Methodology. The present study includes a total of 200 individuals: 100 “individuals with T2DM referred to as “cases” and 100 healthy individuals referred to as “controls”. Extracted RNA was used to synthesise the cDNA for microRNA-330 and E2F1 mRNA expression. Taqman assay method has been used to analyse the microRNA-330 expression in the cases and controls and SYBR green dye was used to study the E2F1 mRNA expression.  Type 2 diabetes mellitus (T2DM) has emerged as an epidemic affecting more than four hundred million people throughout the world. It is a multifactorial disease with range of environmental and genetic factors responsible for its prevalence. In search of novel biomarkers for recording progress of various metabolic diseases, small noncoding RNA in general and microRNAs (miRNAs) in particular have emerged as the most promising biomarkers for diagnosing variety of diseases including diabetes. An increasing number of studies have been published, reporting the quantification of miRNAs in blood of subjects with diabetes and mostly aimed at identifying miRNA modulation in chronic diabetic complications. Due to its association with immune system homeostasis and potential capability to predict diabetes development, the profile of circulating miRNAs may also provide useful information about diabetes pathogenic mechanisms. Thus, the present study aimed to understand the role and expression of microRNA330 and E2F1 mRNA expression in patients with T2DM. p=0.01), increased TG (p=0.0008), and cholesterol (p < 0.0001) in hypertensive T2DM cases as compared to nonhypertensive T2DM cases. Obese patients showed significant increase in TG (p=0.01) and cholesterol (p < 0.0001) as compared to nonobese patients. Similarly, increased TG (p=0.001) and cholesterol (p < 0.0001) was observed in the case of alcoholic patients as compared to nonalcoholic patients. Also, patients with smoking habit showed increased TG (p=0.009p = 0.009), cholesterol (p < 0.0001), and VLDL (p=0.01) as compared to nonsmokers and differences among them was found to be statistically significant. Besides this, significant impact of risk factors like hypertension, obesity, alcoholism, and smoking were observed on microRNA-330 expression and E2F1 mRNA expression. A 7.72-fold increased microRNA-330 and 0.05-fold decreased E2F1 mRNA expression was observed among T2DM cases as compared to healthy controls. Increased expression of microRNA-330 was observed in hypertensive cases , obese cases , and smoking cases  as compared to nonhypertensive, nonobese nonalcoholic, and nonsmoking cases, and differences among them were found to be significant. Decreased expression of E2F1 mRNA expression was observed in patients with alcoholism  and smoking  while patients who were nonalcoholic and nonsmokers showed 0.07-fold increase in expression, and differences among them were found to be statistically significant.  Statistically significant difference was observed in all the selected 5 biochemical parameters among T2DM cases and healthy controls. Risk factors like hypertension were observed to be significantly associated with reduced HDL ( The present study demonstrated that increased level of microRNA-330 and decreased level of E2F1 mRNA expression were found to be associated with pathogenesis of T2DM patients. Risk factors such as hypertension, obesity, alcoholism, and smoking may be were found to be associated with microRNA-330 and E2F1 mRNA expressions, and it can prove a reliable biomarker for T2DM disease progression could be linked to chronic diabetic complications. First mentioned in the Egyptian manuscript about 3000 years ago, diabetes mellitus is one of the oldest diseases known to mankind . Over thIn order to plan adequate prevention of T2DM, the evaluation of traditional biomarkers primarily small noncoding RNA in general and microRNAs (miRNAs) in particular have recently emerged as key regulators of metabolism and metabolic disorders. Growing evidence indicates that microRNAs, which are class of small 20–24 nucleotide extended noncoding RNA, that potentially control the expression of thousands of genes , are alsAn increasing number of studies have been published reporting the quantification  of microRNAs in blood (either plasma or serum) of subjects with diabetes mainly with the aim of identifying circulating microRNA modulation in chronic diabetic complications . Increas2 (obese) and blood pressure ≥140/90 mmHg (hypertensive) were included for the study.The present study was ethically approved (14/08/2015/GKV/IEC/2015) and conducted at institutional ethical committee's Gurukul Kangri University, Haridwar, India. The study was conducted between 2015 and 2019, and all the criteria were followed such as fasting glucose (fasting plasma glucose level of 126 mg/dL or higher) and postprandial glucose (2 h plasma glucose level of 200 mg/dL) were monitored for diagnosis of T2DM disease. Totally 200 study subjects were included in present study: 100 were T2DM patients, and 100 were healthy controls. Written informed consent from all the participants was obtained, and further, 3 ml of peripheral blood sample was collected from all the T2DM cases and healthy control in plain vials and 1 ml in fluoride vials for blood sugar. The samples collected in plain vials were immediately centrifuged at 1500 rpm after collection and stored at -80 °C for further biochemical analysis and cell-free total RNA extraction experiments. A sample of T2DM patients along with healthy controls having BMI >25 kg/mOC, and total RNA from serum was extracted by Trizol reagent using recommended protocol (Invitrogen) and then stored at −80 °C until further processing.Stored serum were thawed at 4A) tailed microRNAs to cDNA using universal RT primer supplied with the manufacturer kit.100 ng from total extracted RNA was used for Polyadenylation and cDNA synthesis using advanced microRNA cDNA Synthesis Kit  by the following manufacturer protocol. Reverse transcriptase enzyme and other essential reagents were added subsequently for cDNA synthesis to switch in poly  for E2F1 mRNA expression following manufacturer protocol.μl reaction volume using the following program: 95 °C for 5 minutes, followed by 40 cycles of amplification, denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 30 seconds. E2F1 mRNA expression was done using SYBR green dye, and GAPDH was used as the internal control to analyse the expression, and the following program was used for amplification: 94°C for 10 minutes, followed by 40 amplification cycles of denaturation at 94°C for 40 seconds, annealing at 58°C for 40 seconds, and elongation at 72°C for 40 seconds. At the end, a melting curve was generated by programming fluorescent measurements every 1°C from 35°C until 95°C to ensure a single PCR product.Quantitative real-time PCR (qPCR) was performed to quantify the expression of microRNAs-330 by advanced Taqman assay using advanced master mix. SNU6 was used as the internal control to normalise the expression. The reaction was performed in a 25-–(ΔΔCt). The Mann-Whitney U test and Student t-test were conducted to observe the significant differences among groups. Results with more than or less than 1 were taken to indicate upregulation or downregulation of microRNA and mRNA expressions. All values have been presented as mean and standard deviation values with p value of <0.05 to be considered as statistically significant.All the statistical analysis was done using Graph Pad Prism version 6.05. QRT-PCR data were analysed by the relative cycle threshold (Ct) method, and every sample was examined in triplicate. MicroRNA and mRNA expression levels were calculated by the relative quantification method using 2The entire demographic characteristics have been mentioned in p < 0.0001), total bilirubin (p=0.0006), LDL (p < 0.0001), TG (p < 0.0001), cholesterol (p=0.02), VLDL (p < 0.0001), blood sugar fasting (p < 0.0001), and post-prandial sugar (p < 0.0001) was observed in T2DM cases compared to healthy control while lower level of HDL was observed in T2DM cases compared to healthy controls (p=0.02).Biochemical parameters were recorded in T2DM cases as well as healthy controls, and a comparison was drawn for each selected parameter. The difference among them was found to be significant . Higher U test and Student's t-test as shown in , cholesterol was 230.3 ± 43.84 (p < 0.0001) among T2DM cases with hypertension, while nonhypertensive patients had lower TG (177.6 ± 42.15) as well as cholesterol (194.8 ± 38.87), and differences among them was found to be significant.Various biochemical parameters were found to be associated with risk factors of the T2DM. For the present study, 5 biochemical parameters, i.e., HDL (high-density lipoprotein), LDL (low-density lipoprotein), TG (triglycerides), cholesterol, VLDL (very-low-density lipoprotein) have been compared against four risk factors, i.e., hypertension, obesity, alcoholism, and smoking. Impact of these four risk factors  has been assessed on each of the five given biochemical parameters. The observed association between them has been observed and evaluated using Mann Whitney shown in . Risk fap < 0.0001). T2DM patients who were obese were also observed to have higher level of TG (209.8 ± 47.14) while nonobese patients had low level of TG (178.9 ± 47.02), and difference among them was found to be significant (p=0.001). Level of cholesterol among obese patients were observed to be high (243.7 ± 43.32) while nonobese patients had lower cholesterol (181.4 ± 14.37) levels, and the difference among them was found to be significant (p < 0.0001). Similarly, two of the other risk factors like alcohol consumption and smoking also reflected significant association with T2DM. T2DM patients who were alcoholic had higher level of TG (213.0 ± 51.01) while nonobese had lower level of TG (186.6 ± 46.32), and difference among them was found to be significant (p=0.01). Further, patients who had habit of smoking showed higher TG level (205.3 ± 51.42) compared to nonsmokers (183.8 ± 16.17), and differences among them were found to be significant (p=0.009). Level of cholesterol among smokers was higher (233.4 ± 47.25) while nonsmokers showed lower level of cholesterol (183.8 ± 16.17), and differences among them was found to be significant (p < 0.0001). Higher level of VLDL was found in smokers (36.21 ± 5.43) while low level in nonsmokers (33.41 ± 5.06), and differences among them was found to be significant (p=0.01).Level of cholesterol among obese patients was higher (258.0 ± 47.45), while in nonobese patients had lower level (192.9 ± 23.52) comparatively, and difference among them was found to be significant . The impact of alcoholism was also seen to be profound on the expression of MicroRNA-330 in persons consuming alcohol as compared to nonalcoholics. Increased expression of microRNA-330 was observed in alcoholics (9.07-fold) as compared to nonalcoholics (6.12 fold) T2DM patients (p < 0.0001). Smoking showed 8.41-fold-increased microRNA-330 expression while nonsmokers had a 6.53-fold microRNA-330 expression, and differences among them were also observed to be statistically significant (p=0.01).The study overall exhibited 7.72 mean fold increase in expression of microRNA-330 in T2DM patients as compared to controls . Associap=0.002), manifesting a decrease in expression of E2F1 mRNA in alcoholic cases. Smoking group of patients also exhibited a decreased expression; it was only 0.03-fold while nonsmokers showed slightly higher expression of E2F1 mRNA with 0.07-fold, and difference among them was found to be significant (p < 0.0001).The impact of selected risk factors, i.e., hypertension, obesity, alcoholism, and smoking, was also observed on E2F1 mRNA expression . Alcoholp = ns) was observed (A negative correlation was observed between microRNA-330 and E2F1mRNA expression in T2DM patients. Correlation coefficient with 0.16 (observed . These pp=0.02) was observed  is one of the most frequently diagnosed metabolic diseases worldwide. T2DM is associated with genetic, lifestyle, and environmental factors which make tissues insulin-insensitive, resulting in high blood glucose levels. T2DM is also connected with severe long-term complications, including cardiovascular disease, renal failure, neuropathy, blindness, and increase in morbidity and mortality . Signifip value of 0.16. Increased levels of microRNA-330-3p can be related to reduction in E2F1 expression potentially and may cause impairment in proliferation of beta cell along with insulin production. It has been shown earlier that cell cycle-related genes involvement is essential to beta-cell compensation [G in 2017 demonstrated that upregulated microRNA-330-3p was observed in plasma of gestational diabetes mellitus patients and advised that microRNA-330-3p could be the essential indicator in GDM outcome for therapeutic goal [A positive microRNA-330 overexpression was observed for cases with diabetes risk factors such as hypertension, obesity, alcoholism, and smoking as compared to those who were nonhypertensive, nonobese, nonalcoholic, and nonsmokers, who manifested comparatively lower expression. E2F1-decreased expression was linked with obesity, alcoholism, and smoking while nonobese, nonalcoholic, and nonsmokers had slightly higher expression comparatively. A negative correlation was observed among microRNA-330 and E2F1 mRNA expression among T2DM patients with a nonsignificant ensation ,18. A sttic goal .E2F1 involved in cell proliferation and metabolic process by coordinating cellular response by acting as regulatory switch . StudiesE2F1 plays a major role in hepatic steatosis. It has been demonstrated that E2F1 essentially involved in hyperlipidemia and hyperglycaemia during resistance to insulin and abnormally increased in type 2 diabetes . A studyIncreased microRNA-330 and decreased E2F1 mRNA expressions were observed to be associated in T2DM patients and were found to be linked with various diabetes risk factors. Increased microRNA-330 and decreased E2F1 mRNA expressions may be associated with pathogenesis, increased risk, and exacerbated disease progression of T2DM cases. These changes in expression of microRNA-330 and E2F1 mRNA are not only reliable biomarkers in the diagnosis of T2DM. MicroRNAs have become the most promising biomarkers for the diagnosis, prognosis, and therapeutic options of a variety of metabolic disorders including T2DM; hence, validation on large T2DM population especially from a developing country like India, with increasing diabetic cases each year, can be of profound significance in adequate treatment and prevention strategies of T2DM.


Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h ( Skeletal muscle aging is associated with numerous factors including (but not limited to) age-associated alterations in circulating hormones, low-grade inflammation, mitochondrial perturbations via free radicals, and transcriptomic alterations due to epigenetic factors . Th+) levels given that NAD+ increases sirtuin activity [Sirtuins are a class of proteins (SIRT1-7) that exist in various subcellular compartments and possess either mono-ADP-ribosyltransferase or deacetylase activity . Researcactivity ,11.Theacrine is a purine alkaloid that is structurally similar to caffeine, and various studies have examined its efficacy as a neuroactive ingredient ,13,14. A5 under standard culture conditions (37 °C in a 5% CO2 atmosphere). Once myoblast growth reached 80–90% confluence ~48 h after seeding, differentiation was induced by removing the growth medium and replacing it with a differentiation medium  horse serum, 1% penicillin/streptomycin, and 0.1% gentamycin). DM was then replaced every 24 h for 7 days to allow for adequate myotube growth.Cell culture methods were performed similarly to other studies published by our laboratory ,19. Brien = 6 treatment wells per time point) or a weight-equivalent of cellulose-only (n = 6 treatment wells per time point) suspended in DM. Notably, we opted not to resuspend the treatments in a vehicle prior to adding these agents to DM  given the potential cytotoxic effects of vehicle compounds. Rather, NAD3 or cellulose were added to DM in 50 mL tubes, tubes were centrifuged for 5 min at 100× g to remove any insoluble material, and the supernatant was used as the treatment media. Notably, the investigators were blinded to treatments until the conclusion of the study and after statistical analyses were performed.Treatments occurred on day 7 post-differentiation for either 3 or 24 h with either 0.45 mg/mL of NAD3  (−ΔΔCT method where 2−ΔCT (housekeeping gene CT—gene of interest CT) and 2−ΔΔCT (or fold change) = . Cyclophilin (Ppia) was used as a housekeeping gene.After all treatments, the differentiation/treatment media were removed, and the cells were washed once with phosphate-buffered saline (PBS). Thereafter, the PBS was siphoned off and then a subset of cells were scraped from a plate and transferred into 250 µL of Trizol for RNA isolation. Following Trizol-based RNA isolation methods, total RNA concentrations were analyzed using a Nanodrop Lite spectrophotometer , and 1 µg of cDNA was synthesized using a commercial qScript cDNA SuperMix  per the manufacturer’s recommendations. Real-time PCR was performed using gene-specific primers and SYBR green chemistry, and all PCR reactions were confirmed to produce only one melt product. Primer sequences can be found in the g for 5 min. After centrifugation, insoluble proteins were pelleted, and the supernatants containing solubilized cell material were placed in new 1.7 mL tubes and stored at −80 °C until the global sirtuin (SIRT) activity and citrate synthase assays were performed as described below.After the RNA scrape described above, 250 µL of ice-cold cell lysis buffer was applied to each well (20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM Na-EDTA, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1% Triton, 20 mM sodium pyrophosphate, 25 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 µg/mL leupeptin; Cell Signaling; Danvers, MA, USA). Plates were then scraped, and the cell slurry was removed and placed in 1.7 mL tubes. Cells were homogenized via micropestles, and homogenates were subsequently centrifuged at 500× Global SIRT activity levels were performed on cell lysis buffer lysates using a commercial assay  similar to previous methods published by our laboratory . Prior tr = 0.84, p < 0.001). The assay principle is based on the reduction of 5,50-dithiobis (2-nitrobenzoic acid) (DTNB) at 412 nm (extinction coefficient 13.6 mmol/L/cm) coupled to the reduction of acetyl-CoA by the citrate synthase reaction in the presence of oxaloacetate. Briefly, protein obtained from lysates was added to a mixture composed of 0.125 mol/L Tris–HCl (pH 8.0), 0.03 mmol/L acetyl-CoA, and 0.1 mmol/L 5,5′-dithiobis-(2-nitrobenzoic acid). All reactions occurred in 96-well plates, reactions were initiated by the addition of 5 μL of 50 mmol/L oxaloacetate per well, and the absorbance change was recorded for 60 s in a spectrophotometer (BioTek Synergy H1). The average CV values for all duplicates was less than 10%.Citrate synthase activity levels were determined in duplicate on lysates from 24 h treatments only; notably, these methods are similar to previous methods used by our laboratory . This meSirt1 mRNA alterations) as well as PGC1-α and NRF2 protein levels . We also opted to interrogate nicotinamide phosphoribosyltransferase (NAMPT) protein levels, given its role in NAD+ synthesis.To validate some of our qPCR targets identified above, we performed follow-up experiments in myotubes derived from C2C12 myoblasts 7 days post-differentiation  . Due to Western blotting was performed in an identical fashion to previous studies performed in our laboratory. For experimental details, readers should refer to ,22. Nota+, NADH, and NADt (NAD+ + NADH) concentrations using an enzymatic kit  and methods previously described by our laboratory [Cells treated with NAD3 or CTL for 24 h were also assayed for cellular NADboratory . Raw absp < 0.050.All statistical analyses were performed using SPSS v25.0 . The 3 h and 24 h data were analyzed independently between treatments using independent samples t-tests. All data are presented in figures and tables as the means ± standard deviation (SD) values, and statistical significance was established as Sirt1 relative to CTL-treated cells (p < 0.001), while not affecting Sirt4 or Sirt6 mRNA expression levels  and 24 h NAD3 treatments increased (p < 0.001) Nfe2l2 mRNA levels relative to CTL-treated cells (p < 0.05) (Data in  < 0.05) b. Howeve < 0.05) c.p = 0.035) , while not affecting the protein levels of SIRT6 (p = 0.097), PGC-1α (p = 0.484), or NRF2 (p = 0.109)  was surprisingly increased with 24 h NAD3 versus control treatments (= 0.035) a. Additi= 0.109) b.+ activates sirtuins as previously discussed; and (c) 24 h NAD3 treatments increased NAMPT protein levels, this being an enzyme involved in NAD+ biosynthesis as discussed later. Data in + concentrations by nearly two-fold  global sirtuin activity was robustly increased by 3 h and 24 h NAD3 treatments; (b) NADSirt1 mRNA levels as well as global sirtuin activity relative to CTL-treated cells. Follow-up experiments on 24 h treated cells indicated NAD3 treatments increased SIRT1 protein levels, NAMPT protein levels, and cellular NAD+ concentrations. However, while certain mRNA markers related to mitochondrial biogenesis were altered with NAD3 treatments, citrate synthase activity levels  were not affected with 24 h NAD3 treatments. These findings are discussed in greater detail below.This is the first study to examine how NAD3, a theacrine-based supplement, affects molecular markers in skeletal muscle cells. Notably, NAD3 treatments increased + concentrations given that decrements in sirtuin activity as well as NAD+ levels have been implicated in tissue aging [+ concentrations after 24 h of NAD3 treatment may be related to the aforementioned effect and critically, this too may be a mechanism that is involved with the robust increase in sirtuin activity observed herein. Collectively, while our findings are limited to an unperturbed cell culture model, these findings are promising given the role that sirtuins have in skeletal muscle; specifically, their role in enhancing mitochondrial function and the transcriptional control of various metabolic genes [As mentioned previously, there is widespread interest in augmenting cellular sirtuin activity and NADue aging . The NADue aging  determinue aging  similarlue aging . The twoic genes .Nfe2l2 mRNA levels, protein levels of NRF2 as well as mitochondrial biogenesis (as assessed through citrate synthase activity) remained unaltered. Although this finding is difficult to reconcile, it may be possible that NAD3, rather than appreciably affecting mitochondrial biogenesis, enhanced mitochondrial function. While we did not assess metrics of mitochondrial function , it is notable that Wang et al. [In spite of the NAD3-induced increase in global sirtuin activity as well as the NAD3-induced increase in g et al.  reported+ concentrations are promising and warrant future research in vivo.There are limitations to the current data. First, these data are in vitro, and muscle biopsy studies in humans are needed to confirm and refine the current findings. Another limitation to these data is that only select biomarkers were interrogated in one cell type. Thus, more studies are needed to extensively assess the molecular alterations induced by NAD3 treatments in human cell lines as well as multiple cell types. Moreover, while there are seven sirtuin isotypes, qPCR was performed on only three of these isotypes due to RNA yield limitations, and follow-up Western blotting was performed on SIRT1/6 due to resource constraints. Notwithstanding, these preliminary findings regarding the effects of a theacrine-based supplement global sirtuin activity, NAMPT protein levels, and cellular NAD+ concentrations. However, and as mentioned prior, human studies are needed to validate the current findings.Although these data are in vitro, this preliminary evidence suggests a theacrine-based supplement can increase SIRT activity, NAMPT protein levels, and cellular NAD


