Cardiovascular disease (CVD) risk factors can be categorized as non-modifiable and modifiable. Modifiable risk factors include some exogenous and behavioral factors that can be easily modified, whereas endogenous modifiable risk factors, such as hypertension, hyperlipidemia, diabetes, or obesity may be modified to a limited extend. An increased prevalence of CVDs as well as their risk factors have been observed in military personnel, as specific military-related stressors are highly correlated with acute cardiac disorders. Military pilots are a subpopulation with great CVD risk due to an accumulation of different psychological and physical stressors also considered to be CVD risk factors. This review presents data concerning CVD risk in military professionals, with a special emphasis on military pilots and crew members. We also discuss the usefulness of novel indicators related to oxidative stress, inflammation, or hormonal status as well as genetic factors as markers of CVD risk. For a correct and early estimation of CVD risk in asymptomatic soldiers, especially if no environmental risk factors coexist, the scope of performed tests should be increased with novel biomarkers. An indication of risk group among military professional, especially military pilots, enables the implementation the early preventive activities, which will prolong their state of health and military suitability. Sedentary time was positively associated with coronary risk, as determined by the 10-year PROCAM risk  [p for trend < 0.0001) increasing with sedentary time independent of leisure time physical activity [The World Health Organization (WHO) defines cardiovascular diseases (CVDs) as a group of disorders of the heart and blood vessels, including coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, and pulmonary embolism. CVDs nowadays are the leading cause of death globally. In 2019, about 17.9 million people died from CVDs, mainly from heart attack and stroke 85%), which are acute events caused by the obstruction of blood flow to the heart (hear attack) and brain (stroke) or by bleeding in the brain (stroke). CVDs are sometimes called ‘silent killers’, as frequently no characteristic symptoms accompany their development until the symptoms of heart attack or stroke appear ,4,5. Apa%, which In aviation, the incidence rate, including both complete and partial incapacitations, ranges from 0.10 to 0.77 per million pilot flight hours in military services and commercial aviation operations . In-fligAirline pilots and the cockpit crew seem to be a subpopulation that is especially prone to CVD appearance. These conditions comprise prolonged sedentary posture; difficulties in moving the limbs; occupational stress; emotional tension; highly demanding tasks, especially during take-off and landing; and the predominance of intellectual activity over physical activity. They also encompass forced operation speed against time shortage; frequent time-zone changes; night-time, meridional, and trans-meridional flights; acceleration; G-loads; noise; vibration; and an unbalanced diet high in saturated fat ,15,16. T2max) in Indonesian military pilots [Investigations of CVD risk in airline pilots are not satisfyingly described in the literature , but Husy pilots . A receny pilots .The Civil Aviation Authority of Malaysia decided that a return to flying duties for a civilian or private pilot, which is the reassessment and recertification, can be completed six months after the cardiovascular event. For a military pilot, however, this waiting period is extended to 1 year. The 4 × 4 aeromedical risk matrix can serve as a very useful tool that can aid in decision making for the assessment and recertification process. Recent studies by Mohammad et al. showed that after coronary artery disease a pilot must be hemodynamically stable with no evidence of significant inducible ischemic left ventricular dysfunction and a minimum of 50% of the ventricular ejection fraction (LVEF). Moreover, a follow-up is recommended at six months after recertification and then annually with a routine noninvasive cardiac assessment .The results of some studies show that there is also an increased prevalence of CVD as well as its risk factors in military personnel, as specific military-related stressors are highly correlated with acute cardiac disorders . MirzaeiMilitary aircrew are of special concern, as they are exposed to significant additional demands when conducting their duties. These demands may be due to the environmental factors associated with flying high-performance aircraft (such as hypoxia or sustained acceleration) and undertaking their activity in a hostile operational environment (from flying over enemy territory to being engaged in air combat maneuvers). They also could be related to merely operating from base environments that are not conducive to the usual maintenance of circadian rhythms or sleep patterns . These pn = 30 participants . In the comparative group, pilots whose 5-year CVD risk scores were lower than 10% were included, which resulted in n = 595 pilots in this group. The authors assessed different CVD risk factors, both endogenous and exogenous, including age, smoking history, diabetes status, history of hypertension, systolic blood pressure, diastolic blood pressure, history of hyperlipidemia, HDL cholesterol, triglycerides, and the total cholesterol–HDL ratio. All investigated risk factors were found to be elevated in the high-risk group of pilots compared to those in lower risk group, with the most pronounced differences observed for hypertension appearance (36.7% versus 6.1%), hyperlipidemia (40.0% versus 10.4%), diabetes (10% versus 0.5%), and tobacco smoking (26.7% versus 13%). Among the investigated high-risk pilot group, nine abnormal resting ECG results were observed. Twenty-six pilots from the high-risk group underwent exercise stress ECGs. In three patients, the exercise stress ECGs were judged to be borderline, and the exercise stress ECG was judged to be positive in four cases. In total, coronary angiography was performed in nine patients from the high-risk group and revealed five patients who had lesions characterized by more than 70% stenosis or occlusions in one or more arteries that were considered significant. The authors emphasized that exercise electrocardiograms, as a diagnostic test to investigate excessive cardiovascular risk in pilots, are not adequate to detect disease or to protect pilots from unnecessary invasive procedures, such as coronary angiography. They suggest the inclusion of laboratory markers and the use of different noninvasive imaging to create a more comprehensive and accurate cardiac investigation algorithm of CVD risk assessment and CVD detection [For many years the “1% rule”, which states that no pilot should have an annual risk of cardiovascular incapacitation exceeding 1%, has served as a possible standard and is endorsed as the threshold of choice by the International Civil Aviation Organization (ICAO), but the methods of determining the risk to an individual pilot vary by jurisdiction . The Fraetection . This suetection . Apart fetection . A revieetection . This pretection . In popuetection . Mulloy etection . Moreoveetection . In 2021etection .trans fatty acid dietary intake, a diet poor in fruits and vegetables, physical inactivity, tobacco smoking, alcohol consumption, and overweight or obesity, all of which are modifiable risk factors. Non-modifiable risk factors include a family history of hypertension, age over 65 years, and coexisting diseases such as diabetes or kidney disease [According to the WHO, hypertension is diagnosed if, when it is measured on two different days, the systolic blood pressure (the pressure in blood vessels when the heart contracts) is ≥140 mmHg and/or the diastolic blood pressure (the pressure in the vessels when the heart rests between beats) is ≥90 mmHg [R = 1.641, p = 0.003) [In studies by Mirzaeipour et al. in Iranian military personnel, hypertension was confirmed as an important risk factor of CVDs (O= 0.003) . In studStudies by Grosz et al. showed that high blood pressure affects 14.7% of Hungarian military pilots , whereasSolovieva et al. compared blood pressures and heart rates in different flight phases  in military pilots of Arctic transport in aviation suffering from essential hypertension and healthy pilots with CVD risk factors but without CVDs. They observed maximum blood pressure and heart rate values that were higher in hypertensive pilots than in the control subjects during each flight phase. The maximum systolic blood pressure during takeoff was 202 mmHg in the first group versus 179 mmHg in the control group. The highest heart rate during takeoff was also recorded in the first group in comparison to the control . The maximum systolic blood pressure and heart rate recorded during the landing in pilots of the first group were 253 mmHg and 150 beats/min, while the values in the control group were 163 mmHg and 141 beats/min, respectively. They concluded that military pilots with hypertension withstand the highest hemodynamic loads during flights in the harsh weather conditions of the Far North and can be permitted to operate airships in the presence of adequately controlled hypertension .2). A diagnosis of overweight is given to a person whose BMI is equal to or greater than 25, whereas a diagnosis of obese is given to a person whose BMI is equal or exceeding 30, despite the age or gender. The main cause of obesity and overweight is an energy imbalance between the calories consumed and the calories expended, resulting from an increased intake of energy-dense foods that are rich in fat and sugars and a decrease in physical activity as a consequence of sedentary work, changes in modes of transportation, or increased urbanization. The prevalence of obesity nearly tripled between 1975 and 2016. In 2016, according to WHO data, 39% of adults aged 18 years and over and 13% of the world’s adult population were overweight or obese, respectively [The WHO defines overweight and obesity as abnormal or excessive fat accumulation that may impair health, based on body mass index (BMI), which relates to a person’s weight in kilograms divided by the square of his height in meters (kg/mectively . Overwei2 (overweight) was observed in 58.1% of examined Serbian military pilots, while 16.2% of them were reported to have BMI > 30 kg/m2, which indicates obesity [Obesity was the most common CVD risk factor diagnosed in the total examined population of the US Army, and its rate was 19.1 . For the obesity . Diabetes, which is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces, affected 422 million of people in 2014, and its prevalence still increases, especially in low- and middle-income countries. Uncontrolled diabetes causes hyperglycemia, which over time, leads to serious damage to many of the body’s systems, especially the nerves (neuropathy), blood vessels (angiopathy), heart, eyes (retinopathy), and kidneys (nephropathy). According to the WHO, the risk of heart attack and stroke is two- to three-fold higher in diabetic patients in comparison with non-diabetic patients . Blood gR = 1.792, p = 0.005) on coronary artery disease risk among all examined risk factors [The rate of abnormal glucose levels diagnosed in the total US Army population was 4.6, whereas the diabetes rate was only 0.8, and both rates increased with age in the total population. For the Air Force population, the values of both rates were similar to the general population, and for military pilots and crew, their values were slightly lower: 