The results of alkaline phosphatase staining, real-time PCR, and Western blot showed that cyclic mechanical stress can regulate the expression level of osteogenic differentiation markers in BMSCs on the surface of TiO2 nanotubes through Wnt/β-catenin. As an important member of the histone acetyltransferase family, GCN5 exerted regulatory effects on receiving mechanical signals. The results of the ChIP assay indicated that GCN5 could activate the Wnt promoter region. Hence, we concluded that the osteogenic differentiation ability of BMSCs on the surface of TiO2 nanotubes was enhanced under the stimulation of cyclic mechanical stress, and GCN5 mediated this process through Wnt/β-catenin.Bone marrow mesenchymal stem cells (BMSCs) play a critical role in bone formation and are extremely sensitive to external mechanical stimuli. Mechanical signals can regulate the biological behavior of cells on the surface of titanium-related prostheses and inducing osteogenic differentiation of BMSCs, which provides the integration of host bone and prosthesis benefits. But the mechanism is still unclear. In this study, BMSCs planted on the surface of TiO In the treatment of orthopedic diseases, the integration relationship between the implant prosthesis and the host bone has always been the main factor affecting the therapeutic effect and the life of the implant. Therefore, the integration of the implant prosthesis and the host bone on the interface and the promotion of host bone regeneration have become a momentous indicator for evaluating prostheses performance. The nature of the interaction between the joint prosthesis and host bone interface is actually the process of bone formation and resorption . Bone me2)  mineralization at the bone–implant interface and induced osteogenic differentiation and increased osseointegration . In this2) . Our pre2) . Many re2) . In the 2) . However devices . By screIn the osteogenic differentiation of BMSCs, the Wnt/β-catenin signaling pathway is one of the most important pathways. The classical Wnt/β-catenin pathway stimulates the expression of alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) to interfere with the whole process . PreviouThe activation and inhibition of signaling pathways can be affected by histone acetylation modification . In the 2 nanotube–modified titanium matrix. On the basis of previous research, we will explore the mechanism on the epigenetic level in-depth and try to provide new insights into the regulation of cyclic mechanical stress on the osteogenic differentiation of rat BMSCs.In this study, we used rat bone marrow mesenchymal stem cells as the research object to observe the effects of cyclic mechanical stress on the expression of GCN5 and the activity of classical Wnt/β-catenin signaling pathway and osteogenesis differentiation of BMSCs on the surface of TiO2. All experiments used cells from passages 3 to 5. The study has been approved by the Ethics Committee of the Second Affiliated Hospital of Anhui Medical University.Sprague Dawley (SD) rats (4 weeks old) were purchased from the Experimental Animal Center of Anhui Medical University  . A total of ∼50 rats were used for various experimental approaches in the current study. The rats were housed in groups (four to five rats per cage). All rats received a standard rodent diet and tap water ad lib. A constant temperature of 21–23°C and humidity of 50 ± 5% were maintained. Following feeding for 1 week, the rats were killed by cervical dislocation and were confirmed to have no spontaneous breathing and heartbeat. All efforts were made to minimize suffering. The rats were immersed in 75% ethanol and sterilized for 20 min. After removing the femur under aseptic conditions, scissors were used to cut both ends of the femur. A 5-ml syringe equipped with a 25-gauge needle was used to rinse the bone marrow in the medullary cavity with α-modified minimum essential medium . The washed cells were collected, cultured in α-MEM supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin , and incubated at 37°C with 5% CO4F at 20 V for 30 min by anodization using a potentiostat. The cathode was a pure platinum foil. After anodization was completed, the sample was rinsed with deionized water.A pure titanium foil with a thickness of 2 mm was cut into a square with a length of 35 mm and used as a substrate. The top and bottom surfaces of the foil were sanded with silicon carbide sandpaper. A pure titanium foil was soaked in absolute ethanol and cleaned with an ultrasonic cleaner. After washing again with deionized water, the foil was air-dried at room temperature. The nanotubes were formed in electrolyte solution containing 0.15 M NH2 nanotubes–modified titanium specimens at a density of 1 × 104 cells/cm2. The cells were cultured for 48 h to allow them to attach and reach approximately 80% confluence, after which the growth medium was replaced, and cyclic mechanical stress was applied. The cyclic mechanical stress parameters used for our manufactured mechanical loading device . The medium was changed every 3 days. The cells were harvested after 7 days of culture for other experiments.To study osteogenic differentiation with DKK1, the density of 1 × 104 cells/cmWe used PCR to amplify Rat GCN5. XbaI and NotI were used to digest the amplicons before inserted into the pCDH lentiviral vector. The sequences of the primers were forward: AGCACTCCCATCTTCAGTCC and reverse: GCTTCCTCTTCTCTCCTGGCAT. The primers for GCN5 shRNA were forward: CCGGGCAGGGTGTTCTGAACTTTCTCAAGAGAAAAGTTCAGAACACCCTGCTTTTTTG and reverse: ATTCAAAAAAGCAGGGTGTTCTGAACTTTTCTCTTGAGAAAGTTCAGAACACCCTGC.Restriction enzymes AgeI and EcoRI were used to digest the PCR product before incorporated into the pLKD vector. Sanger sequencing was performed to verify the inserted fragments. To produce the lentivirus, 293T cell line was co-transfected with two packaging vectors (psPAX2 and pMD2.G) and two plasmid vectors. The supernatant was centrifuged at 1,000 rpm for 10 min to get rid of the cell debris. A 0.45-μm polyethersulfone low-protein-binding filter was used to filter. The titer of the lentivirus was 115 IFU/ml, assessed by a quantification kit . A vector inserted with scrambled GCN5 was used as a negative control.2 nanotubes–modified titanium specimens, and cyclic mechanical stress was applied. On day 7, ALP activity was measured using an ALP kit  according to the manufacturer’s instructions. The results were observed under a stereo microscope.BMSCs were seeded onto TiO® Premix Ex Taq™ . GAPDH served as an internal control, and the expression level relative to GAPDH was calculated using 2−ΔΔCq method. The primer sequences are listed in Total RNA was extracted from the cells with TRIzol  and reverse-transcribed into cDNA using a PrimeScriptTM cDNA Synthesis kit  according to the manufacturer’s instructions. Real-time PCR was performed with SYBRThe cells were washed three times with PBS and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors for 30 min at 4 C. For the Western blot analysis, 15 μL of the sample was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes. The primary antibodies used were rabbit polyclonal anti-GCN5 ; rabbit monoclonal anti-Axin-2 ; rabbit monoclonal anti-β-catenin  and mouse monoclonal phosphorylated β-catenin ; rabbit monoclonal anti-H3K9ac ; rabbit monoclonal anti-RUNX2 ; rabbit monoclonal anti-OCN ; and rabbit monoclonal anti-OPN . For normalization of protein loading, GAPDH antibody  was used. The protein bands were measured with ImageJ 1.48v software and normalized to the corresponding GAPDH bands. The relative density of each target protein normalized to the control was used to represent the changes in expression of target proteins.ChIP assay was performed following the manufacturer’s instructions of a ChIP assay kit . The rabbit monoclonal anti-GCN5  and anti-H3K9ac  were used. The rat IgG  and anti-RNA polymerase II  were used as the negative and positive control, respectively. The precipitated DNA samples were assessed by real-time PCR. Primers were designed from the region of 500 bp distance to the transcription start sites. The primer sequences are listed in p < 0.05 was considered as significant.GraphPad Prism 7.0 (GraphPad Software) was used for statistical analysis. Data are expressed as mean ± SEM. Student's t test was used for comparing two groups, one-way analysis of variance was used for comparing multiple groups, and the least-significant difference method was used as a post-hoc test for multiple comparisons between groups. 2 nanotube–modified titanium specimen surface and related physical parameters (2 nanotubes is about 80 nm (Our previous studies have measured the topography of the TiOrameters . The toput 80 nm . We applut 80 nm . Then thut 80 nm . The resut 80 nm . These rut 80 nm . These iPrevious literature reports that high levels of Wnt signaling, an important signaling pathway that regulates osteogenic differentiation, can be activated in osteogenic differentiation of BMSCs and promote bone formation . In osteWnt signaling pathway activity was observed under mechanical strain and nonmechanical strain. Western blot and real-time PCR were used to detect key molecules in the Wnt/β-catenin signaling pathway: Wnt1, Wnt6, Wnt10a, β-catenin, phosphorylated β-catenin [phosphorylated β-catenin could be regarded as inactive because it was about to be degraded ], and AxAfter applying mechanical strain to the titanium specimen, we detected the expression level of GCN5 in BMSCs through real-time PCR and Western blot. GCN5, one of the important members of the HATs family, played a significant regulatory role in a variety of signaling pathways, especially the Wnt signaling pathway . As the To further demonstrate that the increased level of GCN5 caused by mechanical strain can promote osteogenic differentiation, we transfected the empty vector, the GCN5 lentiviral overexpression vector, and the knockdown GCN5 vector into the BMSCs . ElasticTo further verify that high levels of GCN5 caused by mechanical strain can enhance the Wnt pathway, the levels of markers associated with the Wnt pathway were measured at the same time. In the 0.9% group, the levels of phosphorylated β-catenin were significantly increased and Axin-2 was repressed by the knockdown of GCN5 . The reavia the Wnt pathway. As an inhibitor of the Wnt pathway, DKK1 binds to membrane receptors and prevents Wnt ligands from binding to membrane surface receptor complexes, thereby inhibiting Wnt signaling pathway activity and osteogenic differentiation of BMSCs (We further verified whether GCN5 which was stimulated by mechanical strain regulated osteogenic differentiation of BMSCs of BMSCs . We asseof BMSCs . The Wesof BMSCs . Not onlof BMSCs . Howeverof BMSCs . The resof BMSCs . These r2-modified titanium metal specimen. Moreover, we have demonstrated that cyclic mechanical strain that causes deformation of titanium specimen can promote osteogenic differentiation of BMSCs on the surface of TiO2 nanotube–modified titanium. We set up experiments on the time, frequency, and amplitude of cyclic mechanical strain in the early stage and obtained the optimal parameters for promoting osteogenic differentiation of BMSCs in this device, and applied it to this study. 0.3 and 0.9% represent the degree of compression of the specimen after being subjected to cyclic mechanical stress. As the amplitude of cyclic mechanical stress increases, the osteogenic differentiation of surface cells gradually improved (via the GCN5/Wnt/β-catenin signaling pathway (Titanium and titanium alloys are the most widely used metal materials in orthopedic clinical implants due to the good properties of titanium . In the improved . In this pathway .Epigenetics refers to the activation or inhibition of a gene without altering the DNA sequence . HistoneGCN5 is named after the conventional signaling pathways that regulate amino acid synthesis originally found in yeast . Yeast s2 nanotube–modified titanium specimen aggravates, the expression level of GCN5 gradually increases. Due to the acetylation modification of GCN5, the transcription levels of wnt1, wnt6, and wnt10a and the expression levels of Axin-2 and β-catenin also increased. DKK1 is a specific inhibitor of the Wnt pathway and can inhibit Wnt signaling by competing with the ligand Wnt for receptor LRP6 (The Wnt gene family encodes secreted glycoproteins, which act as ligands for the Frizzled family of transmembrane receptor proteins. In many types of cells, the Wnt/β-catenin signaling pathway plays a role in cell proliferation, cell-fate determination, and cell differentiation. Wnt ligand binding to Frizzled receptor leads to the downregulation of GSK3β. GSK3β can phosphorylate β-catenin and then β-catenin could be hydrolyzed by APC complex. Its downregulation makes β-catenin accumulate in the cytoplasm, which then migrates into the nucleus and when combined with TCF/LEF transcription factors affects the transcription of target genes . The Wnttor LRP6 . DKK1 co2 nanotube–modified titanium specimen surface through GCN5/Wnt. Cyclic mechanical strain promoted the expression of GCN5 and increased the level of acetylation of H3K9, thereby activating the Wnt signaling pathway. This study suggests that applying cyclic mechanical stress to the prosthetic implants could be more effective in promoting osseointegration and improving patient prognosis, and it is a promising prospect for the clinical treatment of bone defects and arthroplasty. This also provides a relevant basis and reference for postoperative rehabilitation training.In conclusion, it was revealed that the cyclic mechanical strain enhanced osseointegration on TiO