Furthermore, cell viability was not reduced when fibroblasts were cultured in DMEM and treated with a hydrogen peroxide (H2O2) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H2O2 was dramatically increased by UVA photosensitization when riboflavin (R) coexisted with amino acids such as tryptophan (T), and found that R with folic acid (F) produced high levels of H2O2 after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN3, which is a singlet oxygen quencher, suppressed only R and T photosensitization. Lastly, we examined the effects of antioxidants , which are singlet oxygen or superoxide or H2O2 scavengers, on R and F or on R and T photosensitization, and found that 1 mM ascorbic acid, Trolox, and L-histidine were strongly photosensitized with R, and produced significant levels of H2O2 during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H2O2 production by UVA photosensitization. These data suggest that a low concentration of R-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H2O2 during UVA exposure.To investigate environmental factors that contribute to ultraviolet A (UVA)-induced oxidative stress, which accelerates the senescence and toxicity of skin cells, we irradiated human fibroblasts cultured in commonly used essential media with UVA and evaluated their viability and production of reactive oxygen species. The viability of fibroblasts exposed to a single dose of 3.6 J/cm Deleterious damage of the skin such as photo-aging and cancers that are caused by chronic long-term exposure to solar ultraviolet (UV) radiation have been extensively documented by a large number of basic and clinical studies ,2,3,4. T2 within two weeks, and suggested a pivotal role for hydrogen peroxide (H2O2) in the induction of cell senescence, which is produced by UVA absorption and possibly by riboflavin, known as a potent UVA chromophore in the medium [The UV spectrum is divided into three groups based on the wavelength: ultraviolet C UVC: 100–280 nm), ultraviolet B (UVB: 280–320 nm), and ultraviolet A (UVA: 320–400 nm) . UVA is 0–280 nm,e medium .2O2 by UVA radiation in vitro in the presence or absence of several UVA photosensitizers, amino acids, and vitamins. We initially confirmed the role of riboflavin, vitamin B2, which is well-known as a photosensitizer [2O2 in Hanks Balanced Salt Solution (HBSS) exposed to UVA radiation. The results showed that riboflavin alone in HBSS produced an extremely small amount of H2O2, but riboflavin coexisting with other chemicals, such as tryptophan, tyrosine, or folic acid, produced high levels of H2O2 in the medium after UVA irradiation at a single dose of 3.6 J/cm2. We then used medium containing all kinds of amino acids and only one vitamin each in Dulbecco’s modified Eagle’s medium (DMEM), and confirmed that riboflavin is a major vitamin that produces H2O2 during UVA exposure. Additionally, we confirmed that tryptophan and tyrosine are major amino acids responsible for the production of H2O2 in the presence of riboflavin. We considered that a singlet oxygen, which is one of the other types of ROS produced by UVA, is an early important type of ROS leading to H2O2 formation, and we showed that NaN3, which is a potent singlet oxygen scavenger [2O2 in medium containing riboflavin and tryptophan. Furthermore, we found that the production of H2O2 in medium containing riboflavin and folic acid was not suppressed by NaN3, which suggests a role for minor type Ⅱ (superoxide) and type I photosensitization in the formation of H2O2. These results indicate that riboflavin, which is present at a low concentration in the medium, may trigger UVA-induced photoreactions by absorbing UVA photon energy, generating triplet states of riboflavin, and transferring its energy to tryptophan and folic acid in the formation of singlet oxygen and/or superoxide, which leads to the production of H2O2 and is toxic to cells. In addition, we investigated the effects of L-ascorbic acid, trolox, L-cysteine, and L-histidine, which are well known ROS scavengers [2O2 by UVA irradiation. The results showed that the addition of ascorbic acid and L-histidine to riboflavin and tryptophan or folic acid in the medium significantly increased the level of H2O2, but, in contrast, L-cysteine suppressed photosensitization by riboflavin. Lastly, our study suggests that the use of sunscreens that contain efficient materials to scavenge singlet oxygen, superoxide, and H2O2 and activators of antioxidant systems within cells is the most effective way to protect the skin against serious damage from solar radiation to maintain youthful and healthy skin without photo-aging and to prevent the formation of skin cancers in later years.In the present study, we aimed to clarify the mechanism involved in the production of Hnsitizer  in the fcavenger , suppresavengers ,23,24,252O2 generated by UVA exposure of DMEM induces the senescence of fibroblasts [2O2 generated in DMEM by UVA exposure is the major type of ROS involved in UVA-induced cell toxicity. The oxidative stress of fibroblasts exposed to UVA in DMEM, which contains amino acids, vitamins, glucose, and buffer components, or in HBSS, which contains only glucose and buffer-related components, were compared. UVA irradiation conditions are shown in roblasts . Therefo2O2 [2O2, we evaluated the production of H2O2 in HBSS containing each of the 14 amino acids and the eight vitamins contained in DMEM exposed to UVA radiation. 2O2 generated when those components were irradiated with UVA. The amount of H2O2 did not increase significantly when UVA was exposed to a mixed solution of 14 amino acids. Exposure of a solution containing eight vitamins to UVA increased H2O2 production, but at significantly lower levels than a solution mimicking DMEM, which contained 14 amino acids and those same eight vitamins , L-histidine, and L-cysteine were used as antioxidants. It has been reported that ascorbic acid and trolox have radical scavenging abilities. L-histidine has a singlet oxygen scavenging ability and L-cysteine has a high H2O2 scavenging ability [2O2 after UVA exposure are comparable  was significantly increased by the addition of 1 mM histidine, which causes strong cytotoxicity. Ascorbic acid showed results similar to histidine  and/or minor type Ⅱ (superoxide) generated by riboflavin photosensitization.One type of photosensitization occurs in medium containing riboflavin and folic acid. Folic acid in aqueous solution easily undergoes oxidative degradation upon UVA exposure to produce a fluorescent pterin derivative . That ptbing UVA . In addibing UVA ,35, and 2O2, and other types of ROS [3, which suggests that the oxidative degradation of tryptophan may be promoted by a singlet oxygen generated at an earlier time by the photosensitization of riboflavin.The other type of photosensitization occurs between riboflavin and tryptophan. Since tryptophan has a maximum absorption wavelength in the UVB region , exposure to UVB is known to produce tryptophan oxides such as FICZ and kynurenine derivatives. These tryptophan oxides have absorption wavelengths in the UVA region, and it has been reported that UVA exposure produces superoxide, Hs of ROS ,37,38. S2O2 produced in DMEM by UVA exposure is dramatically amplified by the following reactions. (1) The photosensitization of type I and type Ⅱ reactions by riboflavin as an initiator oxidizes tryptophan or folic acid present in the medium, (2) photo-activated tryptophan or folic acid oxidizes O2 molecules to produce superoxide or singlet oxygen, and, lastly, (3) H2O2 is generated by the disproportionation of superoxide anions. These reactions that occur in DMEM suggest that a low concentration of photosensitizing molecules may cause high photosensitizing reactions in human skin.These results suggest that H2O2 by UVA-photosensitization. However, ascorbic acid, Trolox, and L-histidine were UVA-photosensitized by riboflavin, even though they have been reported to scavenge a singlet oxygen and superoxide. In addition, it has been reported that ascorbic acid reacts with a singlet oxygen to produce H2O2 and does not reduce the damage caused by UVA exposure of albino hairless mice [Lastly, the effects of antioxidants on the photosensitization between riboflavin-folic acid and riboflavin-tryptophan were evaluated. The results show that L-cysteine dramatically suppressed the production of Hess mice ,40. Takiess mice ,42, foliess mice ,44, and ess mice . Those cIn future investigations, we will try to identify antioxidants that have the ability to scavenge a singlet oxygen and superoxide without photosensitization by photosensitizers such as riboflavin. Furthermore, since this study was performed using DMEM, it will be necessary to analyze more complicated photosensitizing mechanisms in living skin that contains many amino acids and vitamins that are not in DMEM and many photosensitizers such as riboflavin.2. Fibroblasts with PDL less than 10 were used in these experiments.Normal human dermal fibroblasts  were cultured in DMEM  supplemented with 10% normal fetal bovine serum  and 1% antibiotic/antimycotic solution (Sigma) at 37 °C in a humidified atmosphere containing 5% CO2. Fibroblasts were irradiated for 1 h in an ice box for temperature control (3.6 J/cm2).The UVA irradiation protocol has been described previously . Prior t4 cells/well and were incubated for 24 h at 37 °C and 5% CO2. The protocol used to measure cell viability was based on the CellTiter 96® Aqueous One Solution Reagent . After UVA irradiation, the cells were incubated for 3 h at 37 °C in a CO2 incubator. Afterward, the medium was changed to DMEM with 10% FBS and 1% antibiotic/antimycotic solution and 2% CellTiter 96® Aqueous One Solution Reagent, and incubated further for 1 h. After the culture supernatants were transferred to 96-well plates, their absorbance was measured at 490 nm.Cells were seeded in 24-well culture plates at a density of 4 × 10Modified mixtures of DMEM were prepared by adding selected components