3.2 and 0.4, respectively [ factors . In the  factors .Hyperlipidemia refers to the pathological condition characterized by an elevated fasting total cholesterol level (hypercholesterolemia), which may or may not be accompanied by an elevated triglyceride level (hypertriglyceridemia) in the blood, which is recognized as a CVD risk factor . A more R = 1.773, p = 0.002) [Disturbances in the lipid profile of the blood in airline pilots have been investigated as CVD risk factors for years . Mirzaei= 0.002) . Recent = 0.002) . Among P= 0.002) . Hyperli= 0.002) . Grosz e= 0.002) . Detaile= 0.002) . Moreove= 0.002) . The coexistence of some of the aforementioned CVD risk factors is quite common and is defined as metabolic syndrome. Metabolic syndrome causes increased CVD risk and type 2 diabetes risk and a higher risk of death from all causes, including CVDs. Mottillo et al. revealed, that metabolic syndrome is associated with a two-fold increase in cardiovascular outcomes and a 1.5-fold increase in all-cause mortality . What isp < 0.0001) but decreased with the duration of observation . However, no relationship between the prevalence of metabolic syndrome and total flying hours was observed. In addition, no association between metabolic syndrome and work-related stress or shift work was observed. The authors claimed that military flying per se does not seem to contribute in a significant way to metabolic syndrome [In studies by Weber et al. on German military pilots, metabolic syndrome was defined as systolic blood pressure > 130 mm Hg, cholesterol > 200 mg/dL, HDL < 40 mg/dL, triglycerides > 150 mg/dL, and fasting blood glucose > 100 mg/dL. The metabolic syndrome group consisted of 110 individuals meeting these criteria  whose BMI values were significantly higher than those of the healthy individuals and remained higher during the observational period. It should be emphasized that metabolic syndrome was significantly less prevalent in the examined population of military pilots than in the general population and was less prevalent than in other civil or military aviation samples. The prevalence of metabolic syndrome in military pilots in Germany increased significantly with the age of the individual participant  decrease oxygen availability, increase the formation of ROS and reactive nitrogen species (RNS), and deplete the antioxidant system. However, some adaptive mechanisms develop over time. Petraki et al. assessed the effects of a flight simulation on the antioxidative status  as well as the oxidative modifications of proteins and lipids through the measurement of protein carbonyls (PCs) and thiobarbituric acid reactive species (TBARS) of military pilots with differing flying experiences. They observed an adaptation to the flight training in an effort to battle oxidative stress in experienced pilots, revealed by increased levels of GSH . Corsi ep = 0.02) [Atherosclerosis is also considered a chronic inflammatory disease of the arterial wall, with a long asymptomatic phase, which initiates in the place of endothelial injury, where both innate and adaptive immune responses contribute to disease initiation and progression. Vascular inflammation, which is characterized by immune cell infiltration, contributes to atherosclerosis formation by the increased expression of adhesion molecules and their ligands, the extravasation of leukocytes, the activation of immune cells and pro-inflammatory signaling pathways, and an increase in oxidative stress and cytokine production in the arteries. The amplification of inflammation, which is mediated both by T-cell mediated immune responses and the humoral immune response, can lead to plaque rupture. Following this, thrombosis occurs when the thrombus is sufficiently large that it can either partially or completely occlude the coronary vessel lumen and cause an acute event . Inflamm = 0.02) . Due to It is estimated that 50% of patients suffering from coronary heart disease do not have the classical risk factors of hypercholesterolemia, hypertension, smoking, diabetes, obesity, or a sedentary lifestyle, which suggests the importance of genetic background of CVDs . A familLusis et al. proposed a list of candidate genes that should be investigated to evaluate coronary heart disease risk . For somLDLR gene, (2) familial defective apolipoprotein B resulting from a mutation in the APOB gene, and (3) hypercholesterolemia, autosomal dominant, 3, which is caused by one of three missense mutations in the PCSK9 gene. Familial combined hyperlipidemia, which manifests with elevated levels of very low density lipoprotein (VLDL), LDL, APOB, and triglycerides, seems to be related to the APOA1/C3/A4/A5 gene cluster on chromosome 11. Familial hypertriglyceridemia with normal cholesterol levels but higher than normal triglyceride levels (>400 mg/dL) appears to be an autosomal dominant disorder that is connected with more than one locus linked to its transmission (15q11.2-q13.1 and 8q11-q13). Individuals with atherogenic lipoprotein phenotype (ALP) have a large number of small, dense, LDL (subclass pattern B) particles and triglyceride-rich lipoproteins and a decreased level of HDL, and this phenotype is linked to the LDLR locus, mapping to 19p13.3-p13.2 [The risk of coronary heart disease and myocardial infraction is increased by the appearance of autosomal dominant hypercholesterolemia, which occurs in three forms: (1) familiar hypercholesterolemia resulting from a mutation in the .3-p13.2 ,68.APOE gene, encoding an LDL receptor ligand, apoE, and the risk of CHD, and a significant correlation between APOE Ɛ4 and coronary heart disease risk was established. Moreover, the LPL gene encoding lipoprotein lipase, an enzyme responsible for hydrolyzing triglyceride particles and indirectly responsible for the production of HDL-C, was investigated in relation to coronary heart disease, and the diversified influence of different mutations has been established [A number of studies have looked at the association between the ablished ,68.ANRIL, which is a large non-coding RNA of unknown function that was revealed to be expressed in the vascular endothelium, monocyte-derived macrophages and coronary smooth muscle cells, and two protein-coding genes, CDKN2A and CDKN2B, encoding cyclin-dependent kinase inhibitors, were found [PPARG gene, which is located at 3p25-24 and plays a role in adipocyte differentiation, insulin sensitivity regulation, fatty acid metabolism regulation, adipose tissue storage, and free fatty acid reduction, and the CYP1A1 gene, which mediates the oxidative metabolism of exogenous and endogenous molecules, e.g., cholesterol, estrogens, or androgens, are both risk factors of coronary artery disease. Zhang et al. revealed in the Chinese Han population that variants in rs10865710, rs1805192, and rs4646903 were associated with higher coronary artery disease risk. They also found a significant interaction between rs1805192 and rs4646903. The risk of coronary artery disease was the highest in participants with rs1805192- Pro/Ala or Ala/Ala and the rs4646903- TC+CC genotype and was the lowest in those with rs1805192- Pro/Pro and the rs4646903- TT genotype [In 2007, three GWA studies simultaneously reported a locus at chromosome 9p21.3 that was significantly associated with coronary artery disease. At this locus, re found ,70. Fourre found . Howevergenotype .GCLM C-588T (rs41303970), MPO G-463A (rs2333227), PON1 Gln192Arg (rs662), and NAD(P)H oxidase p22phox C242T (rs4673)) and their influence on atherosclerosis. They revealed that the GCLM C-588T polymorphism was associated with elevated atherosclerosis risk , but the associations between the other three polymorphisms did not reach statistical significance. Moreover, they observed a synergistic effect of all investigated polymorphisms, as the atherogenesis risk was significantly greater as the total number of the four concomitant ‘‘pro-oxidant alleles” in each subject was increased . Furthermore, the number of ‘‘pro-oxidant alleles” was a risk factor for a high atherosclerosis risk independent of conventional risk factors (p = 0.0003) [Moreover, some genes involved in the regulation of oxidative stress and the risk of atherosclerosis have been studied to associate the influence of their polymorphism on oxidative stress. Katakami et al. investigated four relatively common genetic variants related to oxidative stress (242T rs46) and theVEGF, ACT, HMGCR, IL-1β, IL-10, and IFN-γ genes) that were previously identified as being associated with an increased risk of CVDs. They revealed that the concomitant presence of the CC genotype of VEGF, the A allele of IL-10, and the A allele of IFN-γ genes was associated with an increased risk of acute myocardial infarction and was more frequent among patients with a positive parental history of acute myocardial infarction, which confirmed that these genes with immune regulatory functions are part of the complex genetic background contributing to familiarity for CVDs [TLR-4 gene and atherosclerosis risk has been proven by the detection of lower levels of pro-inflammatory IL-6 and TNF-α and higher levels of anti-inflammatory IL-10 in carriers bearing the TLR-4 polymorphism. The CD14 gene -260C>T polymorphism has been established as risk factor of coronary heart disease, especially in East Asians. The ICAM-1 gene -469E>K polymorphism was proven to be significantly associated with an increased risk of coronary heart disease in Asian and Caucasian populations. In the case of the TNF-α gene, a pooled analysis revealed that carrying the TNF-α gene A variant conferred a 1.5-fold increased risk of developing coronary heart disease in the Caucasian population. Moreover, the MCP gene -2518A>G SNP, known to increase MCP-1 production, showed an association with an increased risk of CVD in Caucasians [NME4, PNPLA7, GGT5, PTGS2, IGF1R, NT5C2, ENTPD1, and PTEN) could be a sign of elevation of blood pressure for military pilots [Selected genes with immune regulatory functions are part of the complex genetic background contributing to inflammation and atherosclerosis initiation and progression as well as CVDs. Ianni et al. investigated the genetic variations (SNPs) in the promoter region of a number of genes regulating metabolic and immune functions (for CVDs . The assucasians . Zhao ety pilots . p = 0.01) [Among CVD risk factors in both men and women, the levels of sex hormones are considered to be meaningful. Among post-menopausal women, a higher testosterone/estradiol ratio was associated with an elevated risk for CVD incidents and coronary heart disease (CHD), and higher levels of testosterone were associated with increased CVD and CHD, whereas higher estradiol levels were associated with a lower CHD risk . However = 0.01) . One of  = 0.01) . A number of findings confirm that CVD risk may be diminished by the elimination or limitation of modifiable risk factors. Although among soldiers the risk of CVDs is lower than in general population, the identification of the early stages of developing CVDs is currently limited. For a correct and early estimation of CVD risk in asymptomatic soldiers, especially if no environmental risk factors coexist, the scope of the performed tests should be increased with not only additional blood indicators related to oxidative stress, inflammation, or hormonal status but also with genetic studies. The indication of risk groups among military professionals, especially military pilots, enables the implementation of early preventive activities, which will prolong the state of health and military suitability of this professional group.