Cancer is caused by a breakdown of cell-to-cell communication, which results in the unrestricted expansion of cells within a tissue. In many cases, tumor growth is maintained by the continuous activation of cell signaling programs that normally drive embryonic development and wound repair. In this review article, I discuss how one of the largest human protein families, namely FOX proteins, controls the activity of the Wnt pathway, a major regulatory signaling cascade in developing organisms and adult stem cells. Evidence suggests that there is considerable crosstalk between FOX proteins and the Wnt pathway, which contributes to cancer initiation and progression. A better understanding of FOX biology may therefore lead to the development of new targeted treatments for many types of cancer.Aberrant activation of the oncogenic Wnt signaling pathway is a hallmark of numerous types of cancer. However, in many cases, it is unclear how a chronically high Wnt signaling tone is maintained in the absence of activating pathway mutations. Forkhead box (FOX) family transcription factors are key regulators of embryonic development and tissue homeostasis, and there is mounting evidence that they act in part by fine-tuning the Wnt signaling output in a tissue-specific and context-dependent manner. Here, I review the diverse ways in which FOX transcription factors interact with the Wnt pathway, and how the ectopic reactivation of FOX proteins may affect Wnt signaling activity in various types of cancer. Many FOX transcription factors are partially functionally redundant and exhibit a highly restricted expression pattern, especially in adults. Thus, precision targeting of individual FOX proteins may lead to safe treatment options for Wnt-dependent cancers. Wnt signaling is one of the fundamental developmental signaling pathways in the animal kingdom. In adult organisms, it functions as a key regulator of stem cell maintenance and proliferation. Consequently, Wnt signaling is indispensable for numerous biological processes ranging from embryogenesis to tissue homeostasis and regeneration.2+ signaling, Wnt ligands engage FZDs and alternative co-receptors to elicit a β-catenin-independent response. The signaling outcome of these pathways is primarily non-transcriptional, and is best known for regulating cell migration and polarity. Finally, Wnt/Stabilization of proteins (STOP) and Wnt/Target of rapamycin (TOR) signaling promote the stability of proteins other than β-catenin via the Wnt-induced inhibition of the destruction complex, and support proteome maintenance and cell growth in a transcription-independent manner [The basic principles of Wnt signaling are well-understood ,2 Figur. In the t manner .nt1, then known as int-1) as an oncogene activated by viral integration [Given the prominent roles of Wnt signaling in embryogenesis and stem cell regulation, it is unsurprising that dysregulated Wnt pathway activity is associated with numerous serious disorders, especially cancer ,6,7. Indegration . In the egration . Additioegration . Chronicegration , which hegration ,13.2+ signaling promote metastasis as well, by increasing tumor cell migration and invasion [Apart from tumor initiation, Wnt signaling also contributes to cancer metastasis ,14. A hainvasion ,20. In pinvasion ,20. Wnt In light of these findings, it is obvious that the Wnt signaling pathway is a priority target for therapeutic intervention in cancer, and clinical trials are currently underway to assess the efficacy of Wnt modulators in several types of cancer ,22. HoweAlthough the basics of Wnt signaling are well-established, it is considerably less well understood how specific Wnt pathway outputs are generated in different tissues and cell types. For example, whether Wnt signaling elicits canonical or non-canonical downstream effects is mostly determined by the interaction of 19 different Wnt ligands with 10 FZD receptors in humans, but few studies have explored the signaling outcomes of individual ligand/receptor combinations ,25,26,27The current paradigm in β-catenin-dependent gene transcription is that the four TCF/LEF family transcription factors  constitutively occupy Wnt-responsive DNA elements, and that they are kept in an inactive state by Transducin-like enhancer (TLE) family repressors. Upon Wnt pathway activation, β-catenin binds to TCF/LEF and inactivates TLE, thereby enabling target gene transcription . SelectiBesides TLE, numerous other proteins engage β-catenin and TCF/LEF to stratify the Wnt transcriptional response ,31. ThesThe aforementioned examples provide a brief glimpse at the complexity of Wnt signaling at the transcriptional level, and illustrate how tissue and cell-specific Wnt activity is controlled by the action of non-TCF/LEF transcription factors. Below, I review how Wnt signaling is shaped by one of the largest mammalian transcription factor families, the forkhead box (FOX) transcription factors.FOX proteins are highly conserved transcription factors with 44 family members in humans, not counting five duplicated FOXD4-like genes and the FOXO3 variant FOXO3B ,49. TheyGiven these considerations, it is not surprising that FOX transcription factors also act as regulators of Wnt signaling. At the time of writing, approximately half of all mammalian FOX transcription factors have been assigned a role in the Wnt pathway, primarily in cancer cells . Moreoveβ-catenin stabilization and nuclear translocation are central events in canonical Wnt signaling . Thus, aOther FOX family members have been implicated in β-catenin stabilization and nuclear import as well. Overexpression of FOXG1 is associated with apoptosis resistance and metastasis in various cancers ,95. FOXGConversely, other FOX transcription factors may inhibit Wnt signaling by destabilizing β-catenin. Higashimori et al. reported that FOXF2 induces the expression of the E3 ubiquitin ligase IRF2BPL in gastric cancer cell lines . IRF2BPLNon-TCF/LEF transcription factors shape specific Wnt signaling outputs by engaging the Wnt transcriptional complex , and sevA different mode of action has been described for FOXP1, which has contrasting functions in various types of cancer . In lympXenopus development [Xenopus embryos reduced the expression of maternal Wnt target genes, but this was not associated with altered recruitment of β-catenin to TCF binding sites. Thus, the cooperative action of β-catenin/TCF and FOXH1 transcriptional complexes may be required for the induction of specific Wnt targets, at least during embryonic development.FOXG1 enhances Wnt signaling at the chromatin level as well. As with FOXM1 and FOXP1, FOXG1 binds TCF7L2 in addition to β-catenin, and depletion of FOXG1 decreased the recruitment of β-catenin to TCF binding sites in HCC cell lines . The autelopment . Afouda On the other hand, some FOX transcription factors compete with TCF/LEF for β-catenin binding, and act as inhibitors of canonical Wnt signaling. FOXO3 and FOXO4 engage β-catenin via the same domains as TCF/LEF, and thereby inhibit Wnt transcriptional activity while at the same time increasing the expression of FOXO target genes c 85,105,107. UnlLastly, FOXN3, a known tumor suppressor in various types of cancer , also inAnother way in which FOX proteins control Wnt signaling is via the transcriptional regulation of Wnt ligands. Induction of Wnt ligand expression has been linked to disease progression in various cancers, such as ovarian, prostate, and lung cancer ,114,115.Other studies have reported the regulation of individual Wnt ligands by FOX transcription factors. FOXC2, a known oncogene in several types of cancer , inducesXenopus embryogenesis, again working as a downstream effector in the sonic hedgehog signaling pathway [Additional FOX proteins may regulate WNT5A expression. Ma et al. reported that protein levels of FOXE1 and the sonic hedgehog signaling pathway effector GLI2 are concomitantly increased in papillary thyroid cancer (PTC) samples . FOXE1 i pathway . It shou pathway , possiblBesides Wnt ligands, β-catenin itself may be a transcriptional target of some FOX proteins. FOXS1 overexpression decreased the transcription of β-catenin (encoded by the CTNNB1 gene) and two Wnt target genes in gastric cancer cell lines, which was associated with decreased tumor cell proliferation in mice . These fFOXK1 and FOXK2 have a unique mode of action in the Wnt pathway. FOXK1/2 are best known as metabolic regulators, but they have also been implicated in the pathogenesis of various types of cancer ,128,129.Lastly, FOXL1 may regulate Wnt signaling through a distinct mechanism as well. Perreault et al. observed that epithelial cell proliferation was increased in the digestive tract of FOXL1-deficient mice . This inbona fide Wnt targets as well. Christensen et al. reported that FOXQ1 is one of the most highly upregulated genes in CRC, and that its expression correlates with Wnt pathway activity in CRC cell lines [TCF/LEF binding sites are highly abundant in the mammalian genome. It has been shown that Wnt/β-catenin signaling controls the expression of thousands of genes ,135, andll lines  (Figure ll lines ,77, and ll lines . Class All lines ,139, whill lines ,142,143,Finally, a separate mode of reciprocal regulation has been described for FOXM1. Chen et al. identified FOXM1 as a target of Wnt/STOP signaling . In Wnt-As discussed in the preceding chapters, there is mounting evidence that FOX transcription factors are common regulators of Wnt signaling pathways, particularly Wnt/β-catenin signaling. Given that both β-catenin and FOX proteins can be traced back to early metazoan ancestors ,146, it Another unanswered question is whether findings for one FOX protein can be extrapolated to other family members. As outlined above, multiple FOX transcription factors interact with β-catenin and thereby regulate its stability, activity, and subcellular localization . It is nOn the other hand, the role of FOX proteins in the transcriptional regulation of Wnt pathway components may be underappreciated. Our observation that FOXB2 regulates the expression of most Wnt molecules raises the possibility that the majority of Wnt ligands are under transcriptional control by FOX proteins . Many FOIt should be noted that many of the studies referenced here have limitations that need to be considered when interpreting the results. Firstly, FOX proteins are often studied by ectopic overexpression, which may cause them to work in ways that are not physiologically relevant. Secondly, in most cancers, multiple FOX family members are dysregulated at the same time see , which mCould the FOX-dependent regulation of Wnt signaling be targeted for cancer therapy? While it is too early to answer this question with confidence, there are some promising results from preclinical studies that give reason to be optimistic. As discussed earlier, high nuclear β-catenin levels confer apoptosis resistance to colorectal cancer cells treated with PI3K/PKB inhibitors . SimultaThe Wnt pathway and FOX transcription factors are ancient regulators of metazoan cell signaling, and their interaction is intimately linked not only to normal development and tissue homeostasis, but also to cancer initiation and progression. Considering the sheer number of FOX family members, and the fact that many of them have not been studied in great detail, it is highly likely that many important connections in FOX-dependent Wnt signaling still await discovery, and that these findings will further refine our understanding of cell signaling in health and disease.