from the 14 amino acids and the eight vitamins contained in basic DMEM into HBSS (pH 7). The concentration of each component is as follows: Amino acids: 0.084 g/L L-Arginine HCl , Japan), 0.0626 g/L L-Cystine diHCl (TCI), 0.03 g/L Glycine , Japan) 0.042 g/L L-Histidine HCl monohydrate (TCI), 0.105 g/L L-Isoleucine (Wako), 0.105 g/L L-Leucine (TCI), 0.146 g/L L-Lysine monoHCl (TCI), 0.03 g/L Methionine (TCI), 0.066 g/L L-Phenylalanine (TCI), 0.042 g/L L-Serine (TCI), 0.095 g/L L-Threonine (TCI), 0.016 g/L L-Tryptophan (Sigma), 0.12037 g/L L-Tyrosine Disodium Salt Dihydrate , 0.094 g/L L-Valine (TCI). Vitamins include 0.004 g/L Choline Cl (TCI), 0.004 g/L Folic acid (Wako), 0.0072 g/L myo-Inositol (TCI), 0.004 g/L Nicotinamide (TCI), 0.004 g/L Calcium D-pantothenate (TCI), 0.00404 g/L Pyridixine HCl (Wako), 0.0004 g/L Riboflavin (Wako), and 0.004 g/L Thiamine HCl hydrate (TCI).2O2 assay was performed according to a previously described method [2O2 to emit fluorescence. Measurements were performed according to the instructions of the OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit . An ADHP/HRP Working Solution was prepared by adding 100 µM ADHP and 0.2 U/mL HRP in Assay Buffer. Fifty µL UVA-exposed samples, 50 µL ADHP/HRP working solution, and 50 µL of assay buffer were transferred to 96-well plates, and the mixtures were allowed to stand at room temperature for 30 min while being protected from light. The fluorescence intensity of each mixture after incubation was measured using a microplate reader .The Hd method  using 102 for 1 h every day. Cells were re-cultured with fresh DMEM supplemented with 10% FBS after UVA irradiation. The UVA irradiation was repeated a total of 10 consecutive days . Cells 24 h after the last UVA irradiation were used for Western blotting.The protocol of the induction of cellular senescence by repeated UVA exposure has been described previously . Fibrobl® ChemiDocTM XRS+ with Image LabTM Software.Detection of p16 by Western blotting was performed as previously described . UVA-expThe superoxide assay was carried out using a superoxide anion-2-methyl-6-methoxyphenylethynylimidazopyrazynone (MPEC) reaction kit . Light emission was measured using a microplate reader. One hundred µL of each sample exposed to UVA were transferred to 96-well plates and 10 µL MPEC (300 µM stock reagent) were added, which was followed by UVA irradiation for 2 min. After UVA irradiation, the luminescence of MPEC was measured for 1 min. Readings measured at 0, 15, 30, and 60 min from the start of UVA irradiation were compared as the superoxide production ability.3 were UVA irradiated and the concentrations of those chemicals were measured before and after the UVA exposure. The auto-fluorescence intensity of oxidized folic acid was measured  using a micro plate reader.Media containing folic acid, folic acid + riboflavin, or folic acid + riboflavin + NaN3, were UVA irradiated and the concentrations of those chemicals were measured before and after the UVA exposure. Absorption wavelengths were measured using a GeneQuant 1300/10  in a spectrofluorometric quartz cell having an optical path length of 10 mm.Tryptophan and kynurenine were measured using a variable wavelength detector at wavelengths of 280 nm and 360 nm, respectively, according to a previously published protocol . Media c2O2 generated in each solution after UVA irradiation with 3.6 J/cm2 was measured and compared.Antioxidants were added to HBSS containing 1 µM riboflavin and 30 µM folic acid, or 1 µM riboflavin and 100 µM tryptophan, as noted. As antioxidants, 1 mM ascorbic acid (Sigma), 1 mM Trolox (Wako), 1 mM L-cysteine (Wako), and 1 mM L-histidine (Wako) were used. The amount of H2O2 was added to 1 mL of each antioxidant solution, and incubated for 5 min at room temperature. The amount of H2O2 in each anti-oxidant solution after incubation for 5 min was measured using the H2O2 assay.Each antioxidant was adjusted with HBSS so that the final concentration was 1 mM. Ten µL of 10 mM Hp-value of <0.01 is regarded to be statistically significant.Statistical analyses were performed on a minimum of three independent experiments using one-way analysis of the Dunnet II test. A 2O2 during UVA exposure.Our study suggest that a low concentration of Riboflavin-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H


We established that BJJP significantly repressed the proliferation, metastasis and infiltration of HCC cells. Furthermore, BJJP remarkably suppressed HCC cell migration, as well as invasion via epithelial-mesenchymal transition (EMT) by modulating Snail expression, which was associated with the repression of Akt/GSK-3β/Snail signaling axis activation. In vivo HCC xenograft results indicated that BJJP delayed HCC development and efficiently inhibited lung metastasis. Taken together, BJJP was shown to be an effective therapeutic agent against HCC through repression of the Akt/GSK-3β/Snail signaling cascade and EMT.Hepatocellular carcinoma (HCC) is among the most usual cancers globally. In China, Biejiajian pill (BJJP), Traditional Chinese Medicine clinical prescription, is broadly utilized for the prevention and therapy of HCC. However, the mechanisms by which BJJP exerts its effects on the prevention of tumor invasion and metastasis are still largely unknown. In this study,  Liver cancer is the 6th most often diagnosed cancer, as well as the 3rd principal cause of cancer-linked fatalities in the world. It accounted for an estimated 0.84 million new cases, as well as 0.78 million mortalities in 2018 . In ChinAlthough many significant advancements have been achieved for the clinical treatment of HCC, the morbidity and HCC mortality rate remain high. Due to inconspicuous symptoms during the initial period, most patients are diagnosed at the advanced stage, by which the possibility of local treatment methods, such as therapeutic hepatectomy, tumor ablation or tranhepatic arterial intervention, is not available. In terms of treatment, although HCC can be effectively treated using surgical resection and liver transplantation, only 5–15% of patients are fit for surgical resection, as it is only appropriate for early stage patients . The proth residue of GSK-3β, which facilitates GSK-3β ubiquitination and suppresses the degradation of Snail, stimulating Snail protein stabilization, as well as nuclear localization, ultimately promoting EMT  is referred to as the process in which epithelial cells lose their epithelial phenotypic characteristics and gain the phenotypic features of mesenchymal cells. This is manifested by the loss of cell polarity, decreased intercellular adherence and enhanced motor ability. EMT is imperative for the progression of local infiltration and distant metastasis of many tumor cells . Previouting EMT .Traditional Chinese Medicine (TCM) can serve an essential role for the prevention and treatment of HCC. Studies have shown that TCM can relieve clinical manifestations, improve quality of life, enhance immune function, intercept relapse, as well as metastasis, obstruct cancer progression, and extend survival . TCM conrhizoma zingiberis, magnolia officinalis, cassia twig, radix paeoniae alba, rhizoma belamcandae, peach kernel, cortex moutan, rheum officinale, trumpet creeper, semen lepidii, pyrrosia leaf, fringed pink). According to TCM, BJJP can promote circulation, relieve stasis, improve immunity, eliminate pathogenic factors, as well as disperse and soften hard masses; therefore, it is suitable for patients with liver fibrosis and cirrhosis (Biejiajian pill (BJJP), a renowned and canonical Chinese medicinal formula from “The Synopsis of Golden Chamber,” consists of 23 ingredients , 17,500 (dd-ms2); scan range: 100.0–1500.0 m/z; spary voltage: 3.8 kv (Positive); capillary temperature: 300°C; colliding gas: high purity argon (purity ≥99.999%); sheath gas: nitrogen (purity ≥99.999%), 40 Arb; aus gus heater temperature: nitrogen (purity ≥99.999%), 300°C; data acquisition time: 27.0 min. The chromatographic conditions used were as follows: chromatographic column: RP-C18 150 × 2.1 mm, 1.8 μm, Welch; flow rate: 0.300 ml/min; aqueous phase: 0.1% formic acid solution; organic phase: 0.1% formic acetonitrile; needle washing liquid: methanol; column chamber temperature: 35°C; automatic sampler temperature: 10.0°C; injection needle height: 2.00 mm; automatic sampler cleaning settings: both; washing needle volume of automatic sampler: 200.00 μl; soaking time for automatic injection needle cleansing: 3.00 ms; auto injector injection volume: 10.00 μl. The data collected under high-resolution liquid quality were initially sorted using CD2.1 (Thermo Fisher Scientific) and then retrieved and contrasted with the repository .The main components in each batch of BJJP were identified and analyzed using high resolution mass spectrometry. Six different batches of BJJP  were purchased from Sinopharm Zhonglian Pharmaceutical Co., Ltd. . A 1 g sample from each batch was weighed, 1 ml methanol: water  was added, and the mixture was mixed through vortex. Then the resulting mixture was ground for 6 min, then centrifuged for 10 min at 4°C using a centrifugal force of 20,000 × g. We filtered the supernatant through a 0.22 μm filter membrane, and the evaluated the filtrate. The conditions used for mass spectrometry consisted of: ion source: electrospray ionization source (ESI); scanning mode: positive, as well as negative ion switching scanning; detection approach: full mass/dd-msWe obtained 40 Wister rats  weighing 250 ± 20 g each from the Experimental Animal Center of Southern Medical University  2011-0015). The Wister rats were kept at room temperature (21–25°C), 50–60% relative humidity, and under 12 h cycles of day/night. The rats were provided exclusive access to water and food. Experimental operations