Objective: To investigate the effect of Zhilong Huoxue Tongyu capsule (ZLH) in the treatment of cerebral ischemia–reperfusion injury and determine the underlying molecular network mechanism.Methods: The treatment effect of Zhilong Huoxue Tongyu capsule (ZLH) was evaluated for cerebral ischemia–reperfusion injury in middle cerebral artery occlusion (MACO) rat, and the underlying molecular network mechanism was explored by using molecular network analysis based on network pharmacology, bioinformatics including protein–protein interaction (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as molecular docking.Results: The neurological function of rats in the ZLH group was significantly improved compared to those in the NS group (p = 0.000), confirming the positive effect of ZLH for the treatment of brain ischemia. There were 126 intersecting genes screened in ischemia–reperfusion cerebrum that are associated with several important biological processes, such as lipopolysaccharide, and the most important cell component, such as raft, as well as the most important molecular function pointed as cytokine receptor binding. The most important KEGG signaling pathway was the AGE-RAGE signaling pathway in diabetic complications. Moreover, according to the STRING interaction in the PPI network, 10 hub genes including MAPK14, FOS, MAPK1, JUN, MYC, RELA, ESR1, STAT1, AKT1, and IL6 were selected and exhibited in Cytoscape and molecular docking. Lastly, the relation between PPI, GO, and KEGG was analyzed. These findings indicated that multiple hub network genes have been involved in behavior improvement in cerebral ischemia–reperfusion rats subjected to ZLH treatment.Conclusion: Zhilong Huoxue Tongyu capsule improves cerebral ischemia–reperfusion and is associated with multiple network gene expressions. Stroke, one of the three most common causes of death and disability, is increasing with the high-pressure life. About 70% of strokes are derived from ischemic, and the rest are intracerebral or subarachnoid hemorrhages . After sAstragalus, Cassia twig, leech, Panax notoginseng, and chuanxiong, is effective in dispelling wind and dampness and relieving pain for vascular headache and migraine. Among of them, Astragalus can hold the surface for qi and fold sweat; cassia twig can generate muscle, warm the meridians, and pass through Yang Qi; leech can precipitate and break blood clots (Zhilong Huoxue Tongyu Capsule (ZLH) (Sichuan Medicine Z20070528) is an available traditional Chinese medicine (TCM) that could be utilized for the treatment of cerebral stroke . As a cood clots . HoweverNetwork pharmacology is based on core target analysis, network interaction relationship analysis, and description of key biological processes and signaling pathways to explain the disease–target-drug co-association module, which could exhibit the network regulatory relationship between drugs and diseases, so as to uncover the mechanism of pharmaceutical agents , especiaIn this study, we investigated the protective effect and network mechanism of ZLH against cerebral ischemia–reperfusion, by gene network analysis, molecular docking, and animal experiment.n = 12), NS group (n = 10), and ZLH group (n = 11). After 8 h of fasting, the rats were deeply anesthetized with 3% sodium pentobarbital and performed the right common carotid artery occlusion in rats, by inserting a wire into the internal carotid artery. Then, the wound was sutured till the embolization was removed after 1 h  K2020-0004); the animal ethics code is KMMU20220854, and the rats were randomly grouped into the sham group , and 2 ml/day was intragastrically administered to each rat in the ZLH group. The rats in the NS group were given 2 ml/day of saline, and the sham group was fed and watered as usual. All rats in each group were observed until sacrificed at day 5.All rats were assessed 24 h after awakening from anesthesia by using Zea-Longa scores. The scoring criteria were as follows: 1) no signs of nerve damage, 0 points; 2) inability to fully extend the contralateral front paw, 1 point; 3) rotate to the opposite side while crawling, 2 points; 4) falling to the opposite side while standing or crawling, 3 points; 5) inability to walk on their own and loss of consciousness, 4 points. The higher the score indicates the more severe the behavioral disorder of the animal.Astragalus, Cassia twig, leech, Panax notoginseng, and chuanxiong in ZLH were extracted by TCMSP (https://old.tcmsp-e.com/tcmsp.php), according to the condition of oral bioavailability (OB) that was set at > 20 and drug similarity (DL) was at >0.15. After retrieving the active ingredient of the drug, the name of the target protein corresponding to the active ingredient is checked in “related targets” according to the molarity of the active ingredient, and the name of the gene corresponding to the target protein is correspondingly searched in UniProt.The components and related target proteins of https://www.genecards.org/), we downloaded a list of genes related to cerebral ischemia–reperfusion.After entering the keyword “cerebral ischemia-reperfusion” in GeneCards (https://bioinfogp.cnb.csic.es/tools/venny/index.html). We input the gene name using “BI” in List1 and “ZL” in List2 and then constructed the Venny intersection map.The genes involved in cerebral ischemia–reperfusion were screened, and the Venny intersection map with ZLH was constructed  was used to perform the GO enrichment and KEGG pathway analysis of the intersection obtained in Venny. In the Java environment, after the computer was first installed with the activePerl.exe file, org.Hs.eg.db, DOSE, clusterProfiler and pathView Enrichplot, ” we then used the scripts of SymolID, GO, and KEGG file packages to analyze the gene network, which includes the biological processes (BP), cellular components (CC), and molecular functions (MF), and the KEGG signaling pathway that can be obtained.https://cn.string-db.org/) was performed using the intersection derived from the analysis in Venny 2.1.0. The results were then obtained by selecting multiple proteins and entering the intersecting genes in the “list of name” and selecting “Homo sapiens” for analysis.PPI network analysis  type 1, drug mole number and corresponding genes; 2) type 2, disease name and disease gene; 3) type 3, KEGG signaling pathways and corresponding genes; 4) type 4, drug name and mole number; 5) type 5, KEGG signaling pathways. The disease–drug active component–key target–KEGG pathway diagram was constructed rationally by changing its shape, color, and position.A total of 199 genes of components and related target proteins for ZLH were examined , and 1,4A total of 199 genes for ZLH and 1,493 genes for cerebral ischemia–reperfusion were crossed in Venny 2.1.0, and 126 intersecting genes were obtained ; Table 3raft, which plays a regulatory role in the area around cerebral infarction. The most important molecular function is cytokine receptor binding, which activates intracellular enzymes to regulate gene expression , cellular component (CC), and molecular function (MF) were analyzed. The most important biological process was response to lipopolysaccharide; the main component of lipopolysaccharide was the outer membrane of Gram-negative bacteria, leading to the immune reaction. It plays an early warning role in inflammatory response after cerebral ischemia–reperfusion. The most important cell group is pression .The results showed that the top 10 KEGG signaling pathways were as follows: AGE-RAGE signaling pathway in diabetic complications, lipid and atherosclerosis, fluid shear stress and atherosclerosis, IL-17 signaling pathway, TNF signaling pathway, Kaposi sarcoma-associated herpesvirus infection, hepatitis B, HIF-1 signaling pathway, human cytomegalovirus infection, and Chagas disease .In total, 126 intersection genes were obtained from Venny 2.1.0, shown in The top 10 genes were sorted by the degree value, and the circle from the largest to smallest represented the decreasing degree value. As shown, the top 10 hub genes were MAPK14, FOS, MAPK1, JUN, MYC, RELA, ESR1, STAT1, AKT1, and IL6 .Based on the 10 target proteins screened in Cytoscape, molecular docking was carried out with ZLH. The crystal structures of the target proteins were downloaded from the PDB database, formatted by Open Babel, and molecular docking of the target proteins to the ZLH was performed in AutoDock Vina. Finally, PyMOL software was used to process and adjust the color of the large protein, process the ligands and residues, calculate the length of hydrogen bonds, and export the ligands and the overall picture .The disease–drug active component–key target–KEGG signaling pathway diagram was constructed by importing the 44 active ingredients corresponding to ZLH, 126 common cross-targets of cerebral ischemia–reperfusion and ZLH, and the first 10 pathways of KEGG into Cytoscape software. As a result, we constructed the network interaction among all related targets, in which, the closely related pathways were linked in the figure to illustrate the regulatory role of ZLH on cerebral ischemia–reperfusion .To know the relation and link of hub genes with PPI, GO, and KEGG pathway, we compared the position of hub genes in the GO and KEGG pathway. We found that almost all hub gens were involved in BP and selected signal pathways, but only MAPK1 or Mapk14 were involved in CC. These pointed out that 10 hub genes selected from PPI are most importantly related to the findings of GO analysis and signal pathway. In addition, the molecular hub genes like MAPK14, FOS, MAPK1, and JUN may simultaneously locate in different signaling pathways involved in the AGE-RAGE signaling pathway in diabetic complications, lipid and atherosclerosis, fluid shear stress and atherosclerosis, IL-17 signaling pathway, TNF signaling pathway, Kaposi sarcoma-associated herpesvirus infection, hepatitis B, HIF-1 signaling pathway, human cytomegalovirus infection, and Chagas disease, which means that some co-expression genes scattered in different signaling pathways. In turn, the same signaling pathway like the IL-17 signaling pathway includes different genes including MAPK14, FOS, MAPK1, JUN, RELA, and AKT1, simultaneously .p = 0.000, Zea-Longa scores were performed on the sham group, NS group, and ZLH group at 1, 3, and 5 days after surgery. The results showed a significant increase in neurological dysfunction in the NS group compared to the sham group at 5 days after surgery, whereas it reversed in the ZLH-treated group, compared to the NS group (In the present study, we confirmed the effect of ZLH on neurological function repair in brain ischemic rats and determined the gene network in the cerebral ischemia–reperfusion model . We screAfter the brain ischemia model was successfully established and the high-dose treatment of ZLH was used, we found that the score of neurological impairment was higher in brain ischemia, and it is lower in the ZLH group at 5 days. This suggested that the neurological function of cerebral ischemia–reperfusion rats was remarkably improved after high-dose administration of ZLH. Previously, it has been reported that ZLH exhibited a positive effect for the treatment of brain damage , but theNetwork pharmacology can explain the relationship between drugs and gene network of diseases. Through network analysis, different genes regulating the disease and interacting genes on diseases could be found. Network pharmacology also analyzes the mechanism of action of TCM prescriptions on disease, therefore providing a new way to explain the molecular mechanism of TCM efficacy . DespiteThe genes of ZLH were intersected with the genes of cerebral ischemia–reperfusion, and 126 intersection genes were obtained. The GO enrichment and KEGG signaling pathway analysis of the intersection genes showed that these genes were located in lipopolysaccharide as the most important BP. The main component of lipopolysaccharide is the outer membrane of Gram-negative bacteria, which causes immune response and plays an early warning role in the inflammatory reaction after cerebral ischemia–reperfusion . The mosThe top 10 genes with the closest relationship were obtained, and we found that CCND1, ESR1, and JUN were connected with multiple genes in the top 10 pairs . These cAstragalus was docked with MAPK14, AKT1, FOS, IL6, MPK1 MYC, ESR1, JUN, RELA, and STAT1; Dahuang was directly docked with JUN, ESR1, and MYC; Cassia twig was docked with JUN, ESR1, and RELA. However, ESR1 and RELA had no direct interaction with Cassia twig. Therefore, through molecular docking, we provided the vital explanation for the drug directly targeting the molecule (As key nodes, the 10 hub genes were speculated as core genes in cerebral ischemia–reperfusion with ZLH treatment . Each ofmolecule .By constructing a disease–drug active component–key target–KEGG pathway map, it can find the network interactions between the related targets . In thisThe relation of hub genes with PPI, GO, and KEGG pathway was analyzed. Several important genes were linked with the different cellular locations, biological processes, and molecular functions, meanwhile sharing the different signal pathways . MAPK4, In summary, we confirmed the effect of ZLH in protecting the ischemic brain from injury, and by using network pharmacology, molecular docking, and bioinformatics analysis, we explained the net mechanism in cerebral ischemia–reperfusion treated with ZLH. The results revealed that the effect of ZLH in improving neurological function is associated with multiple gene expressions in cerebral ischemia–reperfusion rats. Despite determining the effect of ZLH in treating brain ischemia and explaining the associated network mechanism, we suggest that the follow-up experimental research is absolutely needed to be developed to validate the deep mechanism.