Aims: β-catenin is a critical regulating factor of the Wnt pathway, which is closely linked to tumorigenesis, tumor growth, metastasis, and tumor immunity. Our study focused on exploring the relationship between β-catenin and clinicopathological features, prognosis, as well as infiltrating immune cells and immune scores, so as to illustrate its clinical significance in NSCLC.Materials and Methods: The β-catenin mRNA (CTNNB1) and protein expression data were downloaded from the UALCAN and the UCSC Xena website, respectively. All tumor-immune infiltrating cells’ data were downloaded from the TIMER platform and immune scores were downloaded from ESTIMATE website. The expression of β-catenin protein in our cohort was measured by immunohistochemistry.Results: β-catenin mRNA level was higher in lung adenocarcinoma (LUAD) compared to normal tissues (p < 0.001) and was related to overall survival (OS) (p < 0.001) and post-progression survival (PPS) (both p = 0.049) in LUAD. Aberrant β-catenin protein expression was higher in male and lung squamous cell carcinoma (LUSC) patients (both p = 0.001). Also, it was considered to be a prognosis factor independently (p = 0.034). In addition, β-catenin protein was negatively correlated with CD8+T cells , neutrophils , immune score , stromal score , and ESTIMATE score .Conclusions: Aberrant β-catenin protein expression was evidently higher in NSCLC and might serve as a biomarker for poor prognosis. Most importantly, β-catenin protein might play an important part in tumor immunity and the tumor microenvironment by inhibiting the infiltration of CD8+ T cells and neutrophils. Lung cancer, which is one of the most principal malignant cancers with increasing incidence rates year by year, often occurs in bronchial mucosal epithelium and alveoli ,2. It isβ-catenin is a multifunctional protein, which was first discovered as a mammalian cell adhesion complex in 1990. It can interact with cadherin, participate in cell adhesion, and form cytoskeletons . The imbTumor microenvironment, as the living environment of tumor cells, is composed of various cytokines/chemokines, immune cells, tumor-related fibroblasts, inflammatory cells, and microvessels and has a significant impact on the treatment response and overall prognosis of tumor patients ,18. ReshCTNNB1 (β-catenin mRNA) and β-catenin protein in The Cancer Genome Atlas (TCGA) database was downloaded from the UALCAN  and the UCSC Xena website (http://xena.ucsc.edu/), respectively. All tumor-immune infiltrating cells data  were obtained from the Tumor Immune Estimation Resource (TIMER) platform (https://cistrome.shinyapps.io/timer/). The immune score, stromal score, and estimate score were downloaded from ESTIMATE (https://bioinformatics.mdanderson.org/estimate/). K-M plotter  was applied to analyze the potential prognostic value of CTNNB1 in NSCLC, which was divided into low and high groups according to the median level [The expression of an level .We collected 273 cases of NSCLC postoperative samples from Central South University, The Second Xiangya Hospital. There were 133 cases of LUSC, 140 of LUAD, and 106 non-cancerous lung tissues from 2003 to 2013, including 70 female and 203 male patients with a median age of 56 years. Of the cases, 132 were at clinical stage I and II and 141 cases were at stage III. 154 cases had lymph node metastasis and 119 had no lymph node metastasis. At the last follow-up, 164 patients had survived and 109 patients had died. All patients had complete follow-up and clinical data. None of them had received treatment before surgery. Each case had definite clinical stage and pathological diagnosis on the basis of the Eighth Edition Lung Cancer Stage Classification and the WHO histological classification, respectively .The β-catenin protein with a dilution of 1:100  was examined according to the protocols as described before . In shorThe Immunohistochemical score was independently performed under 200x light microscopy by YZ and SF who were blinded to patients’ clinical information. The score evaluation was as follows : normal p < 0.05 (two-sided) was considered to be statistically significant.SPSS 24.0 was applied to all statistical analyses. The correlation between β-catenin protein level and clinicopathological factors of NSCLC was examined by chi-square test. Besides, the relationship among β-catenin protein, tumor-immune infiltrating cells, and immune score was examined by Spearman’s correlation test. Survival situation was tested by Kaplan-Meier analysis and Log-rank test. Cox hazards model was applied to evaluate the independent prognostic factors of NSCLC. CTNNB1 mRNA expression was up-regulated in LUAD (p < 0.001) but not in LUSC (p > 0.05). We also detected the prognostic value of CTNNB1. Low CTNNB1 mRNA expression was related to better OS  and PPS  in LUAD (p < 0.05), but had no correlation in LUSC (both p > 0.05).In UALCAN database, p = 0.001) compared to female and LUAD patients. It is worth noting that aberrant β-catenin protein expression rate was higher in clinical stage III patients compared to stage I patients, but the statistical difference was weak (p = 0.078). No obvious statistical difference was found in other clinicopathological variables (p > 0.05).In order to explore β-catenin protein expression in our patient cohort, we used IHC to detect the subcellular localization and expression of β-catenin protein in NSCLC and Non-CLT (non-cancerous control lung tissue). β-catenin protein was chiefly distributed in the cell membrane/cytoplasm, as well as in the nucleus . Aberranp = 0.018, p < 0.001, p = 0.002, p < 0.001, In this study, survival status of NSCLC patients with different clinicopathological variables was analyzed through Kaplan-Meier survival analysis. For patients with NSCLC, the overall survival of patients with aberrant β-catenin protein expression was remarkably shorter than that of patients with normal protein expression (p = 0.034), along with clinical stage III (p = 0.003), poor differentiation (p = 0.015), and with LNM (p = 0.019).Moreover, we also studied whether β-catenin protein expression status could be utilized as a prognostic factor for NSCLC patients independently. As shown in + T cells, macrophages, dendritic cells, and B cells. Expression data of β-catenin protein were downloaded from the UCSC Xena website, whose expression level was detected by reverse phase protein array (RPPA). Tumor-immune infiltrating cell data were obtained from the TIMER platform (Tumor Immune Estimation Resource). As shown in + T cells  and neutrophils  in the UCSC had significant negative correlation with β-catenin protein expression level. However, there were no statistical relationships between β-catenin and CD4+ T cells, dendritic cells, macrophages, B cells, or β-catenin protein (All p > 0.05).Firstly, we explored the relationship between β-catenin protein and tumor-immune infiltrating cells, including CD4+ T cells, neutrophils, CD8p = 0.045, r = −0.097), immune score , and estimate score  had a significant negative correlation with β-catenin protein.More and more evidence has shown the importance of immune and stromal cells in the immune microenvironment . Stromalβ-catenin is a multifunctional protein that can interact with cadherin, participate in cell adhesion, and form cytoskeleton tissue ,10. MostIn previous studies, it has been shown that higher β-catenin protein level was closely related to lymph node metastasis and low survival rate in nasopharyngeal carcinoma patients, which can be used as an indicator of prognosis . Besides+ T cells and neutrophils. Meanwhile, β-catenin protein was also negatively related to ESTIMATE score, immune score, and stromal score, suggesting that β-catenin protein has a potential function in tumor immunity, which has been found in other studies [The tumor microenvironment, as the living environment of tumor cells, is composed of various cytokines/chemokines, immune cells, tumor-related fibroblasts, and inflammatory cells ,18. ESTI studies ,36. This+ T cells and neutrophils.In summary, aberrant β-catenin protein expression was dramatically increased in NSCLC and might serve as a poor prognostic biomarker. Most importantly, β-catenin protein might play a key role in tumor immunity and the tumor microenvironment by inhibiting the infiltration of CD8


A hyperbranched phosphorus-containing copper phthalocyanine compound (DOPO-CuPc) was successfully synthesized and used as the flame-retardant additive to prepare flame-retarded epoxy thermosets. The addition of DOPO-CuPc led to a significant enhancement of the flame retardant properties of the epoxy resin. The 15DOPO-CuPc/EP composite obtained a LOI value of 35.8%, and the UL-94 rose from NR to V-0 rating. And the addition of DOPO-CuPc resulted in early decomposition of the epoxy thermoset, but the residual char at 700 °C reached 27.7%. The flame retardant mechanism was further investigated. It was found that DOPO-CuPc could release phosphorus-containing radicals and non-combustible gases in the gas phase to exert gas-phase flame retardant activity. In the condensed phase, the epoxy thermoset formed the expanded honeycomb-like char layer during combustion and the presence of copper phthalocyanine contributed to the stability of the char layer. As with other commonly used engineering plastics, high flammability is one of the major drawbacks4. The flammability of epoxy resins and the resulting fire hazards are a constant threat to people's lives and property, which severely limits their use in applications requiring higher levels of flame retardancy. The addition of halogenated compounds to epoxy resins is a widely used method for preparing flame retardant epoxy resins. Unfortunately, numerous halogenated flame retardants have been listed as persistent organic pollutants (POPs) due to the enormous ecological and human health hazards caused by the products released during combustion5. Recently, researchers are interested in finding eco-friendly flame retardants to alternative halogen-based flame retardants7.As a general thermoset, epoxy resins are widely applied in high-performance engineering fields due to its outstanding mechanical strength, excellent adhesive strength, and superior chemical resistance8. DOPO exhibit higher thermal and chemical stability than other uncycled organophosphorus compounds due to the biphenyl and heterophilic in structure9. In addition, the DOPO structure contains active P–H bonds, which are highly reactive to electron-deficient groups such as olefins, epoxides and carbonyl groups, and can be reacted to generate numerous derivatives12. However, the flame-retardant efficiency of DOPO-based flame retardants containing only phosphorus is generally unsatisfactory. In order to improve the flame retardancy of DOPO-based flame retardants and to reduce their negative effects on other properties of epoxy resin, it is an effective way to combine phosphorus with other flame retardant elements to form a synergistic flame retardant effect. Shang et al. designed a bio-based epoxy resin (TEBA) containing phosphorus and nitrogen13. The results showed that the LOI of TEBA increased by about 64% compared to the original epoxy resin and the UL-94 rating was improved from no rating to V-0. Zhu et al. designed a novel phosphorus-nitrogen containing oligomer (BPOPA) as an additive to enhance the flame retardancy of epoxy resin/ammonium polyphosphate14. It was found that the epoxy composites with 7.5 wt% ammonium polyphosphate and 2.5 wt% BPOPA passed UL-94 V-0 test and with an LOI value of 33.1%. And BPOPA exhibited binary flame retardant activity in both gas phase and condensed phase. You et al. reported a new type of phosphorus-nitrogen flame retardant (TNTP) and applied it in epoxy resin15. It was found that a 5 wt% loading of TNTP can impart satisfactory flame