were carried out as per the Guidelines for the Care and Use of Laboratory Animals of Southern Medical University. The rats were clustered randomly into a negative control arm (NC arm), a low-dose (L arm), a medium-dose (M arm), a high-dose (H arm), and a positive sorafenib control arm (P arm), with eight rats in each arm. BJJP was dissolved in sterilized normal saline and made into a suspension. The L, M and H arm rats were given 0.55, 1.1, and 2.2 g/kg, respectively, of concentrated BJJP through gastrogavage. The NC arm was gavaged with an equivalent amount of normal saline, while the P arm was given 0.03 g/kg sorafenib  in normal saline. Each rat was given 2 ml of the respective solution through gastric gavage twice a day, with a gavage interval of 12 h and a total of 7 consecutive gavages. Blood samples were taken 1 h after the last gastrogavage from the abdominal aorta. The blood samples were allowed to stand for 4 h at 4°C, then centrifugation for 15 min at 3,000 r/min done, and the serum was separated under aseptic conditions. Inactivation of the separated serum done for 30 min in a 56°C water bath. After that, filtration by a 0.22 μm microporous membrane was conducted, and then kept at −80°C until used.2.The MHCC-97H, as well as SMMC-7721 human HCC cell lines were bought from the Cell Bank of Chinese Academy of Sciences . Incubation of the MHCC-97H, as well as SMMC-7721 cells was done at 37°C using Dulbecco’s modified Eagle’s medium  enriched with fetal bovine serum (10%) , as well as penicillin/streptomycin (1%)  in incubator with 5% CO4 cells per well and cultivated for 24 h until they adhered. Incubation of the cells was done in 100 μl of DMEM containing 10% of various concentrations of BJJP or sorafenib serum for 24, 48, and 72 h. For this assay, we used two control arms: cells in the blank control arm were exposed to DMEM containing 10% FBS, while cells in the negative control arm were incubated in DMEM containing 10% rat serum. Thereafter, CCK-8 solution (10 μl) was introduced into every well, cultured for an additional 1 h in an incubator with 5% CO2 at 37°C. A microplate reader  was employed to establish the absorbance  of every well at the wavelength of 450 nm. Cell viability was calculated for each arm using the following formula: cell viability (%) =  × 100.The impact of BJJP on the viability of HCC cells was inspected by a Cell Counting Kit-8 . In brief, we planted the cells into 96-well dishes with 5 × 10MHCC-97H and SMMC-7721 HCC cells were planted in 96-well dishes, and DMEM containing 10% of different concentrations  of BJJP or sorafenib serum were used for 24 h of incubation. After rinsing the cells thrice in PBS, a mixture of Hoechst33342 and PI was prepared according to instructions given in the Hoechst33342/PI Double Stain Kit  and added for staining at 4°C for 30 min. Once again, rinsing of the cells with PBS was done, and images were captured under a fluorescence microscope. Five random regions were selected for observations and the cell apoptosis index was calculated.5 cells per well was done. Subsequently, culturing of the cells at 37°C in an incubator with 5% CO2. When the cells had attained 70% confluence, we removed the original medium, rinsed the cells thrice in PBS, and DMEM containing 10% of different concentrations  of BJJP or sorafenib serum were used for 24 h of incubation. After that, wounds were made on the confluent cell monolayer via scratching by a 20 μl pipette tip. Then, we rinsed the cells thrice using PBS, while discarding any detached cell. Thereafter, growing of the cells in DMEM was conducted. Cell migration was observed under a microscope for 0 h and 48 h in a drug serum culture. The migration area of the cells in each arm at 0 h and 48 h were analyzed using ImageJ software. The formula: cell migration area ratio = (S0h−S48h)/S0h, the cell migration area ratio of the HCC cells in each arm were calculated.Plating of the MHCC-97H and SMMC-7721 HCC cells into 6-well dishes at a concentration of 2 × 105 MHCC-97H, as well as SMMC-7721 cells in 1% FBS DMEM were introduced to the upper chamber, then 24 h incubation at 37°C performed. Thereafter, we used cotton swabs to completely remove cells on the upper surface of the membrane, whereas fixing of the cells that infiltrated into the lower surface was done using paraformaldehyde, and then staining using crystal violet, and the invasive cells enumerated under an inverted microscope  at 200 × magnification.The Transwell invasion test was conducted using 24-well transwell plates and a cell culture insert  with 8.0 μm sized pores along with a transparent PET membrane. After melting and diluting the reagent at 4°C, the Matrigel  was used to coat the filters (50 μl per filter) and 30 min incubation at 37°C conducted. The DMEM containing 10% of different concentrations  of BJJP or sorafenib serum were introduced to the lower chamber. After that, 5 × 10Growing of the MHCC-97H, as well as SMMC-7721 HCC cells in complete medium on chamber slides until they reached 60% confluency. Then, they were exposed to the DMEM containing 10% of different concentrations  of BJJP or sorafenib sera for 24 h. Rinsing of the cells was done thrice using PBS and 15 min fixing by paraformaldehyde (4%) at 4°C on glass cover slips was conducted. After that, permeabilization of the cells using Triton-X 100 (0.1%) for 10 min at 4°C was done. After being washed with PBS, blocking of the cells for 2 h using BSA at room temperature was accomplished, followed by conjugation with the primary antibodies against Phospho-Akt , Phospho-GSK-3β  and Snail  via overnight-incubation at 4°C. The slides were rinsed thrice by PBS and conjugated with Alexa Fluor™ 568 goat anti-rabbit IgG (H+L) secondary antibodies  at room temperature via incubation in the dark for 1 h. Thereafter, staining of the nuclei using DAPI  was performed for 10 min. Finally, we inspected the cells on a fluorescence microscope .® Premix Ex Taq™ II (Tli RNaseH Plus) , the threshold cycle (Ct) was recorded by Light Cycler® 96 System . 2−△△CT method was used to calculated the fold changes of mRNA expression. Data were standardized to the GAPDH control and the primer of GAPDH was brought from Sangon Biotech (Shanghai) Co., Ltd. Primers of Snail, E-cadherin, N-cadherin, as well as Vimentin were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and were shown in MHCC-97H and SMMC-7721 HCC cells were planted in 6-well dishes, and DMEM containing 10% of different concentrations  of BJJP or sorafenib serum were used for 24 h of incubation. The total RNA of these cells was then extracted using TransZol UP Plus RNA Kit  as specification and the concentration and purity of the RNA were detected using NanoDrop™ Lite . Total RNA was reverse transcribed into cDNA by PrimeScript™ RT reagent Kit with gDNA Eraser . According to the instruction of SYBR6) were harvested in serum-free PBS. The right foreleg close to the shoulder of the mice was subcutaneous injected with 5 × 106 MHCC-97H cells. After 3 days, the mice were clustered randomly into six arms (n = 6). Each arm was fed as follows: Normal Control (NC) and Model (X) arm were gavaged with normal saline; high (H), medium (M) and low (L) dose arms of BJJP were gavaged with a high dose (2.2 g/kg), medium dose (1.1 g/kg) and low dose (0.55 g/kg) of concentrated BJJP, respectively. The positive (P) control arm was administered with 0.03 g/kg sorafenib in normal saline once a day. The volume of the tumor was determined using a digital calipers following every two days. This volume was computed using the formula: volume = 0.5 × length × wide (mm3). The mice were sacrificed on day 28, and excision of the tumors done, followed by weighing, and imaging. For the in vivo lung metastasis assay, MHCC-97H HCC cells were fluorescences with lentivirus , a green fluorescent tool with GFP. Orthotopic injection of a 12.5 μl suspension [PBS and Matrigel (1:2)] containing 1 × 106 LV-GFP-MHCC-97H cells into the left liver lobe of BALB/C nude mice was done. After 3 days, we randomly grouped the mice into six arms (n = 5), and were fed as follows: Normal Control (NC) and Model (X) arm were gavaged with normal saline; high (H), medium (M) and low (L) dose arms of BJJP were gavaged with a high dose (2.2 g/kg), medium dose (1.1 g/kg) and low dose (0.55 g/kg) of concentrated BJJP, respectively. The positive (P) control arm was administered with 0.03 g/kg sorafenib in normal saline once a day. Four weeks later, we sacrificed the mice, and harvested the lung and liver. Small animal bioluminescence in vivo imager was employed to inspect the growth, as well as lung metastasis of the liver tumors. Paraffin-embedment of the lung and liver tissues was done, and then staining using H&E for histological confirmation performed.The mice procedures were approved by the Southern Medical University Experimental Animal Ethics Committee (Resolution No.:L2018207). The animal laboratory  provided the four to five weeks old female BALB/c nude mice (15–16 g). These mice were given standard food and were allowed free access to tap water. For the subcutaneous tumor formation experiment, MHCC-97H cells  enriched with protease inhibitor (1%), as well as phosphatase inhibitor (1%)  to extract total protein. After that, the protein concentrations were determined by the BCA Protein Assay Kit . The proteins (30 μg/lane) were fractionated on SDS-PAGE gel (12%) and transfer-embedded onto PVDF (0.45 μm membrane)  membranes. Then, they were blocked using 1×TBST (0.1% Tween-20) enriched with bovine serum albumin (BSA) (5%) at room temperature (RT) for 2 h. Thereafter, overnight-incubation of the PVDF membranes at 4°C with the following primary antibodies: Akt , Phospho-Akt , GSK-3β , Phospho-GSK-3β , E-cadherin (1:1000); Abcam ab227639), Snail , MMP-9 , PCNA , Vimentin , N-cadherin , Cyclin D1 , VEGFA  and GAPDH  was done. After rinsing thrice in 1 × TBST, incubation of the membranes with goat anti-rabbit IgG or goat anti-mouse IgG  was done at RT for 1 h and were then again rinsed thrice with 1×TBST. Visualization of the membranes was conducted using a ECL detection kit , and the relative protein levels were measured using ImageJ software.The effect of BJJP on TNF-α of mice serum was detected using Elisa  assay. After collecting the mouse serum, the experiment was carried out according to instructions given by the manufacturer of the assay kit.p-Akt (1:200), p-GSK-3β (1:400), Snail (1:200), E-cadherin (1:100), N-cadherin (1:200), Cyclin D1 (1:200), KI67) at 4°C overnight. ImageJ software was employed to statistically inspect the immunohistochemical results.Fixing of the solid tumor tissues was done using paraformaldehyde (4%), followed by paraffin embedment, and chopped into 4 μm-thick slides. Deparaffination using dimethylbenzene was performed, and then the segments positioned on slides. Gradient alcohol dehydration was then conducted. Then, the staining of the sections with hematoxylin and eosin was performed for the H&E assay or antigen repaired with sodium citrate and treated according to instruction given by the manufacturer of the UltraSensitive™ S-P Immunohistochemical Staining Kit  Thereafter, the slides were incubated with primary antibodies  and two-way ANOVA were used to determine differences between arms. We used high resolution mass spectrometry to analyze the main components of BJJP, to elucidate the mechanism of action of BJJP against HCC. For data obtained through mass spectrometry, we used the mzCloud, mzVault and ChemSpider databases for the retrieval and comparative analysis, and a total of 2,727 compounds were found to match. Among them, 10 pharmaceutical ingredients, Wogonin, Ursolic acid, Zerumbone, 6-Gingerol, Coumarin, Astragalin, Resveratrol, Rutin, Sinapine and Oleanolic acid, received a mzCloud Best Match score greater than 94. As shown in p < 0.05). These data opined that BJJP suppresses the proliferation and induced the apoptosis of HCC cells.The Hoechst33342/PI fluorescent staining test was employed to inspect if BJJP triggered apoptosis of the HCC cells. As illustrated in p < 0.05). Since MMP-9 and VEGFA have been implicated in the progression and metastasis of various cancers, western blot assessment was employed to measure the expression of MMP-9 and VEGFA in HCC cells treated with BJJP. As shown in in vitro (p < 0.05). These data implied that BJJP inhibited the invasion and metastasis of HCC cells.To analyze the influence of BJJP on HCC cell migration, as well as invasion, we performed the wound healing, as well as Transwell migration assays using MHCC-97H and SMMC-7721 cells. As illustrated in p-Akt (Ser473), p-GSK-3β (Ser9) as well as Snail in both MHCC-97H, as well as SMMC-7721 cells treated with BJJP (p < 0.05). Moreover, the Immunofluorescence assays result implicated that BJJP deceased the nuclear translocation of Snail by regulating GSK-3β.These data implied that BJJP might have inhibited the initiation of the Akt/GSK-3β/Snail cascade. In addition, we further inspected the expression of EMT linked target genes in HCC cells subjected to BJJP. As Snail is a transcriptional repressor of E-cadherin (p < 0.05), while it escalated the expression of E-cadherin, an epithelial biosignature (p < 0.05), validating the EMT inhibition effects of BJJP in HCC cells. All these findings further confirmed the mechanism by which BJJP moderates HCC proliferation, metastasis and invasion via the Akt/GSK-3β/Snail pathway.In EMT, epithelial cells gain the phenotypic features of mesenchymal cells and show decreased intercellular adhesion and increased motility . To detecadherin , we furtin vivo, we created a nude mouse model of MHCC-97H xenograft tumor to assess the impact of BJJP in HCC. As shown in p < 0.05). Moreover, all BJJP treatment arms could decrease the expression of KI67 (proliferation marker), compared with the model arm (p-Akt (Ser473), p-GSK-3β (Ser9), Vimentin, CyclinD1, PCNA, MMP-9, VEGFA and Snail expressions, and increased the expression of E-cadherin (p < 0.05). Additionally, we inspected the p-Akt (Ser473), p-GSK-3β(Ser9), Snail, E-cadherin, N-cadherin and Cyclin D1 contents in HCC tumor tissues using IHC. The IHC results were consistent with that of the western blotting analysis . The tumor inflammatory microenvironment serves a core function in the progression of hepatocellular carcinoma (in vivo (p < 0.05). These data opined that BJJP could repress the carcinogenesis, as well as metastasis of HCC via the Akt/GSK-3β/Snail cascade in vivo.To detect the antitumor influence of BJJP odel arm . In addianalysis . Based oodel arm . The pularcinoma . To explin vitro and inhibited the growth of subcutaneous tumors and pulmonary metastasis of orthotopic transplantation tumors in vivo. Finally, our findings demonstrated that the molecular mechanism, by which the inhibitory effect of BJJP was exerted on the infiltration, as well as metastasis of HCC was related to the inhibition of EMT via the Akt/GSK-3β/Snail cascade.Since the morbidity of HCC has been rising constantly, it is important to inspect potential safe, as well as effective novel treatments for HCC. In China, the primary health care systems, TCM has functioned, and still functions an imperative role because of its unique effects . BJJP isBJJP is a classical Chinese medicine compound composed of 23 kinds of Traditional Chinese medicine. Due to its complex composition, we used high-resolution mass spectrometry to analyze the main components of BJJP to clarify the mechanism of action of BJJP against HCC. By analyzing the results of mass spectrometry, we obtained 10 main drug components in BJJP, such as Wogonin, Ursolic acid, Zerumbone, 6-Gingerol, Coumarin, Astragalin, Resveratrol, Rutin, Sinapine and Oleanolic acid. Studies have shown that the above 10 pharmaceutical ingredients can regulate the occurrence and development of various tumors via different signaling pathways. Among them, Resveratrol inhibits the invasion and metastasis of colon cancer through reversal of EMT via the AKT/GSK-3β/Snail signaling cascade . MoreoveMetastasis is the principal cause of cancer-related fatalities, mainly due to limited efficacy of current anticancer treatments for advanced malignancies. Metastatic cascades include the separation of tumor cells from adjacent cells, local infiltration into surrounding tissues (as either singular cells or groups of tumor cells), and tumor entry into nearby pre-existing or newly formed vascular systems. In this case, these aggressive tumor cells are typically typified by partial loss of epithelial biosignatures and the attainment of a mesenchymal-like phenotype, which is usually associated with migratory and invasive property changes that occur during EMT. EMT is one of the key steps in the metastatic cascade, in which cancer cells lose epithelial properties and acquire mesenchymal phenotypes, enabling tumor cells to be aggressive, motile and anti-apoptotic. In addition, activation of the EMT program affects the immunomodulatory properties and immunogenicity of cancer cells, and is associated with immunosuppression induction and acceleration of tumor progression . In recep-GSK-3β facilitates the Snail nucleation (p-Akt (Ser473), p-GSK-3β (Ser9), Snail, as well as Vimentin expressions, while it could increase the expression of E-cadherin, in vitro, as well as in vivo. The above results revealed that BJJP inhibited HCC proliferation, metastasis and EMT via the Akt/GSK-3β/Snail signaling cascade.EMT serves a pivotal function in the development of metastasis of multiple cancers. Diverse signaling cascades that are atypically activated or inactivated during tumor progression, such as JAK/Stat3, PI3K/Akt, TGF-β/Smad, as well as Wnt/β-catenin, have been found responsible for regulating the EMT process. Previous researches have documented that the Akt/GSK-3β/Snail signaling pathway regulates the invasion and metastasis of various type of tumors, including HCC, via modulating EMT . In thiscleation . Snail, cleation . Moreovecleation . TAMURA cleation . In the in vivo. KI67 is not expressed in stationary or dormant cells at the G0 stage, but is widely expressed in all proliferating cells , and is also a cell proliferation marker that has been applied as a prognostic factor for the diagnosis of different tumor types (It has been chronicled that Cyclin D1 overexpression is a positive modulator of cell cycle progression that induces cell overgrowth, and is an important marker of malignancy . Overexpor types . ImmunohMMPs are a key factor for the acquisition of infiltration, as well as metastasis characteristics of malignant tumor cells. The migration of tumor cells depends on the secretion, as well as activation of MMPs. Studies have documented that MMPs can serve a pivotal role in the EMT of HCC, and MMP-9 is one of the most inspected MMPs in the pathogenesis of HCC EMT. Overexpression of MMP-9 in HCC can increase lymph node infiltration and promote metastasis, leading to a higher TNM stage, dismal differentiation, and poor overall prognosis . MMP-9 sGrowing evidence has shown that chronic and persistent inflammation contributes to cancer development . HCC is In vivo, HCC xenograft data showed that BJJP delayed HCC development and efficiently inhibited lung metastasis. The results of our study indicate that BJJP has the ability to act as an effective treatment agent for HCC.In summary, this study uncovered the mechanism by which BJJP inhibited HCC metastasis and invasion. The mechanism involves the modulation of EMT by modulating Snail expression via inhibiting the initiation of the Akt/GSK-3β/Snail signaling cascade.