Extracorporeal shockwave therapy (ESWT) has been suggested as an alternative treatment for reducing spasticity in patients with cerebral palsy (CP). However, the duration of its effect was rarely known. A meta-analysis was performed to investigate the effectiveness of ESWT at controlling spasticity in patients with CP according to the follow-up period. We included studies in which ESWT was used to manage spasticity in patients with CP, and the effect was compared with that in a control group. Finally, three studies were included. In the meta-analysis, spasticity, measured using the modified Ashworth scale (MAS), was significantly reduced after ESWT compared with that in the control group; however, it was sustained for only 1 month. After ESWT, significant increases in passive ankle range of motion (ROM) and plantar surface area in the standing position were observed compared with those in the control group and sustained for up to 3 months. Although spasticity measured using MAS was significantly reduced for only 1 month, improvement in spasticity-associated symptoms, such as ankle ROM and plantar surface area contacting the ground, persisted for over 3 months. ESWT appears to be a useful and effective therapeutic option for managing spasticity in patients with CP. Cerebral palsy (CP) is a group of disorders that can cause abnormalities in movement, muscle tone, balance, and posture caused by non-progressive disorders of the immature brain . MovemenPrevious studies reported that the severity of muscle spasticity in patients with CP had a significant correlation with the Gross Motor Function Measure ,6. FurthFor the management of spasticity, transcutaneous electrical stimulation, transcranial direct current stimulation, repetitive transcranial magnetic stimulation, acupuncture, cryotherapy, thermotherapy, vibratory stimulation, orthoses, electromyography biofeedback, therapeutic ultrasound, passive movement, and muscle stretching are used in clinical practice ,9,10,11,In addition to botulinum toxin, phenol or alcohol can be injected into spastic muscles, but these are limited by severe procedural pain, persistent pain after injection, and occasional nerve damage ,19. WhenESWT was first used in 1980 to treat kidney stones in urology patients. Shock waves are produced through the rapid propagation of rapidly increased pressure in three-dimensional space, which results in sequences of high-energy biphasic acoustic impulses. Shock waves can be applied by focusing on targeted body tissues without damaging the overall structures . The paiRecently, ESWT has been proposed as an alternative treatment for reducing spasticity in patients with CP ,27,28,29In this study, we investigated the effect of ESWT on spasticity in patients with CP during the follow-up period.We performed a meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We registered the protocol for this meta-analysis on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY protocol no. INPALSY202280066) . We systWe selected studies according to the following criteria: (1) ESWT was conducted for the management of spasticity in patients with CP; (2) the effect of ESWT was evaluated before and after treatment; and (3) the effect of ESWT was evaluated by comparing the results of the experimental and control groups. The exclusion criteria were as follows: (1) anti-spastic medications or botulinum toxin injections were conducted to participants in the control group; (2) the timing of the ESWT effect has not been clarified; (3) previous history of neurosurgery or orthopedic surgery for reducing spasticity; and (4) case reports, review articles, conference presentations or letters, and studies lacking sufficient data or results.2], and adverse effects). Since there was insufficient data in Vidal et al. [Duplicate publications were eliminated before two independent reviewers (MCC and YJC) assessed potentially eligible studies for inclusion in the analysis. The screening was based on a review of the titles and abstracts of the articles. Disagreements were resolved through consensus. After the primary screening, the two reviewers (MCC and YJC) independently investigated the full text of the eligible studies. The collected data comprised the first author’s name, publication year, sample size, demographic data, ESWT protocol, outcome variables , and the evaluation factors included were: (1) adequate sequence generation, (2) blinding, (3) incomplete outcome data, (4) allocation concealment, (5) selective outcome reporting, and (6) other potential sources of bias. The judgment of bias was expressed as “low risk,” “high risk,” or “unclear risk” . In addi2 statistics to measure the degree of inconsistency between the results. When the I2 value was less than or equal to 50%, the pooled data were considered homogeneous, and a fixed-effects model was used for analysis. Conversely, if the I2 value was greater than 50%, the pooled data were considered significant heterogeneity, and a random-effects model was applied. Continuous variables were analyzed, and standardized mean differences (SMDs) and 95% confidence intervals (CIs) were calculated. Statistical significance was set at p < 0.05. A funnel plot and Egger’s test were used to evaluate the publication bias using R version 4.1.2. The publication bias of individual studies was determined based on pooled estimates using funnel plots. We used Egger’s test to determine whether the funnel plot was symmetrical. p < 0.05 was set as indicating the possibility of publication bias.Review management software (RevMan 5.3) was used for the statistical analysis of the pooled data. For each analysis, a heterogeneity test was performed using IA preliminary search of all databases resulted in the selection of 969 potentially relevant studies . A totalThe selected studies included 47 patients with CP. In a study by Amelio et al. , 12 patiVidal et al.’s study was an RCT, and the risk of bias was assessed using Cochrane Handbook 5.1 Assessment Tool. Vidal et al. reported a low risk of bias in random sequence generation . However2 values were > 50%. For outcomes at 2 and 3 months after ESWT, a fixed-effects model was adopted. Analysis of the changes in MAS revealed a significantly larger reduction than that in the control group immediately, 1–2 weeks, and 1 month after ESWT    . However = 0.18) . 2 values were >50%. A fixed-effects model was used for the analysis of the 2- and 3-month outcomes. The ankle ROMs were significantly more increased than those in the control group at all the evaluation time periods   .2 = 87%). Data at 1 week, 1 month, and 3 months after ESWT were analyzed using a fixed-effects model. The plantar surface area contacting the ground during standing was significantly increased after ESWT at all evaluation time periods than that in the control group   .Furthermore, Amelio et al. and Gonkova et al. reported no adverse effects. However, in the study by Vidal et al., small superficial hematomas, petechiae, and light pain occurred during ESWT in three patients.p = 0.211; ROM at 1 month, p = 0.614)  .In our meta-analysis, spasticity measured using MAS was significantly reduced by a greater degree after ESWT than in the control group. However, the reduction in spasticity was sustained for 1 month and undetectable at 2 and 3 months after ESWT. However, after ESWT, significant increases in passive ankle ROM and plantar surface area contacting the ground during standing were found compared with that in the control group. Their increments were sustained for up to 3 months after ESWT. No major adverse effects were reported in any of the included studies.Moreover, when a significant change existed in spasticity, ankle ROM, and plantar surface area contacting the ground during standing, the absolute value of the effect size ranged from 1.94 to 8.13. Based on Cohen’s study, these effect size values are interpreted to significantly reduce spasticity or spasticity-associated symptoms in patients with CP .The pathophysiology of spasticity or hypertonus is complex and not fully understood. It is caused by various spinal and supraspinal pathways as well as multiple neuronal mechanisms that lead to increased reflex excitability. Recent studies have revealed more regarding the pathophysiology of spasticity. Downregulation of the potassium chloride cotransporter, KCC2, in motor neuron membranes appears to be involved in experimentally induced central nervous system injury in rodents . This deEl-Shamy et al. conducted a study to evaluate the effects of ESWT on gait pattern in patients with hemiplegic CP and found that several gait parameters, including cadence, speed, stride length, stance phase time, and cycle time, improved significantly after the application of ESWT . Amelio The mechanism by which ESWT controls spasticity has not been elucidated. However, it has been proposed that ESWT can cause rheological effects, which alter the elasticity and extensibility of the muscles ,37. ESWTIn clinical practice, two types of shockwave generators are used: focused ESWT and radial ESWT. These two types of ESWT differ in physical properties, generation modes, magnitudes of standard parameters applied, and depths attained. Electromagnetic, electrohydraulic, and piezoelectric sources are used to produce focused ESWT. The pressure produced by the focused ESWT rapidly increases, and energy is absorbed as deep as 12 cm . Due to Consensus is lacking on the appropriate or most effective number of shockwave impulses and sessions. However, 2000–3000 shockwaves and strike frequencies of 1–8 Hz applied at weekly intervals are recommended . BleedinIn 2019, Kim et al.  conducteOur meta-analysis was limited because we included only a small number of previous studies. Furthermore, because of the insufficient number of previous studies, we could not perform the analysis according to the shockwave type or application mode. Moreover, the designs of the studies examined in our meta-analysis differed considerably. To obtain more accurate knowledge of ESWT on spasticity in patients with CP, a larger number of well-designed, high-quality studies should be conducted on this topic. In addition, we analyzed only the MAS to measure the degree of spasticity. MAS is the most commonly used tool for assessing spasticity, and alternatives for measuring changes in spasticity after ESWT are lacking. However, although MAS is widely used to measure spasticity, it cannot describe the complex mechanisms of muscle stiffness. Also, MAS is subjective and depends on the experience of physicians who check for spasticity. Furthermore, it is also affected by soft tissue contracture or tightness and not only by spasticity. In future studies, additional tools are needed to evaluate the degree of spasticity. In addition, studies with combined treatment models are necessary to evaluate the effects of combined ESWT and pharmacological interventions.A significantly greater reduction in spasticity, ankle ROM, and plantar surface area contacting the ground during standing was observed in the ESWT versus the control group. While the increase in ankle ROM and plantar surface area contacting the ground during standing persisted for >3 months after ESWT, the reduction in spasticity measured using MAS persisted until 1 month after ESWT. These findings suggest that ESWT is a useful and effective therapeutic option for managing spasticity in patients with CP. To the best of our knowledge, this is the first meta-analysis to investigate the ability of ESWT to reduce spasticity or spasticity-associated symptoms in patients with CP according to the follow-up period.