retardancy and thermal stability to epoxy resins.Currently, phosphorus-containing flame retardants are favored among the various flame retardants due to low toxicity and abundant resources. A typical phosphorus-containing flame retardant, 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO), has been proved to be the effective flame retardant for epoxy resins17. This special conjugate structure imparts exceptional thermal and chemical stability to the phthalocyanine. At the same time, phthalocyanines contain a high content of nitrogen, which shows significant potential for providing flame retardancy and thermal stability to polymers18. On the one hand, it provides thermal and chemical stability to the polymer by using phthalocyanine as an additive. On the other hand, phthalocyanines can be used in combination with other flame retardant elements to provide synergistic flame-retarding effect, such as phosphorus-based flame retardants. In addition, the phthalocyanine molecule can be coordinated with numerous metallic elements to form metal phthalocyanines to satisfy the requirements of different engineering fields due to their strong coordination capabilities20. As far as we know, phthalocyanines have a wide range of applications in various research fields such as solar cells, photoelectric conversion, electrical signal storage, and catalytic materials, but the application in the field of flame retardancy has rarely been reported.Hyperbranched polymers are of increasing interest to polymer scientists because of their unique structural features, synthetic methods, and large number of functional end-groups. In recent years, hyperbranched polymers are widely used as flame retardants for polymers. Phthalocyanines, a hyperbranched organic macromolecular compound with dendritic molecular structure, have attracted attention for their unique two-dimensional co-planar aromatic heterocyclic structureAt present, phosphorus–nitrogen synergistic flame retardant modification has been proved to be a very efficient method to reinforce the flame retardancy of epoxy resins. In this research, we present a DOPO-based hyperbranched copper phthalocyanine (DOPO-CuPc) by grafting reaction of DOPO with copper phthalocyanine and introduced it into the epoxy resin as an additive. The structure of DOPO-CuPc was characterized. The flame retardant properties and thermal stability of the modified epoxy thermosets were evaluated by flammability tests, and the flame retardant mechanism of DOPO-CuPc was analyzed.®. The Bisphenol A type phthalonitrile-based resin containing benzoxazine (BAPh) was purchased from Chengdu Dymatic Fine Chemicals Co., Ltd. Anhydrous cuprous chloride (CuCl) was purchased from Xilong Chemical Co., Ltd. N, N-Dimethylacetamide (DMAc), Methanol and Tetrahydrofuran (THF) were purchased from Chengdu Kelon Chemical Co., Ltd. The 4, 4’-diaminodiphenylsulphone (DDS) was purchased from Wenzhou Shoucheng Chemical Technology Co., Ltd. Diglycidyl ether of bisphenol A  was obtained from Shandong Jiaying Chemical Technology Co., Ltd., China.DOPO was purchased from Adamas-betaDOPO (21.6 g), BAPh (36.9 g) and THF (200 ml) were mixed in a 500 ml four-necked round-bottom flask equipped with a stirrer and a reflux condenser. After stirring at room temperature for 0.5 h, the mixture was reacted under nitrogen protection at 80 °C for 12 h. The solvent was removed using a rotary evaporator, washed with water and dried under vacuum at 60 °C for 12 h. Finally, DOPO-BAPh was obtained.DMAc (200 ml) was added to a 500 ml four-necked round-bottom flask equipped with a stirrer and reflux condenser. Then Bisphenol A type phthalonitrile-based resin containing benzoxazine (3.52 g) and CuCl 0.1 g) were added to the flask, stirred until fully dissolved, and the reaction was kept at 160 °C for 4 h. The product resulting from the reaction was washed, filtered, and dried at 80 °C. The obtained product was then filtered by using methanol at 80 °C and room temperature several times, respectively, and then dried at 70 °C for 12 h in vacuum oven. The final green powder obtained was the target product (DOPO-CuPc)  were conducted on a Perkin-Elmer Spectrum Two FTIR spectrometer. The test was performed by using the KBr pellets method. The detailed conditions are as follows: the spectral resolution was 4/cm, the number of scans was 32, the scan wave number range was 4000–640/cm.The flammability of modified epoxy thermosets was evaluated using limiting oxygen index (LOI) and UL-94. The value of LOI was acquired by using a JF-3 oxygen index meter  according to ASTM D2863. The dimensions of the samples were 130 mm × 6.5 mm × 3 mm. The vertical burning test were determined by using a CZF-3 burning tester  according to ASTM D3801. The dimensions of the samples were 130 mm × 12.7 mm × 3 mm.2. And the dimensions of the samples were 100 mm × 100 mm × 3 mm.The combustion behaviors of the modified epoxy thermosets were recorded using a cone calorimeter  according to ISO 5660-1 at a heat flux of 50 kW/mThermogravimetric analyses (TGA) of the modified epoxy thermosets were con-ducted from a Pyris thermogravimetric analyzer (Perkin-Elmer TGA 4000) under nitrogen atmosphere with gas flow of 20 ml/min. A sample with a weight of about 10 mg was used and heated from 30 to 800 °C at a rate of 10 °C/min. In addition, the samples were dried before the test started.The thermogravimetry-infrared spectroscopy (TG-IR) was studied by connecting a TGA 4000 thermogravimetric analyzer and a Spectrum Two FTIR spectrophotometer through a special tube for transferring the gas. The test conditions of TG-IR are the same as the TGA.The residual chars of the modified epoxy thermosets after cone calorimetric test were carried out using a scanning electron microscope (SEM) (Hitachi S-4800) with an accelerating voltage of 3 kV. Samples were sprayed with gold before the test started.21 and phthalocyanine. And the nitrile groups, P-containing bonds were still detected in DOPO-CuPc, based on the analysis above, the DOPO-CuPc was successfully prepared as designed.The infrared spectra of DOPO, BAPh, DOPO-BAPh and DOPO-CuPc were shown in Fig. 5%), maximum rate of weight loss (Vmax), maximum temperature of weight loss (Tmax) and weight of char yield at 700 °C (Cy700) were listed in Table The thermal stability of the modified epoxy thermosets was studied by TG and derivative thermogravimetric (DTG) in nitrogen. Figure 1 + t2) was reduced to 26 s. With a further increase in the DOPO-CuPc content to 15 wt%, the modified epoxy thermosets achieved V-0 rating and the LOI value increased to 35.0%, and t1 + t2 reduced to 9 s. These results indicated that DOPO-CuPc possessed excellent flame retardant properties.The flame retardant properties of DOPO-CuPc/EP composites were analyzed using LOI and UL-94 vertical burning tests. The results of the tests were presented in Table 2, and the introduction of DOPO-CuPc made the PHRR of epoxy thermosets lower and appear earlier. The PHRR of 5DOPO-CuPc/EP composites was reduced to 565 kW/m2. The continued addition of DOPO-CuPc led to a further decrease in PHRR. The PHRR of 20DOPO-CuPc/EP composites was reduced to 306.9 kW/m2, which was 57.9% lower compared to the neat epoxy resin. The THR of the neat epoxy resin was 88.4 MJ/m2. The THR of DOPO-CuPc/EP composites was reduced by 57.3% compared with that of neat epoxy resin. The results indicated that DOPO-CuPc inhibited the thermal decomposition of epoxy thermosets. In addition, the mass loss rate of the epoxy cured products was reduced from 75.8 to 59.4% with the introduction of DOPO-CuPc. It implied that more residual char was generated during combustion, which inhibited the combustion of the epoxy thermosets and prevented the heat transfer to the matrix interior, proved the flame retardant effect in the condensed phase.To further investigated the flame retardancy of epoxy thermosets, cone calorimeter tests were conducted. The crucial parameters such as the time to ignition (TTI), heat release rate (HRR), peak of HRR (PHRR), total heat release (THR) and total mass loss (TML) were obtained by cone calorimeter. The results were presented in Fig. 22. The blowing-out effect might contribute to flame retardancy of modified epoxy thermosets.In order to better investigated the effect of DOPO-CuPc on the flame retardancy of epoxy resin, the combustion process of epoxy thermosets in UL-94 test was recorded by a digital camera. Figure The analysis of the residual char formed after the combustion of epoxy thermosets could provide a lot of information which reflect the flame retardant mechanism in the condensed phase. The digital photographs of the residual char of the modified epoxy thermoset resin after cone testing were presented in Fig. 23.The residual char of the epoxy thermosets after cone calorimeter testing was collected for SEM to further analysed the microstructure of the surface of residual char. As shown in Fig. 2 (2359/cm), hydroxyl-containing compounds (3500–3700/cm) and aromatic compounds  were detected in the infrared spectra of neat epoxy resin. There were no emerging peaks from the infrared spectra of 20DOPO-CuPc/EP composites, while the ratio of peaks changed. This phenomenon was attributed to the overlap of the newly emerged peaks with the original peaks. Among them, the peaks at 1250/cm (P=O) and 1598/cm (P–O–Ph) were overlapped with the aromatic compounds (CAr–H and CAr–O), respectively25. It indicated that the products of polyphosphate structures were generated in the degradation of DOPO-CuPc, which further reacted with other products to form P–O–Ph bond-containing compounds. Meanwhile, the intensity changing of the absorption peak at 749/cm was the contribution of the DOPO group in DOPO-CuPc. The above results indicated that the DOPO-CuPc/EP composite generated P–O–Ph bond-containing polyphosphate compounds during the degradation.The pyrolysis products of epoxy thermosets were investigated by using TG-IR to elucidate the flame retardant mechanism of DOPO-CuPc in the gas phase. The three-dimensional view of the products generated during the decomposition of the epoxy thermosets and infrared spectra at different temperatures were presented in Fig. 26. These char layers effectively act as heat and oxygen barriers, thus enhancing the flame retardancy of the epoxy resin. Besides, the special cross-linked network structure of copper phthalocyanine result in more aromatic structure in the residual char, which enhances the stability of the char layer. In conclusion, DOPO-CuPc is capable of exerting flame-retarding activity in both the gas and condensed phases.Based on the above analysis, the flame retardant mechanism of DOPO-CuPc is attributed to the flame-retarding effect of gas phase and condensed phase. The flame retardant mechanism of DOPO-CuPc is shown in Scheme A DOPO-based hyperbranched phthalocyanine flame retardant for epoxy resin was obtained via a grafting reaction between DOPO and copper phthalocyanine. Owing to the lower decomposition temperature of DOPO-CuPc, it led to the early decomposition of epoxy resin, but the residual char was enhanced considerably. Besides, the modified epoxy thermosets passed the UL-94 V-0 rating with the LOI value of 35.8% in the loading of 15 wt%. The enhanced flame retardancy of epoxy resins was attributed to the flame retardant activity of DOPO-CuPc in the gas and condensed phases. On the one hand, the generated phosphorus-containing radicals inhibit the combustion process and the non-combustible gas produced dilutes the oxygen concentration. And the blowing-out effect also contributes to the flame retardant activity. On the other hand, the char layer formed by dehydration reaction during combustion acted as a protective char layer in the condensed phase. Furthermore, the presence of copper phthalocyanine reinforces the stability of the char layer.