Because of the rapid development of non-enzyme biosensors, the current state of methods for synthesis of pure and composite/hybrid NiCo2O4 materials and their subsequent electrochemical biosensing applications are systematically and comprehensively reviewed herein. Comparative analysis reveals better electrochemical sensing of bioanalytes by one-dimensional and two-dimensional NiCo2O4 nano-/microstructures than other morphologies. Better biosensing efficiency of NiCo2O4 as compared to corresponding individual metal oxides, viz. NiO and Co3O4, is attributed to the close intrinsic-state redox couples of Ni3+/Ni2+ (0.58 V/0.49 V) and Co3+/Co2+ (0.53 V/0.51 V). Biosensing performance of NiCo2O4 is also significantly improved by making the composites of NiCo2O4 with conducting carbonaceous materials like graphene, reduced graphene oxide, carbon nanotubes , carbon nanofibers; conducting polymers like polypyrrole (PPy), polyaniline (PANI); metal oxides NiO, Co3O4, SnO2, MnO2; and metals like Au, Pd, etc. Various factors affecting the morphologies and biosensing parameters of the nano-/micro-structured NiCo2O4 are also highlighted. Finally, some drawbacks and future perspectives related to this promising field are outlined.Non-enzymatic biosensors based on mixed transition metal oxides are deemed as the most promising devices due to their high sensitivity, selectivity, wide concentration range, low detection limits, and excellent recyclability. Spinel NiCo However, according to some reports, in the presence of urea, metal carbonate hydroxides are initially formed instead of bimetallic hydroxides (OH)2 and Co2(CO3)(OH)2. Subsequent hydrothermal treatment in basic medium followed by calcination at 500 oC formed two-dimensional porous NiCo2O4 nanodisks.Even ethanol as the solvent can also initiate the formation of metal carbonate hydroxides. Two-dimensional porous NiCon et al.    Fig. a and micts) Fig. b in aquets) Fig. c. In purts) Fig. d. Howevets) Fig. e.Fig. 7F2O4 nanosheets on the surface of NiCo2O4 nanocones precursor to give highly ordered 3D hierarchical NiCo2O4@NiCo2O4 core–shell nanocone arrays on nickel foams  template. The KIT-6 template was added into the metal nitrate precursor solution prepared in ethanol. The schematic illustration of the formation of mesoporous NiCo2O4 microspheres is shown in Fig. The morphology, size, shape, and surface area of nanostructures can be designed through template-based synthesis to produce nanostructures with controlled physical, chemical, electrical, and electronic properties essential in notable applications and are also quite different from those of the bulk materials. Generally, three stages, viz., template preparation, directed synthesis of the desired material using the template, and the template removal, are described in the overall growth process of nanostructures . The chen et al.  prepared2O4 submicron spheres with uniform size and mesoporous textual property. These submicron spheres were composed of ultrathin nanosheets with a thickness of a few nanometers. The NaOH solution was used for the in situ removal of silica spheres. Dopamine—a biomolecule containing amine functional groups is capable of self-polymerize under alkaline conditions. It forms a layer of the polydopamine which attracts various metal ions including Co2+ and Ni2+ cations due to strong electrostatic interactions. Further, the alkalinity of the medium results in the formation of –OH–Ni–OH–Co–OH– complex networks. This property has been explored for the synthesis of NiCo2O4 nanostructures by Veeramani et al.  {[Bmim][BF4]}, [Bmim]FeCl4, [Bmim]Cl [BF[Bmim]Cl , and nonlucoside  are also2O4 nanostructures with morphologies hollow nanosphere . . 2O4 nanM−1 cm−2 . The micN  =  3) . As condanowires . Besidesf 0.5 μM .Fig. 25a2O4 is its poor electrical conductivity. However, this limitation can be overcome by forming its composite/hybrid materials. It has been reported that the electrical conductivity and hence the electrochemical biosensing performance of NiCo2O4 can be improved by making its composites with conducting carbonaceous materials like graphene, reduced graphene oxide, carbon nanotubes , carbon nanofibers; conducting polymers like polypyrrole (PPy), polyaniline (PANI); metal oxides NiO, Co3O4, SnO2, MnO2; and metals like Au, Pd, etc. Among these, the carbonaceous materials are considered to be potential candidates as compared to others due to their excellent electrical conductivities, good mechanical strength, thermal and chemical stabilities, and resistance to oxidation–reduction reactions. Besides, these carbonaceous materials provide a large specific surface area for better adsorption of analytes, which ultimately results in very high sensitivity and very low detection limits.One of the main disadvantages of using bare NiCo2O4 due to its high specific surface area of 2670 m2 g−1 and excellent conductivity .38.38\docum2O4 nano-/microstructures are reviewed. Research has proved that urea can be electrochemically oxidized by NiCo2O4 nano-/microstructures 2 at the time of urea electro-oxidation [2O4 nanostructures prepared via facile chemical deposition method showed higher catalytic activities, lower overpotential, and more tolerance toward urea electro-oxidation as compared to Co3O4 [2O4/3D graphene/ITO exhibited high sensitivity of 166 μA mM−1 cm−2, a linear range of 0.06–0.30 mM, and a low detection limit of 5.0 µM with a very small response time of 1 s through non-enzymatic detection method [2O4/3D graphene in the CV as compared to NiCo2O4/CNT-modified electrode confirmed the superiority of 3D graphene as a matrix material for electrode fabrication. The higher oxidation current potential was attributed to the highly porous nature and excellent conductivity of the interconnected 3D graphene matrix [− ions [Recently, Amin et al.  exploredxidation . Mesoporto Co3O4 . NiCo2O4n method . Furthere matrix . Since o [− ions .2O4-modified electrodes. Rutin, a flavonoid substance, is used as anti-bacterial, anti-viral, antiproliferative, antioxidants, antagonists, and anti-inflammatory. It also controls the blood pressure and vascular fragility including hereditary hemorrhagic telangiectasia, haemangiomas, vitamin C deficiency, etc. [2O4-decorated reduced graphene oxide for the electrochemical sensing of rutin using differential pulse voltammetric (DPV) technique. Flake-like NiCo2O4 sheets anchored on the wrinkled reduced graphene oxide sheets through electrostatic interaction prevented the self-agglomerations. The wide linear range of 0.1–150 μM and a low detection limit of 0.01 μM were observed along with excellent anti-interference capabilities. The strong synergism between reduced graphene sheets and mesoporous NiCo2O4 resulted in increased redox peak current and decreased potential difference. During electro-oxidation, rutin is converted into 3′,4′-diquinone with the release of two H+ ions and two electrons /peptide@g-C3N4 nanocomposite-modified GCE. 3,4,9,10-perylene tetracarboxylic acid (PTCA) was used to connect the peptides and g-C3N4. The modified GCE exhibited increased DPVs peak currents when the trypsin concentration was increased from 10−10 to 10−4 mg mL−1. Kaur et al. [2O4/Nano-ZSM-5 nanocomposite-modified GCE. Wide linear ranges were 1–1200, 0.6–900, 0.9–1000, and 0.9–1000 μM, while the corresponding detection limits were 0.8, 0.5, 0.7, and 0.7 μM for ascorbic acid, dopamine, uric acid, and tryptophan, respectively. Simultaneous detection was possible as the anodic oxidative peak currents were observed at different applied potentials, i.e., 0.158, 0.394, 0.561, and 0.820 V, respectively, for ascorbic acid, dopamine, uric acid, and tryptophan in DVP plots at a scan rate of 20 mV s−1.Trypsin, a serine protease secreted from the pancreas, has also been widely studied recently as it used for identifying and determining the amino acid sequence in protein and peptide, particularly at the C-terminus and as a specific biomarker for diseases like chronic cystic fibrosis, chronic pancreatitis, cancer, and many pathological changes , 309. Lir et al.  studied 2O4 nano-/microstructures and the presence of any other component along with NiCo2O4 significantly affect the biosensing efficiency. Comparative analysis revealed better electrochemical sensing of glucose by one-dimensional nanofibres and nanorods and two-dimensional nanosheets like morphologies of NiCo2O4 than other morphologies. Doped and composites/hybrid NiCo2O4 nano-/microstructures exhibit superior sensing parameters that bare NiCo2O4. In particular, graphenic nanomaterials due to their excellent conductivity and large surface area significantly elevate the I–V characteristics to many folds. These materials also accelerate the rate of heterogeneous electron transfer, i.e., the transfer of electrons from/to electrode to/from bioanalyte molecules [Detailed comparison from Tables olecules .Table 2E2O4 nano-/microstructures with versatile morphologies and their subsequent use for the development of biosensors for efficient non-enzymatic sensing and detection of biomolecules such as glucose, H2O2 and urea are comprehensively reviewed. As compared to NiO and Co3O4, the NiCo2O4 nanomaterials showed better electrochemical sensing as adjudged by broader redox peaks with larger area coverage in the CV curves. The biosensing efficiency of the NiCo2O4 nano-/microstructures can be improved by engineering the morphology, specific surface area, porosity, doping and by making composite/hybrids with various carbonaceous materials, conducting polymers, metal oxides, non-metals and metals. These materials not only improve the mechanical, thermal, and chemical stability but also modulate the bandgap energies, electronic and ionic conductivities, dispersion behavior, avoid aggregation of the NiCo2O4 nanomaterials and provide short electron and ion diffusion pathways. All these factors contribute to better electrocatalytic behavior with excellent sensitivity, selectivity, and long-term stability of the spinel NiCo2O4 nano-/microstructure-based biosensors. It is hoped that this review will provide basic ideas as well as new insights for future research and progress in this field.Herein, various strategies for the synthesis of spinel NiCo2O4nano-/microstructures need to be addressed for further improvement. Some of these issues have been identified herein.Despite extensive research in this area, many issues that impede the practical application of NiCo2O4 nanomaterials are controlled by factors like temperature, pH of the reaction solution, precursor concentration, solvent nature and quantity, presence of the growth directing agents and templates, etc. It is, therefore, one of the major challenges to design large-scale and low-cost morphology controlled synthesis of the NiCo2O4 nanomaterials for next-generation biosensors.Structural features of the NiCo2O4 nano-/microstructures with other hybrid materials or conductive substrates to designing NiCo2O4 composite/hybrids is found to improve the intrinsic characteristics like low electronic conductivity and wide bandgap and hence the biosensing behavior of NiCo2O4. However, still, more in-depth understanding is required to correlate the synergism between the components of the composite/hybrid materials.Rational combination of NiCo2O4 nano-/microstructure-based biosensors are other issues that need to be addressed and solved. The high cost of electrochemical work stations restricts the practical applications of these sensors. In this regard, portable and wearable sensing devices will be promising. The toxicity issues of the NiCo2O4 nano-/microstructures and other components are very rarely discussed in the literature. Future research thus should also focus on studying this important issue.Cost-effectiveness, easy to manufacture, recyclability, sensor disposal, and biocompatibility of NiCo2O4 nano-/microstructure-based sensors is affected by factors like working temperature, pH of the medium, scan rates, and applied potential. The optimization of these parameters is rarely addressed. Studying the specificity of NiCo2O4-based sensors from competitive assays requires an appropriate protocol because it is reported that the sensor can detect glucose, H2O2, urea, trypsin, etc.The biosensing behavior of the NiCoDue to complex structures of the biomolecules, the interface and mechanistic studies at the surface of the nanomaterials are still undefined. The redox transformation of the molecules during electrocatalytic biosensing is also a debatable issue. Therefore, future work should focus on elucidating the interaction mechanism between nanomaterials and biomolecules on the electrode surface, to fabricate a new generation of biosensors.