Soldiers in the military are exposed to numerous stressors, including some that are of an extreme nature. The main objective of this military psychology research study was to evaluate soldiers' occupational stress. Even though several tools have been developed to measure stress in this population, to date, none have focused on occupational stress. Hence, we developed the Military Occupational Stress Response Scale (MOSRS) to provide a tool to objectively measure soldiers' occupational stress responses. An initial pool of 27 items was assembled from the literature, existing instruments, and interviews with soldiers. Of those 27, 17 were included in the MOSRS. The scale was subsequently completed by soldiers from one military region, and exploratory factor analysis (EFA) and confirmatory factor analysis were conducted using Mplus8.3 and IBM SPSS 28.0 software, respectively. A total of 847 officers and soldiers were selected for scale testing, and 670 subjects were retained after data cleaning and screening according to the set criteria. After performing the Kaiser–Meyer–Olkin (KMO) and Bartlett's test, principal components analysis (PCA) was appropriate. The PCA yielded a three-factor model  with the items and factors strongly correlated. The confirmatory factor analysis revealed loads ranging from between 0.499 and 0.878 for each item. The Cronbach's α coefficient of the MOSRS was between 0.710 and 0.900, and the Omega reliability was between 0.714 and 0.898, which were all higher than the critical standard value of 0.7, indicating that the scale has good reliability. Analysis of the discrimination validity of each dimension revealed that the scale has good discrimination validity. The MOSRS demonstrated sound psychometric characteristics with acceptable reliability and validity, suggesting that it could be used to assess occupational stress in military personnel. The military profession exposes personnel to a large number of stressors, including some that are very extreme and more intense than those experienced in other human activities. While the research category of extreme combat stress reactions causing casualties has not attracted attention from psychologists, it has attracted great attention from psychiatrists. Military psychiatrists primarily treat and permit the wounded members to return to work. A review of the literature found that military stress research focuses heavily on the fields of combat stress and post-traumatic stress disorder (PTSD) and less on daily stress from military occupations.Regarding the relationship between occupational stress and physical and mental health, numerous studies have been focused in the same direction, examining the close relationship between occupational stress and physical health. Kawakami and Tsutsumi  summarizThe question of how to objectively and accurately evaluate these factors should form the basis of further study on the relationship between stress and work environment, performance, and physical and mental health. There is also a need to compare different occupational stress levels and take effective intervention measures to optimize the work stress level. A universally applicable, objective, and accurate assessment tool needs to be established to assess the stress experienced by military personnel.Based on the study of occupational stress in military personnel in their home country and abroad, foreign military personnel most commonly use occupational stress questionnaires intended for the general population, such as the Occupational Stress Inventory—Revised (OSI-R)  were selected using the convenience sampling method for interviews. The entries and sentences of related stressors were extracted from the stress event database and compiled based on the occupational stress structure of the military inside and outside of the military.A total of 847 soldiers were selected to test the scale, and 670 subjects were retained after cleaning and screening according to the standards set in the previous stage. Among them, 335 were randomly selected for exploratory factor analysis (EFA) (sample 1 data), and 335 were randomly selected for confirmatory factor analysis (sample 2 data). According to the data cleaning and screening criteria, subjects with response times of <500 s and those who answered linearly and regularly were excluded.A total of 335 subjects were included in sample 1. This data included information on the age of personnel, ranging from between 17 and 42 years (22.97 ± 4.263 years), and the length of military service, which ranged from 1 to 24 years (1.10 ± 0.294 years). The sample included 303 men (90.4%) and 32 women (9.6%) who were divided into the following groups: conscripts, noncommissioned officers, and officers, of which two did not fill in the category of rank. There were 161 conscripts (48.1%), 126 non-commissioned officers (37.6%), and 46 officers (13.7%). Their education level were divided into the following five categories: junior high school, senior high school, junior college, bachelor's degree, and master's degree or above, among which five people did not fill in the education level category. There were 43 junior high school students (12.8%), 158 senior high school students (47.2%), 73 junior college students (21.8%), 52 undergraduate students (15.5%), and 4 students with master's degrees or above (1.2%).Sample 2 included 335 subjects, including data on the age range of the personnel, which ranged between 17 and 34 years (22.69 ± 3.412 years) and the length of military service, which was between 1 and 15 years (3.87 ± 3.354 years). There were 289 men (86.3%) and 46 women (13.7%) whose grades were categorized into conscripts, non-commissioned officers, and officers, one of which did not fill in the grade category. There were 178 conscripts (53.1%), 118 non-commissioned officers (35.2%), and 38 officers (11.3%). Their education levels were divided into the following five categories: junior high school, senior high school, junior college, bachelor's degree, and master's degree or above. There were 42 junior high school students (12.5%), 154 senior high school students (46.0%), 95 junior college students (28.4%), 40 undergraduate students (11.9%), and 4 students with master's degrees or above (1.2%).The effects of the occupational stress response were divided into physiological response, psychological response, and behavioral response. The items of the occupational stress response scale for military personnel were chosen as follows: some occupational stress response items were taken from existing theoretical research literature; items from existing stress response scales that were suitable for the actual situation of the military arms were extracted or rewritten appropriately; and items were developed according to the results of the interview outline.Microsoft Excel was used for data entry. After data entry, Statistical Package for the Social Sciences (SPSS) version 28.0 (IBM) was used to manage, describe, statistically analyze, and project the data of sample 1, and EFA was conducted. The confirmatory factor analysis was performed on the data of sample 2 using Mplus8.3.The scale was set to four points from 0 to 3. The higher the score, the greater the pressure value. There was no reversal clause. Descriptive statistical analysis was used to analyze the items in terms of minimum and maximum, average, standard deviation, kurtosis, and skewness.t-test, the decision value of each item was between 7.541 and 19.396, which is higher than 3 and meets the requirements.As shown in p-value of < 0.01, the difference was extremely significant, with a KMO value of 0.918, close to 1, allowing for PCA.As shown in 2 = 2.247, root mean square error of approximation (RMSEA) = 0.061, 95% confidence interval (CI) (0.051–0.071), comparative fit index (CFI) = 0.942, Tucker–Lewis index (TLI) = 0.932, standardized root mean square residual (SRMR) = 0.048, and model fit indicators have a good fit, indicating that the Military Occupational Stress Response Scale (MOSRS) has good structural validity.Confirmatory factor analysis showed that χAs shown in As shown in As shown in In this study, the factor structure of a new scale—the MOSRS—was explored through exploratory and confirmatory factor analyses. The results showed that the scale included the following three factors: a physiological response, a psychological response, and a behavioral response. Physiological responses mainly included physical symptoms and physical discomfort caused by occupational stress and were biased toward physiological and pathological symptoms. Psychological responses primarily included cognitive symptoms and negative emotions resulting from work stress, while behavioral responses mainly focused on behaviors and negative styles of coping with work stress.PCA supported the three-factor model of the MOSRS and showed that the factor structure was clear and had high structural validity. The three-factor model was created based, in part, on the research of Schmitt , who repIn the past, stress scales for soldiers have focused on external stressors including events and effects in the environment rather than on subjective and internal feelings (Fevre et al., Although the development of the scale has achieved good reliability and validity, which reflects occupational stressors and occupational stress responses, there are still some limitations of this study that we acknowledge and additional areas of research to be explored are as follows:First, the subject group selected for this study was relatively homogenous, and only officers and soldiers of one military department participated. Hence, it is not known how generalizable our findings are to other populations. In the future, additional research using the MOSRS should be undertaken with other military arms and divisions to further evaluate the scale's value and effectiveness.Second, after objectively and effectively evaluating the professional stresses faced by soldiers, measures should be taken to guide officers and soldiers in conducting effective stress management. Future studies, which are outside the scope of the current study, may develop military occupational stress assessment manuals and establish officers' and soldiers' occupational stress files, according to different occupational pressure levels. In addition, effective intervention measures may be implemented to maximize the potential of the military workforce in their careers, improve the officers' and soldiers' management of stress and their stress responses, and optimize their work performance and physical and mental health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.QW and ZY: conceptualization, methodology, software, investigation, formal analysis, and writing—original draft. RQ: data curation, visualization, and investigation. SC: resources and supervision. XZ: conceptualization, funding acquisition, resources, supervision, and writing—review and editing. ZH and WX: supervision and writing—review and editing. All authors contributed to the article and approved the submitted version.