Murraya koenigii (M. koenigii), Micromelum minutum (M. minutum), and Clausena indica (C. indica) are three varieties of curry leaves in the family Rutaceae. They have been widely used in Ayurvedic medicine worldwide in the treatment and prevention of various diseases. Earlier findings provide strong evidence to support the three curry leaf species' potent pharmaceutical and biological effects, including antioxidant, antidiabetic, anti-inflammatory, and antitumor activities. Various parts of these plants, such as leaves, seeds, flowers, and fruit, contain constituents responsible for the modulation of numerous biological processes. Leading constituents of curry leaves play a crucial role in diabetic and anticancer management by regulating various molecular pathways, including Bcl-2, Bax, NF-κB, and TNFα, according to in vitro and in vivo models established. Therefore, this review summarizes the current knowledge on research achievements made in terms of phytoconstituents, their structures, biological activities, and pharmacological actions with clinical studies of curry leaves up to date. The review also emphasizes the necessity for comprehensive research studies on the pharmacological actions and the mechanisms of selected phytochemicals of M. koenigii, M. minutum, and C. indica to validate their efficacy as potent herbal remedies for many ailments. Murraya koenigii (M. koenigii), Micromelum minutum (M. minutum), and Clausena indica (C. indica) available in most Asian countries. Their phytochemical constituents play a role in disease management through the managing of many metabolic pathways.The use of plant-based natural products in the treatment and prevention of diseases and health enhancement due to nutritive importance and pharmacological benefits has led to the significant attention of the scientific community and the general public nowadays. Medicinal plants are readily available and provide a cost-effective source with lesser side effects to develop new drugs , 2. PlanM. koenigii, M. minutum, and C. indica. Of the three, M. koenigii is a fascinating house plant grown in Asia and native to Sri Lanka, Bangladesh, and India, which had used curry leaves or “karapincha”  for centuries. The dark green fresh leaflets of M. koenigii are widely used in Asian cooking mainly for their aroma and versatile medicinal properties [M. koenigii are used to treat diabetes, chronic fever, dysentery, and diarrhea [M. minutum is also used as a flavoring agent and is reported to have medicinal value in the southern part of Thailand and many other Asian countries [M. minutum roots are used to cure ringworms and to regulate menstruation. Other parts of M. minutum are used as carminatives, purgatives, and expectorants [M. minutum are used traditionally to treat toothache and teething issues in babies, skin irritations caused by scabies, and as a remedy for stomachache and headache [C. indica is famous as a folk medicine in many Asian countries. Leaves and roots of C. indica treat various health issues, such as flu, colds, joint dislocation, bone fractures, headaches, colic, and rheumatism. Moreover, C. indica fruits are widely used in Vietnam and South Indian cooking mainly due to their aroma [M. koenigii, M. minutum, and C. indica discussed in the review, have somewhat common morphological characteristics and taxonomic positions as indicated in M. koenigii, M. minutum, C. indica are shown in Rutaceae, the family of flowering plants, is composed of 160 genera and a few herbaceous perennials [operties , 8. Furtdiarrhea . M. minuountries . In partctorants , 12. Theheadache . C. indiir aroma , 15. TheM. koenigii and M. minutum in traditional medicines and home remedies has attracted the scientific community, but limited studies have been conducted to evaluate their pharmacological and medicinal efficacy. Despite the importance of C. indica in folkloric remedies, its pharmacological potential and chemical constituents have been rarely identified. Therefore, this review will shed light on the major bioactive compounds of M. koenigii, M. minutum, and C. indica and their pharmacological effects highlighting the potential in the key medicinal activities through the modulation of various biological pathways. The major pharmacological activities of the M. koenigii, M. minutum, and C. indica are discussed in detail in this review  and have been thoroughly explored using different techniques, including mass spectrometry, Nuclear Magnetic Resonance Spectroscopy (NMR), Infrared, and ultraviolet spectroscopy. Alongside these studies, pharmacological activities of the isolated compounds have been carried out via in vitro and in vivo studies combined with animal models.Several studies have contributed to understanding the chemical constituents and therapeutic actions of M. koenigii, M. minutum, and C. indica and their pharmacological activities. All three varieties have nutritive value owing to the presence of a variety of essential phytochemicals, minerals, and trace minerals present primarily in their leaves and seeds. Phytochemical studies of the leaves, roots, seeds, and stem bark of M. koenigii, M. minutum, and C. indica have yielded many essential metabolites such as terpenoids, polyphenols, coumarins, alkaloids, flavonoids, carotenoids, vitamins, and nicotinic acid, as summarized in the supplementary information Tables Tables 2M. koenigii, M. minutum, and C. indica, have demonstrated a wide range of common pharmacological effects such as antihyperlipidemic, antimicrobial, antioxidant, and antihyperglycemic, and apoptotic activities [M. koenigii and M. minutum, have demonstrated remarkable cytotoxic anticarcinogenic activities. In addition, M. minutum has shown a remarkable effect as a poultice for ringworms [M. koenigii, M. minutum, and C. indica contain various phytoconstituents that play an essential role in disease management via modulation of different cell signaling pathways  diabetes is reported as 90% of the diabetes population in the world, being the most prevalent form of diabetes that is referred to as non-insulin-dependent diabetes. Type 2 diabetes may be owing to obesity and unhealthy lifestyles among individuals. While insulin production is present in this form, the extent of produced insulin may be inadequate to meet the body's needs or, in some cases, may be due to insulin resistance development . RecentlM. koenigii leaf extracts on hyperglycemic activity. Rats have been selected as suitable animal models after inducing diabetes with different drugs, alloxan or streptozotocin (STZ), to mimic human hypoglycemic conditions. Several studies on the antihyperglycemic activity of M. koenigii in various solvents with varying degrees of polarities are reported. The rationale behind using solvents of varying polarity is to search for more promising drugs to manage hypoglycemic conditions. Water is the most common solvent used because of M. koenigii's common use in aqueous food preparations and decoctions.In this regard, a large amount of work has been done using animal models to study the effectiveness of M. koenigii leaves demonstrated a substantial decrease in the blood glucose levels of diabetic mice. Notably, no effect on the blood glucose levels of normal treated mice was observed, and the aqueous extract exhibited a significant reduction of the blood glucose level in rats at a dose of 200 mg/kg. This observation indicates a promising therapeutic effect on diabetes by M. koenigii with fewer or no undesirable side effects [M. koenigii in treating hyperglycemia [A study with alloxan-induced diabetic rats administrated with the aqueous extract of  effects . Severalglycemia –40.M. koenigii leaves has shown an effect on reduced blood glucose levels. Also, it enhances the effect of insulin by extending its therapeutic value in a study with healthy Wistar rats [M. koenigii with insulin was achieved, and restoration of glucose homeostasis alone or in combination with other antihyperglycemic agents was suggested [M. koenigii with insulin indicates a clinical potential of developing a safe therapeutic approach to diabetic management that needs to be validated for the long run. A study was undertaken to evaluate the protective effects of M. koenigii leaves aqueous extract against beta-cell damage in streptozotocin-induced diabetes rats. The results showed that administration of M. koenigii leaves effectively brings about its antihyperglycemic effect via secretion of insulin secretion from the regenerated beta cells and remnant beta cells [M. koenigii leaves extracts on plasma insulin and blood glucose levels in alloxan-induced diabetic rats. These researchers had successfully achieved a significant reduction (P < 0.05) of blood glucose levels of diabetic rats treated with methanol and aqueous extracts compared to the diabetic control group. A significant rise in plasma insulin was observed on the 43rd and 58th days of treatment in methanol and aqueous extracts suggesting stimulated secretion of insulin from the beta cells of pancreatic cells [M. koenigii leaves as dietary constituents in controlling hyperglycemia in normal rats, alloxan- or STZ-induced mild diabetic rats, or moderate diabetic rats [M. koenigii leaves did not affect any profound hypoglycemic effect in normal rats, as stated in previous findings [M. koenigii on alloxan-induced diabetic rats, where results revealed that the extract exhibited hypoglycemic effects [M. koenigii enhanced insulin secretion from the pancreas [Fresh juice of tar rats . A succeuggested . Therefota cells . Anotheric cells . Another effects . Anotherpancreas .M. koenigii on hemoglobin glycation and alloxan-induced diabetic rats with a blood glucose level of 13 mmol/l. Alloxan-induced diabetic rats were treated orally with a crude aqueous extract (500 mg/kg body weight), and the outcome of the study indicated a significant (P < 0.05) decrease in blood glucose and glycated hemoglobin levels compared to the diabetes control group [Raised glycated hemoglobin concentration in plasma is the clinical hallmark of poorly controlled diabetes . Glycateol group .in vitro protein glycation inhibitory potential of M. koenigii aqueous leaf extract. The results revealed that, in the presence of M. koenigii aqueous leaf extract, migration of BSA under nondenaturing conditions was retarded compared to the control [in vitro protein glycation inhibitory potential of the M. koenigii aqueous leaf extract.Inhibition of hemoglobin glycations is considered to be one of the therapeutic approaches that delay or prevent the progression of major diabetic complications. A method was developed with bovine serum albumin (BSA) using polyacrylamide gel electrophoresis under native conditions (PAGE) to investigate the  control . This woM. koenigii leaves. The study results showed that ethanolic extract successfully regulated the blood glucose level and managed neuropathy [Diabetic peripheral neuropathy, a peripheral nerve disorder, develops as a late manifestation of long-standing or uncontrolled diabetes. A study was made to evaluate neuropathy management in male Wistar rats by the ethanolic extract of uropathy . StudiesM. koenigii. The increased latency time in streptozotocin-induced diabetic rats suggested pain perception