 1H–1H COSY, HSQC, and HMBC data of compound 3 resulted in the full assignment of the 1H and 13C NMR data, as shown in 1H–1H COSY spectrum determined the presence of one spin coupling system (from H–1 to H–4 and H3–14), as shown in 2–3 to C–1, C–2, C–4, C–5, and C–14; from H–4 to C–2, C–5, and C–15; from H–6 to C–5, C–8, C–10, C–11, C–13, and C–15; and from H–8 to C–6, C–9, and C–10 established the planar structure of compound 3  effects + has been detected, and it is predisposed to lose an H2O (m/z 18) due to the instability of the peroxy ring, resulting in a characteristic fragment ion at m/z 233.1569. The presence of the carbonyl group in the structure leads to a further loss of the CO group (m/z 28) to afford an ion at m/z 205.1588. More importantly, the inverse Diels–Alder (DA) reaction is readily available for the specialized structure of nardosinane-type sesquiterpenoid, and the ion at m/z 233.1569 could further produce a characteristic fragment ion at m/z 191.1429 in the clue of cleavage of C3H6 (m/z 42). In addition, losses of C3H4 (m/z 40) and CH2 (m/z 14) from the ion at m/z 233.1569 afforded the ions at m/z 219.1377 and m/z 193.1229, respectively. Similarly, a series of ionic fragments were further produced at m/z 177.0905, 163.0750, 149.0960, 135.0805, 123.0442, 109.1010, 95.0854, and 81.0698. Interestingly, keto–enol tautomerization may occur throughout the MS spectrometric cleavage process of a nardosinane-type sesquiterpenoid. Surprisingly, nardosinone and 2–deoxokanshone L could both generate a fragment of 2–deoxokanshone M in their cleavage processes.All identified compounds belong to the nardosinane-type sesquiterpenoids; therefore, they share some common ionic fragments in the similar fragmentation pathways. Take nardosinone as an example : A quasi5) +  and 191.1066 [M-H]− .2–Deoxokanshone L (3): Write amorphous powder. c 0.65, MeOH); UV λmax (MeOH) (log ε): 200.2 (2.35) nm, 297.1 (2.55) nm; IR (KBr) νmax cm−1: 3379, 2968, 2925, 2349, 2337, 1607, 1571, 1462, 1384, 1227; ECD  λmax (Δε): 209.8 (−1.1), 246.2 (−0.61), 291.6 (2.79), 328.4 (−2.97); 1H NMR  and 13C NMR : see m/z: 251.1647 [M+H]+ .X-ray crystallographic analysis of (1): A suitable colorless crystal (0.16 × 0.13 × 0.1 mm3), afforded by slow evaporation of a mixture solution (methanol: water = 2:1) of (1), was mounted on a Rigaku Xtalab P200 diffractometer equipped with CuKa radiation (λ = 0.154184 nm). The intensity data were collected using CrysAlisPro 1.171.39.46 software at 294 K. Crystal data: C12H16O2, molecular formula = 192.12, cell parameters: a = 12.7239 (5) Å, b = 8.0557 (4) Å, c = 40.0828 (10) Å, α = 90°, β = 93.402 (3)°, γ = 90°, V = 4101.2 (3) Å3; monoclinic space group P21, Dcalc = 1.192 g/cm3, Z = 2, F (000) = 1596.0; absorption coefficient μ = 0.684 mm−1. A total of 113,981 reflections were collected  in the range of 6.628° ≤ 2θ ≤ 155.356°. The final indices [I > 2σ (I)] were R1 = 0.0614, wR2 = 0.1781; final R indexes [all data] R1 = 0.1095, wR2 = 0.2247; goodness-of-fit F2 = 1.017; flack parameter = −0.07(14). The structure was solved by direct methods using the Olex2 software package (https://www.olexsys.org/olex2/). The crystallographic data of 2–deoxokanshone M has been deposited at the Cambridge Crystallographic Data Center (Deposition number: CCDC 2215280).ECD Calculation of (1 and 3): As reported in our previous study [1 and 3, 100 excitation states at the B3LYP/6–311+g  level were calculated using an IEFPCM solvent model in MeOH with a half bandwidth of 0.45 eV. The calculation results of different conformers were Boltzmann averaged to simulate the ECD spectra after UV correction, which were finally extracted using SpecDis 1.70.1 software.us study , stochasUsing the method previously described , the intAs reported in a previous report , a CCK-8® H class plus system , equipped with a column heater, sample manager, quaternary solvent manager, and photo-diode array (PDA) detector, was employed to undertake the chromatographic separation using an Acquity UPLC BEH C18 column  at 35 °C. The mobile phase comprised acetonitrile (A) and 0.1% formic acid aqueous solution (B). The samples were detected at a wavelength of 270 nm with the flow rate set at 0.3 mL/min and an injection volume of 3 µL, and the gradient program was set as follows: 0–22 min, 18–26% (A); 22–30 min, 26–95% (A), respectively.A Waters Acquity UPLCY–axis) versus the corresponding concentration (X–axis). The limits of detection (LOD) and quantification (LOQ) of the tested compounds were calculated as concentrations where the signal–noise ratio (S/N) was about 3 and 10, respectively. The calibration plot was linear for 5 evaluation components, and a linear relationship with the r2 values being greater than 0.9995 was observed in all cases. The linear relationship, LOD, and LOQ values are detailed in For quantitative analysis, linearity was evaluated by analyzing six different concentrations of the standard solutions . Each linearity sample was injected in triplicate. The calibration curve was constructed as a linear regression analysis of the peak area . The chromatographic condition of UHPLC was employed as described in Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in vitro conditions were prepared according to the Chinese Pharmacopoeia  . For SGFAn accurately weighed amount of 20.0 mg of nardosinone was dissolved in 40 mL of 50% aqueous methanol, and the solution was then incubated at 80 °C for 108 h. Similarly, each accurately weighed 20.0 mg amount of nardosinone was dissolved in 40 mL of SGF, SIF, SGF–A, and SGF–B fluids, respectively, and incubated at 37 °C for 108 h. It is worth noting that due to the poor water solubility of nardosinone, it does not dissolve in simulated gastrointestinal fluids at room temperature; instead, it is in a suspended state. Therefore, an equal volume of methanol was added to dissolve and configure the sample before further tests or analyses. Moreover, during the incubations, the concentrations of nardosinone, isonardosinone, desoxo-narchinol A, 2–deoxokanshone L, and 2–deoxokanshone M were calculated based on the regression equation at each point of time  using the above-established UPLC method. The stability study of nardosinone was repeated three times to guarantee the reliability of the results. In addition, trivalent 1.0 mg of nardosinone was dissolved and incubated in 2 mL of 50% aqueous methanol (80 °C), SGF (37 °C), and SIF (37 °C), respectively, until it completely degraded to afford the incubation end products before being diluted five times with methanol for the UHPLC-DAD/Q-TOF MS analysis.p < 0.05 were considered statistically significant.The data were presented as the mean ± standard errors of the mean (SEM). The statistical differences were analyzed using GraphPad Prism 8.0 software using a one-way ANOVA test. Values of 12 norsesquiterpenoid featuring an α, β-unsaturated enol group in its structure, and it possesses significant vasodilatory activity without any anti-neuroinflammatory activity. Nardosinone is more susceptible to temperature and pH changes, and the degradation products slightly vary in high-temperature and simulated gastric and intestinal fluids conditions. Furthermore, the transformation pathway of 2–deoxokanshone M was reasonably deduced, and the MS fragmentation pattern of nardosinone and its plausible degradation pathway were proposed accordingly. Our study preliminarily sheds light on the stability of nardosinone. Future investigations are still needed for the analysis on the metabolites of nardosinone and the components that are absorbed in the blood. This study of the degradation products and patterns of nardosinone will facilitate the identification of the components of N. jatamansi extracts or fractions and the elucidation of dynamic mechanisms, and these findings may contribute to providing reasonable guidance for the preparation, production, and quality control of nardosinone, N. jatamansi, and the prepared patent Chinese medicines that contain N. jatamansi.Four main degradation products of nardosinone in refluxing boiling water were isolated and identified as 2–deoxokanshone M, desoxo-narchinol A, 2–deoxokanshone L, and isonardosinone. Among them, 2–deoxokanshone M represents a new C


Ignatzschineria sp. strain RMDPL8A was sequenced and analyzed. This draft genome sequence was 2,175,527 bp long, with a GC content of 45.12% and 1,890 protein coding genes.The genome of  Schineria was first described by Tóth et al. in 2001 and later replaced with the genus Ignatzschineria in the order Lysobacterales by Tóth et al. in 2007 (Ignatzschineria (class Gammaproteobacteria) are Gram-negative, aerobic, nonmotile, non-spore-forming bacteria with a regular rod shape  and incubated at 30°C for 48 h. Cells were harvested and high-quality genomic DNA was extracted using the following method. A single bacterial colony was suspended in 500 μL of Tris EDTA (pH 8.0), followed by bead beating for 2 to 3 min. Then, proteinase K (20 mg/mL) was added, and the suspension was incubated at 55°C for 2 h with occasional shaking. A 100-μL volume of 0.5 M NaCl was added, and the suspension was incubated for 30 min at 72°C. DNA was extracted using phenol-chloroform-isoamyl alcohol (25:24:1), washed with 70% ethanol, and dissolved in 100 μL Tris-EDTA buffer (pH 8.0). An intact band of DNA was observed using ethidium bromide gel electrophoresis . The genomic DNA was sequenced on the Illumina MiSeq platform using paired-end (2 × 250-bp) technology with v2 Illumina chemistry . The genN50, 1,848,963), with a GC content of 45.13%, 100% completeness, and 0% contamination. Among the 1,957 total genes, 1,909 coding sequences and 1,890 proteins were identified. In addition, 41 tRNAs and 2 rRNAs (one 16S rRNA and one 23S rRNA) were found. Three clustered regularly interspaced short palindromic repeat (CRISPR) arrays were found in the genome using CRISPRCasFinder (The total length of the genome sequence was 2,175,527 bp; sequencing yielded 3 contigs (asFinder .JAPPWA000000000. The version described in this paper is version JAPPWA010000000. The associated BioProject and Sequence Read Archive accession numbers are PRJNA907172 and SAMN31953526, respectively.This whole-genome shotgun sequencing project has been deposited at DDBJ/ENA/GenBank under the accession number