Even though Italian Occupational Physicians (OP) are increasingly involved in the managing of overseas workers, their knowledge, attitudes, and practices (KAP) in travel medicine are mostly undefined. We, therefore, permed a KAP study specifically targeting the management of travelers’ diarrhea (TD) by OP. A total of 371 professionals  completed in 2 rounds (2019 and 2022) a specifically designed web questionnaire that inquired participating OP on their knowledge status (KS), risk perception, and management of TD through pre- and post-travel advice and interventions. Multivariable odds ratios (aOR) for predictors of a better knowledge status were calculated through regression analysis. Eventually, the majority of participants (53.4%) had participated in the management of cases of TD in the previous months, but only 26.4% were reportedly involved in pre-travel consultations. The overall knowledge status was unsatisfying , with substantial uncertainties in the management of antimicrobial treatment. Interestingly, only a small subset of participants had previously prescribed antimicrobial prophylaxis or treatment . Main effectors of a better knowledge status were: having a background qualification in Hygiene and Public Health , having previously managed any case of , and having higher concern on TD, reported by acknowledging high frequency  and severity  of this disorder. As the adherence of participating OP to official recommendations for TD management was unsatisfying, continuous Education on Travel Medicine should be improved by sharing up-to-date official recommendations on appropriate treatment options for TD. Escherichia coli (ETEC) has been historically recognized as the most common cause of TD, but in recent years its prevalence has declined worldwide, with increased occurrence of cases associated with enteroaggregative E. coli (EAEC) and enteropathogenic E. coli (EPEC) [Travelers’ diarrhea (TD) has been defined as the increase in the frequency of bowel movements during a trip abroad, up to three or more loose stools per day ,2,3,4. Ci (EPEC) . TD occuDuring the last decade, global improvements in sanitation and hygiene infrastructures have led to a substantial decline in TD incidence rates ,6,8,9,10In fact, while fatal cases have been only anecdotally reported , TD is aTD has acquired a distinctive significance in occupational settings, as individuals afflicted by TD during their travels are also precluded from participation in daily work-related activities ,11,13,14In this regard, it should be stressed that our understanding of knowledge, attitudes, and practices (collectively KAP) of medical professionals on TD is mostly based on surveys from professionals working in academic or tertiary care units specialized in infectious/tropical diseases and travel medicine ,27,28,29In a preliminary study that we performed shortly before the inception of the SARS , we specThis cross-sectional questionnaire-based study was originally designed as a follow-up study to our original survey on GPs in the Emilia Romagna region of Italy  (see STR(a)A summary of the aims of the survey;(b)A link to the full informed consent, whose acceptance through a specific dichotomous question (yes vs. no) was mandatory for receiving;(c)The direct link to the questionnaire ;(d)A preliminary question about the respondent’s occupational settings, i.e., whether he/she was actually working as OP or not.In both rounds of delivery, the principal investigator (MR) initially contacted the administrators of the recipient group, requesting their preventive authorization for sharing the study invitation. The latter included:The questionnaire was made available to all group members receiving the questionnaire and agreeing with the participation during the time period mentioned above and once more between 15 July and 15 August 2022, but only participants who reportedly worked as OP at the time of the survey were able to complete it.The questionnaire was previously validated in a preliminary study performed on GPs , and it Demographic characteristics of the participant were retrieved, including age, gender, seniority, and any previous personal experience with TD ,36,37. MEscherichia coli—ETEC, Shigella spp., Vibrio cholerae, Norwalk virus (NoV). All included items had been previously designed through accurate analysis of similar studies and Guidelines of the International Society of Travel Medicine available at the time of the pilot study (2019) [Participants were inquired about some common misconceptions about TD through a series of 20 true–false statements  and 3 multiple-choice questions focusing on the etiology of watery diarrhea, dysentery, and gastrointestinal syndromes with diarrhea and vomiting  ,26. A su ,26. A sy (2019) ,38,39,40Perceived risk represents a significant effector for eventual behaviors ,37,41,42We specifically inquired OP participating in our study whether, in the previous 12 months, they had recommended any antimicrobial prophylaxis to travelers and antimicrobial treatment in suspected cases of TD. Both items were assessed by means of a fully labeled 5-point Likert scale that ranged from “never” , to “rarely” , “sometimes” , “often” , and “always” .A preliminary check on collected data was performed by two researchers who independently ensured their consistency, while the primary investigator examined unclear responses to arbitrarily determine the correct answer. Partially completed questionnaires or questionnaires lacking demographic data were excluded from eventual analyses.p < 0.100 for rejecting normal distribution), while categorical variables were reported as percent values. Normally distributed continuous variables were compared by means of Student’s t-test or ANOVA, where appropriate, while not normally distributed continuous variables were compared through Mann–Whitney or Kruskal–Wallis tests for multiple independent samples. Similarly, the association between continuous variables was assessed through Pearson’s correlation coefficient  or Spearman’s rank correlation coefficient .In the descriptive analysis, continuous variables were initially expressed as mean ± Standard Deviation (SD) and tested for normal distribution by means of the D’Agostino and Pearson omnibus normality test  was assessed by calculation of Cronbach’s alpha coefficient of reliability on the items of the knowledge test. Values of Cronbach’s alpha range from 0 to 1, and although the standard for considering the estimate as “good” or conversely “unacceptable” are entirely arbitrary, many methodologists recommend a minimum value ranging between 0.65 and 0.8 (or higher in many cases) .p value < 0.05 and corrected for the following factors: gender, age, having previously managed any RD case and having participated in the survey during 2022 vs. 2021. Adjusted Odds Ratios (aORs) with their respective 95% confidence intervals (95% CI) were calculated accordingly .Association of individual factors , personal attitudes , individual practices  with the higher knowledge status was initially evaluated through the Chi2 test. A multivariable regression analysis was then modeled with all variables that in univariate analysis were associated with higher knowledge status with a The study was conducted according to the guidelines of the Declaration of Helsinki. A preventive ethical review and approval were waived for this study because of its anonymous, observational design and due to the lack of clinical data about patients that could configure the present research as a clinical trial. Participants were also guaranteed that retrieved data would be stored only for the time required by data analysis. The study, therefore, did not configure itself as a clinical trial, and a preliminary evaluation by an Ethical Committee was not required, according to Italian law .As shown in In this regard, it should be stressed that while the parent group 2019 only included OP, the discussion groups targeted in the second round of 2022 was open to all medical professionals, and the actual number of the active OP was unknown at the time of the survey.p = 0.096). The average seniority as OP of respondents was 13.3 years ± 11.7, and again no substantial differences were identified between respondents in 2019 vs. 2022 . However, the share of participants reporting a professional experience as OP ≥ 10 years at the time of the survey increased from 69.1% in 2019 to 75.8% in 2022 (p = 0.002).In summary, a total of 371 participants were included in the analyses . The meap = 0.003).Of them, 13.5% reportedly worked as General Practitioners alongside their main activity as OP, with no differences between 2019 and 2022, while the share of professionals having a background in Hygiene and Public Health (10.5% for the sample as a whole) was greater among participants from 2019 (14.0%) than in those from 2022 . On the contrary, the share of respondents having managed at least one case of TD in the previous 12 months decreased from 56.4% in 2019 to 47.7% in 2022 (p = 0.035), for a pooled estimate of 53.4%. Similarly, participants having been involved in preventive consultations in the previous 12 months decreased from 32.1% in 2019 to 15.6% in 2022 .Focusing on the previous professional interaction with TD, around 1/3 of participants were previously affected by this disorder (36.1%), with no substantial differences among participants recruited in 2019 (35.0%) compared to 2022 , being substantially skewed and not normally distributed .As shown in p < 0001; Q10 100% vs. 91.4%, p = 0.001; Q18 100% vs. 88.9%, p < 0.001). Some significant uncertainties were reported on the epidemiological features of TD, as only 27.2% had knowledge that in the last decades, the global incidence of TD is actually decreasing , but up to two-thirds of participants correctly identified in Southern Asia as the area at highest risk for TD , and the wilderness travelers as individuals at particularly high risk for developing this disorder . Interestingly, the awareness of the high risk for TD in Southern Asia decreased from 74.5% in 2019 to 53.1% in 2022 (p < 0.001).The determination of Cronbach’s alpha resulted in an estimate of 0.707, suggesting an acceptable internal consistency of the questionnaire. Single statements are reported in p = 0.006), and 62.3% correctly recalled the proper use of loperamide and analogous in managing of TD , but only 29.1% had any familiarity with the use of bismuth subsalicylate .As regards treatment options, most respondents correctly acknowledged fluoroquinolones as antibiotics that can be used as first-line options for TD , while only one third of participants understood that these antibiotics are not specifically recommended for patients at high risk of complications . Moreover, around 60% of participating OP correctly identified rifaximin as a preventive option for patients at high risk for complications  and as a therapeutic option for severe, non-dysenteric TD . Alternative non-antimicrobial treatment was similarly associated with a mixed pattern of understanding, as 59.0% acknowledged the lack of consolidated evidence for recommending the use of probiotics as a preventive option , and particularly anti-rotavirus immunizations  immunizations for preventing TD. In fact, knowledge gaps appeared concerning TD etiology. Even though 59.8% of the sample correctly identified the etiologic role of bacterial infections , only 35.6% identified in Shigella spp. the most frequently reported cause of dysentery , while a larger share of participating OP improperly recalled this role for ETEC (36.9%), followed by Campylobacter spp. (10.8%), Vibrio cholerae (7.3%), NoV (5.7%), and amoebic infections (3.8%). Similarly, only 40.7% of OP correctly identified ETEC as the main cause of watery diarrhea (Q13), followed by Campylobacter spp. (19.9%), NoV (12.9%), Vibrio cholerae (11.3%), and eventually Shigella spp. (9.7%), and amebic infections (5.4%). Finally, only a