in the tested animal model. Therefore, the ethanolic extract of M. koenigii significantly decreased the glycemic level and prevented the progression of neuropathy in the diabetic animal model [M. koenigii is under clinical trials. One clinical research indicated a transient reduction in fasting and postprandial blood sugar levels in non-insulin-dependent diabetes mellitus patients provided with 12 g of M. koenigii leaves powder supplementation [M. koenigii, only a few compounds have been investigated on their antidiabetic potential. A study reported an in vitro antidiabetic activity in L6-GLUT4myc myotubes and in vivo studies of streptozotocin-induced diabetic rats with six major carbazole alkaloids, koenidine, murrayazoline, koenimbine A, koenidine, mahanimbine, and O-methylmurrayamine, extracted from M. koenigii leaves. The results revealed the potential use of koenidine in managing type II diabetes with insulin resistance [The loss of pain perception occurs in diabetic patients due to damage of nerves. An experiment was made to investigate the pain threshold of the streptozotocin-induced diabetic rats after administering a dose of 300 and 500 mg/kg ethanolic extract of al model . Furthersistance .M. minutum seeds ethanolic extract with coumarin and microminutinin has been shown to lower serum glucose levels in diabetic rats. A significant decrease in glucose levels was observed after administering coumarin (100 mg/kg) and microminutinin (25 mg/kg). The in vivo study further demonstrated the potential of using M. minutum seeds in treating hyperglycemia [C. indica roots has been investigated by bioguided isolation of the ethyl acetate extract. The isolates, dentatin, clausine K, and nordentatin, demonstrate anti-α-amylase activity suggesting the hypoglycemic activity of C. indica roots [C. indica fruit identified coumarins as major compounds demonstrating anti-α-amylase activity [C. indica and M. minutum, too, could be promising sources of antidiabetic agents.The effect of glycemia . Similarca roots . A recenactivity . TherefoM. koenigii, C. indica, and M. minutum play a pivotal role in preventing and treating diabetes mellitus. Even though the exact molecular mechanism in this vista is not understood fully, it was considered that their constituents play a vital role in the modulation of various cell signaling pathways, including insulin secretion. Many of the experiments discussed above are attributed to the presence of coumarins, carbazole alkaloids, and their derivatives in the M. koenigii, C. indica, and M. minutum extracts, as they are considered to be potent antihyperglycemic agents [ß cells, in addition to the enhanced secretion and sensitivity of insulin from the pancreas, as suggested by various studies. Umbelliferone and osthole are the most common coumarins studied to modulate molecular pathways in diabetes and its complications [The active compounds of c agents . Carbazoications .γ (PPARγ) is an essential nuclear hormone receptor acting on the transcription of genes  involved in glucose disposal. A study finding reveals that umbelliferone upregulated the expression of surface GLUT4 gene and PPAR γ in adipose tissue, enhancing insulin sensitivity leading to increased peripheral glucose uptake [γ, increasing insulin sensitivity, and facilitated the synthesis and storage of fat [Peroxisome proliferator-activated receptor e uptake . Ostholee of fat . The acte of fat . In anote of fat .M. koenigii represents a promising candidate with potential use for the management of diabetes mellitus. Therefore, M. koenigii represents the clinical potential of developing a safe therapeutic drug confirmed with further preclinical and clinical research. Although plenty of evidence demonstrates that coumarins and other metabolites are effective against hyperglycemia, there had been no adequate investigations reported on the involvement of C. indica and M. minutum in hyperglycemia.Keeping in view the tremendous antidiabetic studies and availability of literature, M. koenigii and M. minutum have been reported to have a potential role as a remedy for cancer and inflammation. The preliminary approach for any potent anticancer compound from natural sources is an investigation of in vitro cytotoxicity studies on various cell lines. Numerous studies have been carried out on isolating natural compounds and understanding their antitumor and cytotoxic activities. M. koenigii and M. minutum also have been successfully addressed in this regard.Cancer involves multiple genetic alterations, unlike other monogenic disorders. The onset of cancer typically consists of a series of mutations in the genome resulting in uncontrolled cell proliferation, lack of apoptosis, and alterations in epigenetic regulations. Nearly ten million new cancers are diagnosed annually, making it the second most common cause of mortality worldwide . TypicalM. koenigii, C. indica, and M. minutum play an essential role in the modulation of various signaling pathways in the cell. These constituents upregulate the tumor suppressor genes p53 and downregulate the genes responsible for developing cancer, such as NF-κB. Furthermore, various carbazole alkaloids and coumarins of these curry leaves activate the cyclooxygenase pathway and apoptosis.Constituents Some chemotherapeutic drugs have proved to be highly cytotoxic agents to normal cells, as seen in clinical studies. However, the preferential cytotoxicity against malignant tissues is crucial in a suitable treatment. Plants and their derivatives play an essential role in the cytotoxicity studies searching for an alternative for chemotherapeutic drugs with selected toxicity towards cancerous cells. Active compounds in a plant extract target cell lines and significantly affect cytotoxicity.M. koenigii in the breast cancer cell line MDA-MB-231 with an IC50 of 14.4 μg/mL [M. koenigii and M. minutum, and its analogs have shown significant cytotoxic and anticancer activities in prostate cancer studies. Prostate cancer, the most commonly diagnosed cancer in men worldwide, is associated with alterations in androgen receptor functions. Studies have shown the functions of mahanine related to the androgen receptor, inhibiting ligand-dependent and -independent transactivation leading to a significant decrease in the expression of androgen-regulated genes. Furthermore, mahanine results in proteasomal degradation by compromising the stability of the androgen receptor [An experimental study reported the cytotoxic activities of alkaloid extract from .4 μg/mL . Mahaninreceptor . Anotherreceptor . The duaM. minutum executed apoptosis through the intrinsic pathway in human leukemia U937 cells [M. minutum leaves methanol extract composed of six coumarins including minutin A, minutin B, 8,4″-dihydroxy-3″,4″-dihydrocapnolactone-2′,3′-diol, 8-hydroxyisocapnolactone-2′,3′-diol, 8-hydroxy-3″,4″-dihydrocapnolactone-2′,3′-diol, and clauslactone E reported a significant cytotoxic activity against lung adenocarcinoma (A549 and SBC3) and leukemia (K562 and K562/ADM) cell lines [M. minutum leaves.In another study, mahanine isolated from 37 cells . A studyll lines . FurtherM. minutum roots and its isolates were reported to have inhibitory effects against the KB cell line and weak cytotoxicity against the NCI-H187 cell line providing a basis for the use of M. minutum roots in the treatment of cancers [in vivo efficacy to promote various isolates of M. koenigii and M. minutum to be used in treatments in carcinoma.Specific cytotoxicity activities of the extracts of  cancers . The varApoptosis is a typical biochemical function that induces the committing of cell death of pointless cells. Resistance towards apoptosis is a crucial factor of tumor cells. Therefore, any search for the new chemotherapeutic agent that can induce apoptosis is enhanced by understanding apoptotic pathways. The two main pathways of apoptosis are intrinsic and extrinsic. External death signals trigger apoptosis in extrinsic pathways, and intrinsic pathways are mediated by Bcl-2-associated X protein (Bax) and Bcl-2 antagonist. Any alteration in Bcl-2 and Bax causes the development of cancers .M. koenigii- and M. minutum-derived extracts and phytochemicals, including carbazole alkaloids such as mahanine, mahanimbicine, girinimbine, mahanimbine, and coumarins. A valuable study on the effect of mahanine in prostate cancer reported the induction of apoptosis in cancer cells. Several bioactive compounds, including girinimbine, a carbazole alkaloid isolated from M. koenigii, have been reported to potentially inhibit inflammation and induce apoptosis in human colon cancer cells (HT-29) in vitro [in vivo in a 24-hour treatment of zebrafish embryos, indicating a significant distribution of apoptotic cells in embryos. These findings strongly suggest a potential for girinimbine to be further examined for its use in treating cancer as a chemotherapeutic agent. Another in vitro study of girinimbine on A549 lung cancer cells about apoptotic mechanistic pathways has demonstrated a successful induction of the early phase of apoptosis as indicated by stability assays [in vitro and in vivo studies [Several investigations provided evidence of the proapoptosis functions of the in vitro . The indy assays . Further studies , 66.via regulating the intrinsic pathway by the downregulated Bcl-2 protein expression and upregulated Bax [0/G1 phase arrest [These findings strongly represent the notion that girinimbine has a significant involvement in both intrinsic and extrinsic pathways of apoptosis. Moreover, girinimbine upregulates the p53 the key tumor suppressor protein, and the cyclin-dependent kinase (CDK) proteins, p27 and p21. The overexpression of CDK proteins inhibits abnormal cancer cell proliferation and promotes cell cycle arrest at the G1 phase. Additionally, girinimbine activated apoptosis ated Bax . Anothere arrest . In thisM. koenigii, has been studied to evaluate the efficacy of the inhibition of MCF7 breast cancer cells through apoptosis [in vitro. One possible mechanism of koenimbine in apoptosis in breast cancer cells could be the downregulation of the Wnt/β-catenin self-renewal pathways. Similarly, koenimbine has remarkably inhibited cell proliferation of prostate cancer stem cells in a recent study [Koenimbine, another dietary component isolated from poptosis . This stnt study .C. indica roots were used to purify few compounds, including dentatin, nordentatin, and clausine K, with anticarcinogenic properties [C. indica is reported to be a rich source of phytochemical components such as sesquiterpenes, coumarins, and carbazole alkaloids which might have a strong potential for anticancer activity. Nevertheless, there is still room for studies on C. indica, which needs to be screened for the novel, cost-effective, and efficacious anticancer agents.In a previous study, operties . In addiκB transcription factor, has a pivotal role in human cancers. The overexpression of NF-κB induces antiapoptosis and angiogenesis and promotes tumors. Several stimuli such as cellular and environmental stresses, DNA damage, growth factors, and cytokines activate NF-κB. Therefore, the inhibition of NF-κB is crucial in the prevention and control of cancer. A study evaluated the chemopreventive