Currently, diabetes has become one of the leading causes of death worldwide. Fasting plasma glucose (FPG) levels that are higher than optimal, even if below the diagnostic threshold of diabetes, can also lead to increased morbidity and mortality. Here we intend to study the magnitude of the genetic influence on FPG variation by conducting structural equation modelling analysis and to further identify specific genetic variants potentially related to FPG levels by performing a genome-wide association study (GWAS) in Chinese twins.DZ = 0.20, 95% CI: 0.04–0.36) was much lower than half that of the MZ twin correlation . For the variation in FPG level, the AE model was the better fitting model, with additive genetic parameters (A) accounting for 67.66% (95% CI: 60.50–73.62%) and unique environmental or residual parameters (E) accounting for 32.34% (95% CI: 26.38–39.55%), respectively. In the GWAS, although no genetic variants reached the genome-wide significance level (P < 5 × 10− 8), 28 SNPs exceeded the level of a suggestive association (P < 1 × 10− 5). One promising genetic region (2q33.1) around rs10931893 (P = 1.53 × 10− 7) was found. After imputing untyped SNPs, we found that rs60106404 (P = 2.38 × 10− 8) located at SPATS2L reached the genome-wide significance level, and 216 SNPs exceeded the level of a suggestive association. We found 1007 genes nominally associated with the FPG level (P < 0.05), including SPATS2L, KCNK5, ADCY5, PCSK1, PTPRA, and SLC26A11. Moreover, C1orf74 (P = 0.014) and SLC26A11 (P = 0.021) were differentially expressed between patients with impaired fasting glucose and healthy controls. Some important enriched biological pathways, such as β-alanine metabolism, regulation of insulin secretion, glucagon signaling in metabolic regulation, IL-1 receptor pathway, signaling by platelet derived growth factor, cysteine and methionine metabolism pathway, were identified.The final sample included 382 twin pairs: 139 dizygotic (DZ) pairs and 243 monozygotic (MZ) pairs. The DZ twin correlation for the FPG level (rThe FPG level is highly heritable in the Chinese population, and genetic variants are significantly involved in regulatory domains, functional genes and biological pathways that mediate FPG levels. This study provides important clues for further elucidating the molecular mechanism of glucose homeostasis and discovering new diagnostic biomarkers and therapeutic targets for diabetes. Diabetes, as a chronic and metabolic disease, can cause serious damage to the blood vessels, heart, kidneys, nerves and eyes. This condition is one of the leading causes of death worldwide, and higher fasting plasma glucose (FPG) levels, even if below the diagnostic threshold of diabetes, can also lead to increased morbidity and mortality. Diabetes and higher-than-optimal FPG level together leaded to 3.7 million deaths from 1980 to 2014 worldwide [ADCY5, G6PC2, MADD, TCF7L2, GCK, XIRP2, VPS16, PTPRA, etc. [The FPG level is affected by both genetic and environmental factors. Currently, the magnitude of genetic impact on FPG variation has been researched in some studies. And the heritability of the FPG level varied, with 0–0.77 in Europeans –8, 0.16–RA, etc. –27. HoweChinese population are different from other ethnic populations in the aspect of genetic constitutions. Genetically related individuals (e.g. twins) will greatly increase the power of genetic association analysis and effectively identify the genetic variants potentially associated with complex traits . Here, wThe final sample consisted of 382 twin pairs: 139 dizygotic (DZ) pairs and 243 monozygotic (MZ) pairs. The median (interquartile range) age for all twins was 50 (45–57) years, and the median (interquartile range) FPG level was 5.10 (4.59–5.80) mmol/L  was much lower than half of the MZ twin correlation , suggesting the genetic effect on the FPG level , with additive genetic parameters (A) accounting for 67.66% (95% CI: 60.50–73.62%) and unique environmental or residual parameters (E) accounting for 32.34% (95% CI: 26.38–39.55%), respectively.As Table The median age of 139 DZ twin pairs was 49 years (interquartile range: 45–56 years), and the median FPG level was 5.14 mmol/L (interquartile range: 4.60–5.90 mmol/L) , as illustrated by the Manhattan plot , with 17, 1, 4, 1, 4 and 1 SNPs located on chromosomes 2, 5, 6, 8, 10, and 13, respectively (Table P = 1.53 × 10− 7) on chromosome 2q33.1 at SPATS2L.The quantile-quantile (Q-Q) plot is shown in Fig. lot Fig. .a. HowevP = 1.53 × 10− 7- 6.94 × 10− 6) were located at or close to SPATS2L which could moderate the protein expression of β2-adrenergic receptors [SPATS2L was nominally associated with FPG level (P < 0.05) in the subsequent gene-based analysis.As shown in the regional association plot Fig. , one proeceptors . AdditioP = 2.38 × 10− 8), located at SPATS2L reached the genome-wide significance level (P < 5 × 10− 8), as illustrated by the Manhattan plot  with the FPG level  with previously reported FPG-associated SNPs found in relevant GWASs, we found that 8 SNPs rs7684538, rs2367204, rs7186570, rs861085, rs1402837, rs2302593, rs4869272 and rs492594 could be replicated , a total of 1007 genes were nominally associated with the FPG level (P < 0.05). The top 20 genes ranked by P-values are shown in Table BRAT1, TSPO, SLC2A12, KCNK5, PTPRA, ADCY5, PCSK1, and VPS16, were found.Although none of the genes reached the genome-wide significance level P < 2.6 × 10− 6,P < 0.05) were found, and the top 30 pathways are shown in Table β-alanine metabolism, regulation of insulin secretion, glucagon signaling in metabolic regulation, IL-1 receptor pathway, signaling by platelet derived growth factor (PDGF), cysteine and methionine metabolism, etc.A total of 719 biological pathways were nominally associated with the FPG level (emp-C1orf74 (P = 0.014) and SLC26A11 (P = 0.021) were differentially expressed between the two independent groups.The gene expression levels of 25 genes in patients with impaired fasting glucose (IFG) and healthy controls  in mainland China , 30–34.SPATS2L. SPATS2L might indirectly affect FPG levels by regulating the protein expression of β2-adrenergic receptors [SPATS2L was the topmost gene in the gene-based analysis. Thus, SPATS2L may serve as candidate gene to be further validated and a potential biomarker for diabetes.Even no SNPs reached the genome-wide significance level, 28 SNPs showed suggestive evidence of an association with the FPG level. We found one promising genetic region (2q33.1) where 17 suggestive SNPs were linked to eceptors  that coueceptors , 36. In SPATS2L, that has been discussed above. Furthermore, more than 200 SNPs were found to reach the level of a suggestive association. We compared our results with previously reported SNPs [Post-imputation analysis revealed that one SNP, rs60106404, was significantly associated with the FPG level. This SNP is located at an important gene, ted SNPs , 37–40 aBRAT1 deficiency could lead to increased glucose consumption [TSPO expression plays an important role in maintaining healthy adipocyte functions, and the activation of TSPO in adipocytes could improve glucose uptake [SLC2A12, a member of the solute carrier family, catalyzes the uptake of sugars through facilitated diffusion [KCNK5 could influence the homeostasis of glucose by regulating insulin secretion [PTPRA gene is a member of the protein tyrosine phosphatase (PTP) family. PTPRA might play a role in insulin signaling as a negative regulator and further influence glucose homeostasis [PTPRA and FPG levels has previously been reported [ADCY5 plays a role in the normal regulation of insulin secretion [ADCY5 has been previously reported to be associated with FPG levels [PCSK1 is prohormone convertase 1/3 (PC1/3), which is essential to activate some peptide hormone precursors involved in regulating glucose homeostasis [VPS16 with FPG levels has been previously reported [In the gene-based analysis, 1007 genes were nominally associated with FPG levels. Several interesting genes might influence FPG levels through the following mechanisms: (1) sumption ; (2) TSPe uptake ; (3) SLCiffusion ; (4) theecretion ; (5) theeostasis . Moreovereported ; (6) ADCecretion , which mG levels , 27; (7)eostasis , and itsreported , its funC1orf74 and SLC26A11 were differentially expressed. SLC26A11 was involved in the transport of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds, as indicated by the GeneCards database, while the mechanism of C1orf74 involved in blood glucose metabolism still needs to be explored.In addition, we tested the gene expression levels of several top genes in IFG cases and healthy controls, and found that β-alanine could significantly decrease glycolytic metabolism and change glycolytic-related gene expression [β-cells. When pancreatic IL-1R signaling is absent, the whole-body glucose homeostasis is disrupted [The pathway enrichment analysis identified some important FPG-associated biological pathways: (1) pression ; (2) glupression –51; (3) isrupted ; (4) in isrupted –55; (5) isrupted ; (6) PIPisrupted , 58.The strength of twin samples in our study was observed. The variation of human phenotype may be due to effects of genetic structure, gender, age and certain environmental exposures. Twin samples, as genetically related individuals, will highly increase the power of genetic association analysis and effectively find the genetic variants potentially associated with complex traits . Hence, Nevertheless, this study also has some limitations. This study was with a relatively small sample size because of the difficulties of recruiting and identifying qualified twin pairs. However, our results could still provide useful clues for hypotheses to be further replicated and validated while exploring the molecular mechanism of diabetes. Considering that the genetic influence on FPG variation is expected to be comprised of a lot of SNPs, a meta-analysis with a larger number of samples will be an ideal and desirable method.Our study has confirmed the significant contribution of genetic effects on FPG variation. The FPG level is highly heritable in the Chinese population, and some genetic variants are involved in regulatory domains, functional genes and biological pathways that mediate FPG levels. The results provide important clues for further elucidating the molecular mechanism of glucose homeostasis. The potential candidate biomarkers of FPG level presented here merit further verification.The main materials and methods of this study were similar to our previously published studies , 60.Briefly, we collected samples through the Qingdao Twin Registry (QTR) , 62. AllBriefly, we firstly genotyped DNA samples, and then conducted quality control analysis . 1,365,1DZ) was much lower than half of that of MZ twins (rMZ), the ADE model was taken into account.Genetic analysis were conducted by using Mx programme . The FPGα and degree of freedom by the Mx programme.In the classical twin design, the phenotypic variation was decomposed into that due to additive genetic (A), dominant genetic (D), and unique environmental or residual (E) influences. Standard structural equation modeling (SEM) methods were used to estimate the A, D, and E components while adjusting for age, sex, and BMI. We performed a likelihood ratio test to compare the performances of the full ADE model and its nested model, i.e., AE model. And the better fitting model was chosen according to the parsimonious principle and a lower Akaike’s information criterion (AIC) value . The pow− 8) was set as genome-wide statistical significance, and the commonly used threshold (1 × 10− 5) was adopted for suggestive hits [GEMMA  was adopive hits –70.P < 2.63 × 10− 6 was set as genome-wide statistical significance.We performed this analysis by using VEGAS2 tool , 72. TheWe used the PASCAL tool to compute the gene and pathway scores . IndividP-value < 0.05 as statistically significant.The blood samples of 8 subjects  were sequenced to obtain the gene expression data. The cases were defined as those with FPG ≥ 6.1 mmol/L, i.e., IFG status, and the healthy controls were defined as those with FPG ≤ 4.7 mmol/L. Then, the expression levels of 25 genes, i.e., the genes where the top SNPs were located and the top 20 genes in the VEGAS2 analysis, between the two independent groups were compared by the Wilcoxon rank sum test. We set the Additional file 1.  Descriptive statistics for twins.Additional file 2.  Phenotypic correlation coefficients  with effects of covariates in twin pairs.Additional file 3. Summary of the imputed SNPs with a P-value < 1 × 10− 5 for association of the FPG level in GWAS.Additional file 4. Comparison between our imputed SNPs  and other previously reported SNPs in GWAS.Additional file 5. The characteristics of subjects in validation analysis.Additional file 6. The questionare of survey on health status of elderly twins in Qingdao city.