quarter of participants  correctly associated NoV with a syndrome characterized by diarrhea and vomiting (Q15), whereas the majority of respondents identified the main etiology in other pathogens, such as Shigella and Campylobacter spp , followed by ETEC (12.9%), Vibrio cholerae (11.9%), amoebic infections (8.6%), and Cryptosporidium spp (1.6%).When dealing with available vaccination strategies, many participants acknowledged the availability in Italy of a typhoid vaccine , while substantial uncertainties were reported on the reliability of anti-cholera . On the contrary, only one-third of all respondents acknowledged TD as a severe or even highly severe condition (32.9%), and even though the corresponding share increased in 2022 compared to 2019 (36.0% vs. 31.3%), the difference was not substantial (p = 0.364).Overall, 60.1% of participants perceived TD as a condition that in overseas workers can be acknowledged as frequent or highly frequent, with no substantial differences between 2019 (60.5%) and 2022 , with no substantial differences between estimates for 2022 (46.5% ± 18.0) and 2019 .Corresponding RPS was estimated to be 47.1% ± 19.5 . As shown in p < 0.001): in other words, individuals characterized by a better understanding of TD reported a higher risk perception of this disorder.As shown in p < 0.001), reporting a background qualification in Hygiene and Public Health , recognizing TD as a frequently reported , and severe disorder  among overseas workers.In univariate analysis , a betteIn multivariable analysis, the outcome variable of better knowledge status on TD was assessed through a binary logistic regression model that included the following explanatory variables : male geEventually, the higher knowledge status was positively associated with reporting a background qualification in Hygiene and Public Health , having managed any case of TD in the previous 12 months , recognizing TD as a frequently reported  and severe disorder .In our cross-sectional study on 371 Italian OP, the actual knowledge and risk perception of sampled participants on TD were far from optimal, suggesting that substantial formative interventions on travel medicine should be implemented in order to improve the capability of these professionals to properly cope with the requirements of overseas workers. As suggested by previous studies on frontline healthcare professionals, knowledge status is critical for guaranteeing the appropriate spreading of preventive measures and updated management options for TD ,47,48,49While the better understanding of TD in professionals having a background qualification in Hygiene and Public Health can be explained through the greater familiarity of those professionals with infectious diseases ,40,50,51According to the current legal framework ,55,56,57Unsurprisingly, TD represented a considerable issue not only for professionals actively involved in pre-travel consultations: even though the share of respondents who had managed at least one case of TD in the previous 12 months was greater among professionals participating in pre-travel consultation than in those who were reportedly not involved (79.6% vs. 44.0%), the resulting amount of time spent in these activities was substantial 44.0%; . On the S. typhi, V. cholerae, and rotavirus, none of these agents is acknowledged as a major cause of TD [S. typhi vaccination is only recommended for travelers to endemic settings [In fact, available options for primary prevention through effective vaccines were affected by extensive misunderstandings. Even though reasonably effective vaccines have been made available against se of TD , and S. settings ,3,5,21.Moreover, only a very limited subset of recruited OP actually recommended antimicrobial treatment either as a preventive or treatment option ,12,29,48Nonetheless, participants were improperly reluctant to rely on antimicrobial treatment , with figures that were hardly comparable to our previous study on Italian GPs , and alsThe knowledge gaps on bismuth subsalicylate and probiotics may be particularly informative about the shared unfamiliarity with updated recommendations on the management of TD among the recipients of our survey. Some evidence suggests that bismuth subsalicylate may be an appropriate option in the management of mild symptoms of TD, eventually delaying or even avoiding the requirements for antibiotic treatments ,3,5,21. Limits. Despite the potential significance of this study, several substantial shortcomings and limits must be addressed and discussed.First of all, the present study has been designed as an Internet-based survey, whose shortcomings are therefore shared by our report ,76. EvenSecond, even though we implemented a very flexible and reliable design for the knowledge test and risk perception assessment ,38,39,43Third, not only was our study designed as a cross-sectional study, but our sample was also of limited size and should be only cautiously interpreted as representative of the Italian National level ,31,32. IFourth, it is important to stress that the collected data were not externally validated. Consequently, there were no reliable estimates on the TD cases that were actually treated by participating OP. More precisely, how the recommendations on the managing of TD have been actually shared with the affected workers and then implemented by potential recipients and shared with other healthcare providers still remains unclear.Last but not least, the inception of the SARS-CoV-2 pandemic has led to the dichotomization of our study in two distinctive “rounds”. In order to avoid duplicated questionnaires, in 2022, we opted for sharing the questionnaire in discussion groups that were well distinctive from the original one. However, this choice has led to the potential inconsistency of the study populations, as otherwise suggested by some differences in the composition of the sample, particularly the share of professionals having a background in Hygiene and Public Health, and by the heterogeneous referral of several items at knowledge test. Such issues can be explained as an indirect consequence of the pandemic, whose requirement for professionals having a background in Hygiene and Public Health has reasonably led some professionals to shift from private practice in Occupational Medicine to other roles in Public Health settings ,81,82. SIn conclusion, our data collectively hint towards active involvement of sampled Italian OP in pre-travel advice of overseas workers. Interestingly, the tasks they claimed seemingly exceeded the usual interventions of OP: even though Occupational Medicine is mostly acknowledged as a diagnostic and preventive branch of medicine, as well as recalled by the ICOH Code of Ethics (third edition 2014), they not only participated in pre-travel advice but also guaranteed post-travel management. As most of them are actively involved in the management of TD, improving the core competencies of OP on travel medicine represents an urgent need in a globalized world that is rapidly recovering from the constraints of the SARS-CoV-2 pandemic. This appears particularly needed, as substantial knowledge gaps were identified in the management of anti-microbial therapy. Again, the unbalance between the core curriculum of specialization courses in Occupational Medicine and their actual tasks may both explain such gaps and suggests that focused informative interventions on appropriate etiological  prophylaxis and treatment of infectious diseases may be of some interest for the management of overseas workers. In times that require talking about global health, our study underlines the importance of the OP, increasingly called to play a crucial role in the protection of health and safety of workers, notably including overseas workers, and call for implementing specifical training programs on travel medicine and focused anti-microbial prophylaxis and therapy, both in graduate schools of Occupational Medicine and through Continuous Medical Education interventions.


Rotating shift work places a serious burden on nurses’ physical and psychological health. Gastrointestinal (GI) symptoms are a common complaint among shift workers. This study assessed GI symptoms and identified the associations between dietary habits, psychological status, and sleep quality among rotating shift nurses. Data from 125 female nurses in rotating shifts who worked at two tertiary hospitals in South Korea were collected using a questionnaire that included the Gastrointestinal Symptoms Questionnaire; the Dietary Habit Questionnaire; the Depression, Anxiety, Stress Scale (DASS)-21; and the Pittsburgh Sleep Quality Index (PSQI). All participants experienced various GI symptoms, and 47% of them complained of at least one severe GI symptom. There were significant differences in GI symptom scores according to the status of depression, anxiety, stress, and sleep quality. In multiple linear regression analysis, the factors associated with an increase in the occurrence and severity of GI symptoms were poor sleep quality and morbid anxiety and stress. The model explained power at 43.2%. As most nurses in rotating shifts experience GI symptoms, they should receive counseling and training programs at work to alleviate psychological symptoms, improve sleep quality, and pay more attention to their health status as well as GI symptom management. Shift work, defined as an irregular and unusual work schedule outside the day working times ,2,3, is Gastrointestinal (GI) symptoms and dysfunction, including diarrhea, flatulence, constipation, bloating, and abdominal pain, most commonly occur among shiftwork nurses ,16,17,18Shift workers experience changes in meal patterns, including skipping meals, consumption at unconventional times, and increased high-fat intake and caffeine consumption ,7,14. ThThe shift system of Korean nurses generally includes day, evening, and night shifts and is based on a rapidly rotating shift , while nThis study was a cross-sectional and secondary analysis using part of a study conducted to identify the health problems of hospital nurses in South Korea in 2020 . A totalThe appropriate sample size for this study was 109, calculated using the G-power program for multiple linear regression with a priori sample size estimation. We computed it as estimated to have a medium effect size of 0.15, significance level of 0.05, and power of 0.80, with eight predictive variables based on previous studies ,4,15,18,2). Job-related characteristics were the department of work, work experience, and the average workdays of day, evening, and night shifts per month.The demographic characteristics examined were age, marital status, living situation, regular exercise, and body mass index -21 downloaded from the DASS Website . The thrSleep quality was measured with the Korean version of the Pittsburgh Sleep Quality Index (PSQI-K) ,39. The p-values. Demographic and job-related characteristics and outcome variables were summarized using descriptive statistics with frequencies, proportions, means, and standard deviations (SD). We compared differences in the total GI symptom score by categorizing the outcome variables using independent t-tests and one-way ANOVA, and the Bonferroni test was used for post hoc analysis. Variables that were not normally distributed or had a sample size of a group lower than 15 were used to conduct non-parametric tests, including the Mann–Whitney or Kruskal–Wallis test. We used the independent t-test, chi-squared (χ2) test, and Fisher’s exact test to compare the outcome variables between the two groups with and without at least one severe GI symptom. Multiple linear regression was performed to identify variables associated with the severity of GI symptoms in the participants.Data were analyzed using SPSS version 25.0 , with a 0.05 level of significance and two-sided n = 102, 81.6%). Of the participants, 78.4% were single and 37.6% lived alone. Approximately