activity of M. koenigii leaf extract on 4T1 breast cancer cell-challenged mice. The results confirmed those aqueous leaf extracts significantly prevented tumor formation compared to the untreated group of mice. The activation of NF-κB is induced by the cytokines cytokineIL-1β and pleitropic cytokine IL-6 was found to be reduced in the M. koenigii-treated 4T1 breast cancer cell-challenged mice [The transcription factor, NF-ged mice .Cardiovascular diseases are the number one cause of death in the world. According to WHO, cardiovascular diseases in the elderly population have created 18 million deaths in the year 2016, resulting in 31% of deaths in the globe . AtherosM. koenigii leaf extracts. The biochemical response of M. koenigii was studied on various types of cholesterols in a study with Albino rats. These researchers fed Albino rats a diet fortified with 20% coconut oil with 10% M. koenigii leaf feed for three months. An increase in HDL and reduced LDL, VLDL, and total serum cholesterol levels were observed in the experiment [M. koenigii aqueous leaf extract of 300 mg/kg on the biochemical response in normal and STZ-induced severe diabetic rats were evaluated. The results confirmed a decrease of 22.9% and 37.1% of triglyceride level and 19.2% and 30.3% in total cholesterol in normal and diabetic rats, respectively, in one month [Various studies are undertaken to evaluate the antihyperlipidemic potential of the periment . In anotne month .M. koenigii leaf on lowering blood cholesterol levels could be ascertained by various experimental conditions, including the study's duration, the type of animal model or the form of M. koenigii leaf used, or the solvent used for the extraction. Another finding showed that obese diabetic ob/ob mice given 80 mg/kg aqueous M. koenigii extract for ten consecutive days have significantly decreased blood cholesterol levels from 277.6 ± 16.6 mg/day (day 0) to 182.0 ± 15.3 mg/day [M. koenigii leaves lowered body weight gain in high-fat diet-induced obese rats at oral administration of 30 mg/kg/day.The potency of 3 mg/day . In addiM. koenigii aqueous extract (180 mg/kg body weight) on albino Wistar rats treated with Voltral tablets to increase the level of CK-MB [M. koenigii compared to the group treated with atenolol, a drug available in the market as a beta blocker, further suggesting a potential role of M. koenigii in lowering CK-MB in myocardial infarction.Creatine kinase (CK-MB) is a well-known heart enzyme and a cardiac biomarker found primarily in the cardiac muscle. A study assessed the effect of of CK-MB . A signiM. minutum seed, a rich source of coumarins [M. minutum seeds in rats. However, no report on the potency of C. indica on treating or reducing cardiovascular biomarkers was found in the literature.oumarins , has beeoumarins . In thatth century's rising demand for new antimicrobial agents. However, most new antimicrobial agents fail after many clinical trials and treatments due to the side effects and rapid resistance development. Traditionally, M. koenigii, M. minutum, and C. indica are well known for their health benefits and are rich in antimicrobial activities owing to the presence of a variety of essential phytochemicals and many secondary plant metabolites, such as tannins, terpenoids, alkaloids, flavonoids, phenols, and quinines present in their leaves and seeds [The occurrence of microbial drug resistance of many microorganisms and their complications are growing worldwide. Therefore, antibiotic resistance has become one of the major issues since the late 20nd seeds .in vitro. Therefore, aqueous extracts of widely used culinary spices, including M. koenigii, were screened in a research study against several microorganisms found on edible fish to identify their antimicrobial activities [M. koenigii has shown a significant antibacterial activity towards various pathogenic bacterium types found in edible fishes. The minimum inhibitory concentration (MIC) of M. koenigii on Staphylococcus aureus (S. aureus) was 250 μg/ml and 500 μg/ml on Escherichia. coli (E. coli), Lactobacillus spp., Salmonella typhi (S. typhi), and Shigella spp. Both methanol and aqueous extracts of M. koenigii showed antibacterial activity among all the herbs extracts tested in the study suggesting the use of M. koenigii in cooking to eradicate the respective bacteria and as a food preservative.The biodeterioration of food due to microorganisms causes a significant loss to crop from vegetables, grains to fish, and poultry. Many plant extracts have been reported to display antifungal, antibacterial, and insecticidal properties tivities . M. koenM. koenigii on seed-borne fungi species. M. koenigii was reported to have a remarkable mycelial growth inhibitory effect against all the seed-borne fungi used in the experiment. However, the highest inhibition of mycelial growth was observed in Alternaria alternata [M. koenigii in a study [An investigation was carried out to screen twenty medicinal plant extracts against seed-borne fungi in seeds, causing weight and quality losses leading to the reduction of commercial value. This study was aiming at the efficacy of lternata . In view a study . AcetoniM. koenigii plant parts, bark extract is the least common among researchers. Few investigators have used the agar cup-plate method in accessing the antibacterial activity of different microbial species of the bark and leaf extracts. Bark extracts have shown a significant inhibitory activity compared to the leaf extracts of M. koenigii, and the most susceptible species was S. aureus [M. koenigii in different solvents, acetone, chloroform, ethanol, and methanol, have been studied for their antimicrobial activities. The study results indicated that the acetone and methanol extracts had higher antimicrobial activity than other solvents [In evaluating the antioxidant property of . aureus . Leaf ansolvents .in vitro antibacterial potential of the aqueous and ethanolic leaf extracts of M. koenigii against bacterial species accountable for endometritis. Bacillus sp., Salmonella sp., Staphylococcus sp., and Corynebacterium spp. are responsible for endometritis in domestic cows. Ethanol and aqueous extracts of M. koenigii showed a potent antimicrobial effect towards pathogens under study. The ethanolic extract showed a better response than the aqueous extract [Another study revealed the  extract .M. koenigii leaves are rich in many phytochemicals, serving as antioxidant and antimicrobial agents [M. koenigii have been extensively explored recently. An experiment was made to evaluate the antifungal activity of methanol and ethanol extracts and the essential oils of M. koenigii leaves. The results indicated that all the extracts showed antifungal activity and methanol extract had the highest activity and essential oils with a moderate antifungal activity [Essential oils extracted from plants have been recognized for their antioxidant and antimicrobial attributes for decades. Essential oils of l agents . The antactivity .M. minutum is reported to be a rich source of antimicrobial compounds. Antibacterial potential of leaves, stem bark, and root bark ethanolic extracts of M. minutum against E. coli and S. aureus was investigated to validate its current use as alternative medicine in a study [S. aureus were significant when compared with E. coli. Among the three extracts, leaf extracts showed the highest zone of inhibition towards both bacteria species due to the prevalence of highly cytotoxic coumarin compounds and highly antimutagenic carbazole alkaloids. For instance, mahanine could explain the increased inhibitory activity of leaf extracts [M. minutum showed partial activity towards S. aureus and inactivity towards E. coli in this study. a study . The inhextracts . HoweverM. minutum is rich in sesquiterpenes germacrene, germacrene-B, δ-elemene, and ß-caryophyllene, having a potential antimicrobial activity [M. minutum essential oils was investigated at a concentration of 0.25%—1% (v/v) in a study. The results showed significant antimicrobial activity at 1% v/v towards all the species under study [The essential oil from activity . The anter study .C. indica leaf essential oil extracts from South India, and significant activity against B. cereus, S. typhi, and P. vulgaris was observed [E. coli and S. aureus. Another experiment was made to evaluate the MIC of essential oils from C. indica from Vietnam towards selected Gram-negative and Gram-positive bacteria. The results showed significant antibacterial activity against E. coli and S. aureus. In addition, the MIC of various fungi strains was also tested, and interestingly, the essential oil was inactive for all the tested fungi species [E. coli and S. aureus are contradictory in the studies conducted in South India and Vietnam, signifying a difference in their chemical profiles. The essential oil from C. indica from Vietnam leaf contains terpenes, mainly terpinolene, and myristicin, as the major constituents [A study was performed on the antibacterial activity of observed . However species . Therefotituents . At the tituents .M. koenigii or M. minutum fruits, but the antimicrobial activity of C. indica fruits has recently been reported [C. indica, have shown significant antimicrobial effects against the Gram-positive bacteria, and no activity was reported for the tested fungi and yeast species [M. koenigii, M. minutum, and C. indica observed in literature are tabulated in Hardly any literature was found on the antimicrobial activity of reported . Flavono species . MicroorMedicinal plants are excellent natural antioxidants that can be used to prevent diseases such as cancer, heart diseases, strokes, and inflammation . The secM. koenigii plays a vital role among the traditionally important medicinal plants utilized in Asian ayurvedic practices due to their health benefits and therapeutic actions [M. koenigii are reported to be a rich source of plant polyphenols, flavonoids, and alkaloids, possessing a high antioxidant potency [M. koenigii (2691 μmol of ascorbic acid/g sample) among green leafy vegetables [in vitro antioxidant property of leaf extracts of M. koenigii has been studied in different solvents/solvent mixtures such as water, methanol, hexane, water : ethanol, or chloroform with DPPH, Fe3+ ion reducing method, and hydroxy radical scavenging activity [M. koenigii leaf extracts to determine the total phenol content and DPPH assay. The 50% ethanol extract showed the highest scavenging ability and total phenolic content [ actions . Various potency –100. A rgetables . The in activity . All sam content .2O2 scavenging method has been used to screen the total antioxidant activity of M. koenigii acetone and petroleum ether leaf extracts. The mature leaf acetone extracts had a relatively greater scavenging activity by 50% compared to young leaves in petroleum ether [M. koenigii leaves in different solvent extracts, benzene, ethyl acetate, petroleum ether, acetone, methanol, and ethanol. It was evident that the benzene layer had the highest radical scavenging power, followed by ethyl acetate and petroleum ether [M. koenigii in different solvents.The Hum ether . A studyum ether . This obum ether . DiffereM. koenigii powder as an antioxidant in in-ground goat