half of the nurses had regular exercise, and the average BMI was 21.2 kg/m2 (SD = 2.8). The largest number of participants worked in the general ward , followed by intensive care unit nurses , and emergency room nurses . The mean work experience was 64.5 months (SD = 35.1), and most nurses  had worked for under 120 months. The average workdays per month were 6.3 days (SD = 2.1) for day shift, 6.6 days (SD = 2.0) for evening shift, and 5.7 days (SD = 1.2) for night shift , and most participants were under 30 years old (ht shift .n = 59) complained of at least one severe symptom. n = 102, 81.6%) were the most prevalent, followed by abdominal rumbling , bloating , heartburn , and postprandial fullness . The main GI symptoms that nurses complained of were empty feelings , bloating , abdominal rumbling , and postprandial fullness .All participants experienced at least one GI symptom, and 47.2% (p = 0.671), and caffeine consumption group, who consumed caffeine more than one cup per day  than those in the other groups. When the differences in dietary habits between nurses with (n = 59) and without (n = 66) at least one severe GI symptom were compared, the mean caffeine consumption per day was significantly higher in the group with severe GI symptoms . In addition, nurses with severe GI symptoms consumed less healthy foods (p = 0.017). In particular, there were significant differences in the frequency of protein (p = 0.020) and vegetable (p = 0.020) intake between the two groups . There were no significant differences, although the total score of GI symptoms was higher in the alcohol consumption group, who consumed alcohol more than three times per week , anxiety (p < 0.001), and stress (p < 0.001), the higher the GI symptom score. In addition, the mean GI symptoms total score was significantly higher in the poor sleep quality group than that in the good sleep quality group . In comparison with severe symptom and without severe symptom groups, the mean scores for depression , anxiety , and stress  were significantly higher in nurses with severe GI symptoms. The prevalence of mild or moderate depression (74.6% vs. 30.3%), anxiety (62.7% vs. 22.7%), and stress (28.8% vs. 6.0%) was higher in the group with severe symptoms. The sleep quality of nurses with severe GI symptoms was significantly poorer than that of the other group , while the proportion of nurses with poor sleep was not different between the two groups .As shown in p = 0.088) and unhealthy dietary habits  were excluded as predictive factors because they were not correlated with the GI symptom score in the Pearson’s correlation coefficient analysis. We categorized the depression, anxiety, and stress variables as the normal, mild and moderate groups to determine whether psychological distress that occurred was more important than a numerical score of psychological status, and then converted them to dummy variables. Factors associated with an increase in GI symptom total score were the prevalence of anxiety , stress , and sleep quality . The most influential variable was sleep quality; poor sleep quality increased the severity of GI symptoms. The predictive model explained 43.2% of the power   .In South Korea, many nurses experience rotating shift work over an extended period ,32,40 anIn this study, all rotating shift nurses had at least one GI symptom; 47% complained of more than one severe GI symptom, and the most frequent GI symptoms were an empty feeling, abdominal rumbling, bloating, heartburn, and postprandial fullness. Prior research indicated that the prevalence of GI disorders was 72.3% among Egyptian shiftwork nurses, and the most frequent symptom was reflux syndrome . Among IOur findings showed that nurses with severe GI symptoms consumed more caffeine per day and less protein and vegetables. In other studies ,44, GI sIn this study, the prevalence of depression, anxiety, and stress was 51.2%, 41.6%, and 16.8%, respectively. A higher degree of psychological distress, including depression, anxiety, and stress, was linked to a higher GI symptom score, and the prevalence of psychological distress was significantly higher in nurses in rotating shifts who complained of severe GI symptoms. This is similar to the finding that FBDs are associated with higher levels of depression and anxiety . These rOur finding that GI symptoms are associated with poor sleep quality is similar to previous findings ,18. HoweThrough multiple linear regression analysis, we found that the severity of GI symptoms was likely to increase among nurses in rotating shifts who had anxiety (β = 0.21) and stress (β = 0.17) problems and had poor sleep quality (β = 0.36). According to a study by Ibrahim et al. , the occPoor sleep quality was a crucial predictive factor for worsening GI symptoms in this study and has been shown in previous studies to be an important factor associated with GI problems ,15,18. MThe results of this study should be interpreted with caution due to several limitations. First, this study was conducted with rotating shift nurses at only two tertiary hospitals; therefore, it may not be generally representative of nurses in South Korea. Second, as the reading of results relied only on subjective data using questionnaires, there might be a gap between self-reported GI disorders and actual GI problems in nurses. In addition, there was a limitation in that detailed dietary habits such as mealtime, frequency, and quantity of each nutritional intake were not obtained due to the investigation using categorical variables. Finally, the impact of the shift work environment could not be confirmed because this study focused on identifying an association between dietary habits, psychological distress, and sleep quality with GI symptoms. Despite these limitations, this study is significant and suggests a need for efforts to reduce the prevalence of high GI symptoms among nurses. Moreover, it presents management directions and emphasizes the importance of establishing an organizational system for GI symptoms and health promotion by comprehensively confirming the influence of psychological distress and sleep quality on GI symptoms.We conducted a descriptive cross-sectional study in two tertiary hospitals in South Korea to identify the association between GI symptoms, eating habits, psychological problems, and sleep quality among nurses in rotating shifts. Many nurses working rotating shifts experience severe GI symptoms. They are influenced by comprehensive variables, such as lifestyle changes and a decline in the homeostasis regulation of physical, psychological, and sleep status caused by circadian rhythm misalignment. In particular, this study showed that the crucial factors related to the severity of GI symptoms are poor sleep quality, anxiety, and stress. We found that GI symptoms, which are often ignored for being are too familiar, must be a principal health problem that demands concern among nurses in rotating shifts. It is likely that these could be improved through the management of sleep and psychological conditions. To improve the health status of nurses in rotating shifts, focus should be placed on continuously identifying the factors that influence GI symptoms and developing a comprehensive program based on changes in lifestyle, psychological status, and sleep quality. Additionally, provisions and policies must be established to ensure a better workplace environment for nurses.


MDM2 is the principal inhibitor of p53, and MDM2 inhibitors can disrupt the physical interaction between MDM2 and p53. The half-life of p53 is very short in normal cells and tissues, and an uncontrolled increase in p53 levels has potential harmful effects. It has been shown that p53 is frequently mutated in most cancers; however, p53 mutations are rare in retinoblastoma. Therefore, therapeutic strategies aimed at increasing the expression levels of wild-type p53 are attractive. In this minireview, we discuss the potential use of nutlin-3, the prototype small molecule inhibitor that disrupts the MDM2-p53 interaction, for the treatment of retinoblastoma. Although p53 has pleiotropic biological effects, the functions of p53 depend on its sub-cellular localization. In the nucleus, p53 induces the transcription of a vast array of genes, while in mitochondria, p53 regulates mitochondrial metabolism. This review also discusses the relative contribution of p53-mediated gene transcription and mitochondrial perturbation for retinoblastoma treatment. MYCN and MDM2  are the first synthetic molecules that act as potent and selective MDM2 inhibitors . It has in vitro . Nutlins of MDM2 . Nutlin- of MDM2 . However of MDM2 .The first clinically tested molecule is RG7112 IC50 ~20 nM), a new member of the nutlin family  nM, a ne, 23. FurThe imidazopyrrolidinone siremadlin is an advanced molecule that has been tested in patients with solid tumors and hematological malignancies harboring no p53 mutations. In a phase I clinical study involving patients with AML, siremadlin has demonstrated promising results . MoreoveNFRSF10B/TRAIL-R2 -monoubiquitinated proteins are stable in the cytosol and this modification acts as a signal for intracellular trafficking , which is present in the mitochondrial genome, thus, inhibiting respiration and inducing ROS . For such diseases, recent studies have focused on improving mitochondrial functions to restore vision, with therapeutic approaches directed to ameliorate mitochondrial membrane potential and stability, ROS production, mitochondrial fusion and fission, mitochondrial biogenesis, mitophagy, apoptosis, and mitochondrial DNA (mtDNA) transcription . RetinobKORB1 RPE cells, mitochondrial respiration associated with proton leakage is reduced during the electron transfer  region in 60 Rb tumors . The autHK1, and altered expression of Krebs cycle-related genes, suggesting reduced dependence on glycolysis and altered Krebs cycle, together with a preference for fatty acid metabolism when compared to the average of all pediatric retinal cells  for subconjunctival administration, and using this formulation, 2000- and 20000-fold higher intravitreal nutlin-3a levels can be achieved compared to oral and intravenous administration  and p53TKO mice . Results showed that the activation of p53 was higher and the response was overall better in mice constitutively expressing MDMX than in mice lacking p53, confirming that nutlin-3a activates p53 in cells with high MDMX levels   Clinical applications of MDM2 inhibitors are limited due to drug resistance as a result of mutations in MDM2, p53, or other proteins involved in the response pathways, or due to the off-target effects . To reduvia the p53 pathway  with inhibitors of DNA repair agents (B02), and this combination has synergic effects on p53-dependent cell death leading to a reduction in Y79 or RB1021 cell-derived tumors, but not in WERI-Rb1 cell-derived tumors in xenograft models . This dr pathway . These r pathway . Another pathway . However pathway . Moreove pathway , with loRecently, retinal organoids have been generated from patient-specific induced pluripotent stem cells (iPSC), which may help in further advancement of the preclinical research , 89, 90.We believe that the development of new preclinical models together with recent results from clinical trials, may open new opportunities for the treatment of retinoblastoma and encourage the use of MDM2 inhibitors.Conceptualization, GZ. Writing original draft preparation, GZ, RV, and AR. Writing review and editing, GZ, AR, EZ, SA, SA-S and RV. All authors contributed to the article and approved the submitted version.This work received no external funding. Funds for open access publication fees were received by RV as local funding from University of Ferrara (FIRD program).http://www.editage.com) for editing and reviewing this manuscript for English language.We would like to thank Editage (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.