meat and precooked goat meat. The results confirmed that the M. koenigii powder at 0.2% (w/w) acts as an effective inhibitor for oxidation products formed during raw ground and precooked meat during storage [M. koenigii plant as a whole or various isolates from its bark, leaves, roots, or fruits with various methods and solvents, suggesting the pronounced antioxidant activity of M. koenigii [Another study was made to investigate the efficacy of  storage . Many otkoenigii , 85, 105M. minutum bark extract and the active fractions isolated in methanol fraction by preparative TLC method. M. minutum exhibited significant antioxidant activity with IC50 values of 54.3 and 168.9 μg/mL, respectively [An important study was made based on evaluating the antioxidant activity of ectively . The ideC. indica fruits was evaluated. The results have showed that the highest total phenolic content was observed in the methanol extract [50 values, 0.12 and 0.26 mg/mL, respectively.The potential antioxidant activity of methanol, hexane, ethyl acetate, and water extracts of  extract . FurtherMost of the isolated antioxidant active components in plant extracts of various solvents have been identified; antioxidant activities are diverse. These differences might be due to the extraction of curry leaves in a single solvent instead of fractionating them into different solvents. Furthermore, the various extraction methods and the extraction time and temperatures employed might have different levels and compositions of antioxidant compounds extracted, which further leads to the need for phytochemical characterization.M. koenigii, M. minutum, and C. indica, which have rich sources of bioactive metabolites. Their natural origin, low cost, and minimal adverse effects have attracted the scientific community to develop nutraceuticals and plant-based drugs. Clinical studies confirmed the pivotal role of M. koenigii and M. minutum in the prevention of various diseases. The amass quantity of information that has been accumulated in this regard has allowed us to understand the molecular mechanisms underlying these activities fully or partially in potential treatments. Although C. indica has been proven to be medicinally important, least or no attention was received by scientists. Therefore, further studies are essential for elucidating the role of the individual phytochemicals of C. indica in eliciting various pharmacological effects. The pharmacological effects of these three species differ on the part of the plant or fruit used. Furthermore, the geographical, seasonal, and other variations among countries and regions also influence the chemical composition. In closing, M. koenigii, M. minutum, and C. indica have proven to be promising candidates in both the drug designing by natural sources and the development of nutraceuticals. We believe that the current review provides a cumulation of information that will shed light innovatively for future investigation on the use of underutilized plant materials to discover new drugs with the aid of animal models and clinical trials.In recent years, the exponential growth of plants' use in medicine has been observed due to their remarkable pharmacological activities. The present review identified three of such important plants,


In contrast, pre-treatment of ICG-001 before IR had no effect on renal Wnt/β-catenin signaling or the progression of AKI to CKD. Mechanistically, in vivo expression of exogenous Wnt1 before IR suppressed the expression of proapoptotic proteins in AKI mice, and reduced inflammatory responses in both AKI and CKD mice. Additionally, exogenous Wnt1 inhibited apoptosis of tubular cells induced by hypoxia-reoxygenation (H/R) treatment in vitro. To conclude, the present study provides evidences to support the preventive effect of Wnt/β-catenin activation on IR-related AKI and its subsequent progression to CKD.Studies suggest that Wnt/β-catenin agonists are beneficial in the treatment of acute kidney injury (AKI); however, it remains elusive about its role in the prevention of AKI and its progression to chronic kidney disease (CKD). In this study, renal Wnt/β-catenin signaling was either activated by overexpression of exogenous Wnt1 or inhibited by administration with ICG-001, a small molecule inhibitor of β-catenin signaling, before mice were subjected to ischemia/reperfusion injury (IRI) to induce AKI and subsequent CKD. Our results showed that  Acute kidney injury (AKI) is a clinical condition due to a rapid loss of renal function by various injuries, such as drugs, ischemia, sepsis, and toxins . AKI is Wnt/β-catenin signaling plays fundamental roles in organogenesis, tissue homeostasis, and development of various diseases, including kidney diseases . β-CatenRenal ischemia-reperfusion injury (IRI) is considered the major cause of AKI. Therefore, renal IRI animal model has been commonly used for the research of AKI and AKI-CKD progression . Both phad libitum. Animal studies were approved by the Animal Ethical and Welfare Committee at Southern Medical University. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals.Male C57BL/6 mice weighing approximately 22–25 g were purchased from Vital River Laboratory Animal Technology , and maintained in the animal facility  at Southern Medical University  with water and food The induction of AKI and CKD in mice was described in our previous studies . BilaterTo evaluate the preventive effect of Wnt/β-catenin activation on AKI, mice were injected with 1 mg/kg hemagglutinin (HA)-tagged Wnt1 expression vector  or empty vector (pcDNA3) 2 days before BIRI using the hydrodynamic-based gene transfer technique as reported previously . Blood aTo evaluate the effect of early Wnt/β-catenin activation on the progression of AKI to CKD, mice were either subjected to daily hydrodynamic tail vein injection of Wnt1 expression plasmids at 1 mg/kg or daily intraperitoneal injection of ICG-001  at 5 mg/kg for 3 days before UIRI. Blood and kidney tissues were collected 11 days after UIRI. In our preliminary study, 2 days of Wnt1 injection could induce the expression of Wnt1 in AKI mice (data not shown). It should be noted that 3 days of Wnt1 injection was chosen to ensure and lengthen the expression of Wnt1 during the progression of AKI to CKD.Human proximal tubular epithelial cell line (HKC-8) was provided by Dr. L. Racusen at the Johns Hopkins University . Cell culture were performed as previously described . HKC-8 cCells were trypsinized, collected, and washed with PBS. PE Annexin V staining was performed according to the manufacturer protocol. A cytometric analysis was performed with a flow cytometer  to measure the apoptosis rates by detecting the relative amount of PE Annexin V positive and 7-AAD negative cells. Each assay was performed in triplicate.Creatinine levels in serum and urine as well as blood urea nitrogen (BUN) levels were measured using an automatic biochemical analyzer .Kidney sections and immunohistochemical staining were performed as previously described . PrimaryWestern blot analysis was performed as described previously . PrimaryTotal RNA isolation and qPCR were carried out as previously described. The mRNA levels of various genes were normalized with β-actin. The primer sequences of the genes are listed in P < 0.05 was considered significant.All data were expressed as mean ± SEM. The data were statistically analyzed using Sigma Stat software . Comparison between groups was made using one-way ANOVA, followed by the Student–Newman–Keuls test. via hydrodynamic tail vein injection at 2 days before BIRI  or empty vector (pcDNA3) ore BIRI . Serum lore BIRI . Consistore BIRI . Notablyore BIRI . Taken tin vivo expression of exogenous Wnt1 before IR. Nuclear factor-kappa B (NF-κB), particularly its heterodimer form p65/p50, plays a critical role in regulating the inflammatory response in kidneys of AKI mice. Western blot analysis showed that both p65 and its phosphorylated form (p-p65) were markedly increased in kidneys of mice after IRI, whereas such alterations were prevented by exogenous Wnt1 , collagen I, collagen III, and α-SMA was markedly induced in kidneys of mice at 11 days after UIR . Such alCompared with Sham mice, the mRNA expression of inflammatory genes, such as IL-1, IL-6, and TNF-α, was significantly up-regulated in kidneys of mice at 11 days after UIR . Such alTo further pinpoint the role of Wnt1 in AKI, we established HKC-8 tubular cell models of AKI using hypoxia-reoxygenation (H/R) treatment. Flow cytometry was employed to detect the apoptosis, and revealed the elevation of apoptosis rate in HCK-8 cells after H/R treatment . ExogenoAlthough AKI is increasing in incidence and is the major risk factor for progression to CKD, there are no effective therapies for AKI prevention and treatment. A number of situations, such as heart surgery, intra-tubular deposits, and administration of nephrotoxic drugs, could lead to the development of AKI . This hiin vivo expression of exogenous Wnt1 before IR could protect mice against AKI and prevent the progression of AKI to CKD. In vivo expression of exogenous Wnt1 at 2 days before renal bilateral IR was able to activate renal β-catenin, leading to reduced renal apoptosis and inflammation in AKI mice (1 day after IR). Additionally, in vivo expression of exogenous Wnt1 at 3 days before renal unilateral IR inhibited endogenous Wnt/β-catenin signaling and prevented the development of renal fibrosis in CKD mice (11 days after IR). In contrast, in vivo expression of exogenous Wnt1 at 5 days after renal IR was shown to induce β-catenin activation and accelerate AKI to CKD progression  signaling, and is currently in clinical trial for various cancers. We previously showed that administration of ICG-011 at 5 days after IR restored kidney function and impeded the progression of AKI to CKD, as evidenced by reduced renal fibrosis . AdditioMechanisms underlying the protective effect of Wnt/β-catenin may involve the modulation of apoptosis and survival pathways. Genetic ablation of tubule β-catenin promotes tubular apoptosis after IRI or folic acid treatment, whereas activation of Wnt/β-catenin suppresses pro-apoptotic proteins . In the In conclusion, the present study provides the evidence to support the preventive effect of Wnt/β-catenin activation on IR-related AKI and CKD, though more work is needed to elucidate the exact mechanisms by which exogenous Wnt1 protects against AKI and CKD. Given the dual role of Wnt/β-catenin signaling in AKI and CKD, the present study highlights the importance of timing when using Wnt/β-catenin modulators for the prevention and treatment of AKI. Future studies using more pharmacologic and genetic approaches as well as the optimal treatment timing and duration are warranted to investigate the effect of Wnt/β-catenin agonists in AKI prevention and treatment.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Ethical and Welfare Committee at Southern Medical University.XH, YZ, and LX conceived and performed the experiments. DW, TG, FL, and YL conducted the experiments. TG, YL, and LX drafted the manuscript. All authors participated in the experiments, and gave final approval to the version submitted for publication.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.