TP53 targeting in the future.Treatment of osteosarcoma, apart from chemotherapy modifications, has not changed for approximately 30 years. Similarly, as in other tumors, mutations in the TP53 gene are often observed in osteosarcoma. In this article, we highlight the possibility of targeting p53 in the treatment of osteosarcoma. We collected data on mutations in this gene founded in patients-derived samples. We describe animals with TP53 dysfunction, which may constitute preclinical models. We put emphasis on several molecules which act on p53 protein or its activity. We also highlight gene therapy approaches. Although many of the therapies are at an early stage, they offer hope for a change in the approach to osteosarcoma treatment based on TP53 gene is mutated in 50% of human tumors. Oncogenic functions of mutant TP53 maintain tumor cell proliferation and tumor growth also in osteosarcomas. We collected data on TP53 mutations in patients to indicate which are more common and describe their role in in vitro and animal models. We also describe animal models with TP53 dysfunction, which provide a good platform for testing the potential therapeutic approaches. Finally, we have indicated a whole range of pharmacological compounds that modulate the action of p53, stabilize its mutated versions or lead to its degradation, cause silencing or, on the contrary, induce the expression of its functional version in genetic therapy. Although many of the described therapies are at the preclinical testing stage, they offer hope for a change in the approach to osteosarcoma treatment based on TP53 targeting in the future.The  Osteosarcoma (OS) is a primary malignant tumor of the skeleton, characterized by the formation of immature bone or osteoid tissue by the sarcoma cells that develop mostly in the long bones. Localized (non-metastatic) OS, if treated in specialized sarcoma centers with surgery along with pre- and postoperative chemotherapy, is cured in 60–70% . Still, TP53 are very similar in sarcoma and multiple cancers. In the sarcoma field xenograft models expressing mutant TP53 in pleomorphic rhabdomyosarcomas, the tumor profile was even more aggressive and with greater metastatic potential than p53 null or p53 wild-type tumors. In particular, p53G245C and p53R273H mutations were reported to promote a more aggressive phenotype with an enhanced malignant potential, high cell viability, and proliferation, enhanced cell migration [TP53 is a promising therapeutic target in OS. Selected therapies aimed at p53 reactivation are expected to suppress OS [The oncogenic functions of mutant igration ,11,12. Apress OS . The devpress OS ,15.TP53 in OS and its potential as a therapeutic target. We review different types of experimental therapeutic approaches, including gene therapy and pharmacological modulation of p53. We also review the OS animal models particularly useful for studying TP53-targeted treatments.This article aims to review the current knowledge of the role of TP53 gene spreads over 13 exons mapped to region 17p13.1 [TP53 encodes at least 12 different isoforms [The  17p13.1 . TP53 enisoforms . Isoformisoforms . In normisoforms ,20. In iisoforms , a proliisoforms ,22,23,24The p53 protein is a central hub for activation of numerous stress-induced pathways, including DNA damage, senescence, metabolism; it is also engaged in cellular death, cellular reprogramming, and drug resistance. Below, we outline these areas of the p53 activity in the cell .Activation of p53 is stimulated by DNA damage and is dependent on ATR/CHK1 and ATM/CHK2 signaling. Oxidizing conditions could also stimulate p53 activation through stress-activated kinases  independently from the DNA damage response ,26.SLC7A11 membrane transporter, the activity of GPX4, and in consequence, accumulation of ROS [The p53 protein is engaged in programmed cell death. It activates several genes encoding apoptotic proteins  ,33,34,35n of ROS .p53 regulates transcription of cellular senescence marker p21, which mediates p53-induced cycle arrest and promotes senescence, including nucleotide-associated senescence ,45,46,47wt p53 [wt p53 also reduces the number of transcription factors necessary to generate iPSCs from embryonic fibroblasts [p53 plays an important role in inducing pluripotency. Silencing of p53 resulted in increased reprogramming efficiency in murine and human cells ,51. The wt p53 . The actroblasts ,53.GLUT1 and GLUT4 [TIGAR gene was confirmed in osteosarcoma Saos-2 and U2OS cell lines [PFKFB3 and PFKFB4 genes, leading to inhibition of glycolysis and an increase of ribonucleotide generation via the pentose phosphate pathway [SCO2 (Synthesis of Cytochrome c Oxidase 2) gene [p53 is a regulator of various metabolic pathways. It reduces glucose uptake, represses transcription of nd GLUT4 . The binll lines . Elevatell lines . p53 rep pathway ,57. p53  2) gene .In p53-deficient tumors, restoration of p53 activity results in an inflammatory reaction involving neutrophils, macrophages, and NK cells . Alteredwt p53 were more sensitive to cisplatin than A431 cells (bearing p53 R273H) [TP53 gene is associated with resistance to preoperative chemotherapy  [ARF [Altered p53 proteins may modify tumor-transformed cell response to treatment. U2OS cells with active 3 R273H) . Radiolotrexate) . Chemosete) [ARF . This prte) [ARF ,68.TP53 gene is mutated (somatic mutations) in over 50% of human neoplasms, the mutations occur mainly in the DNA-binding domain, but over 20% are outside it [TP53 mutations, the second TP53 allele is deleted [The tside it . Most oftside it . In 60%  deleted .TP53 gene may exert three possible effects. First, if transformation occurs in two alleles or when it appears in one allele but the second one is lost, it depletes the ‘guardian’ p53 function. Secondly, if mutated p53 controls its co-expressed wild-type form preventing its binding to DNA, such a situation is called the dominant-negative effect. Thirdly, mutated p53 acquires a new activity absent in the native form of protein, i.e., gain-of-function [TP53 gene mutations were found in the studies on OS patients  positions in P53 protein, based on datasets The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and International Agency for Research on Cancer (IARC). The most frequent amino acid substitutions in these codons are classified as non-functional ,79. MutaTP53 gene occur in about 70% of the cases [TP53 gene lead to Li-Fraumeni syndrome, in which different tumors appear in people of different ages—very early (infants or young children) in endoderm and mesoderm-derived cells (this includes OS) but much later, in old age, in endoderm derived cells [Li–Fraumeni syndrome (LFS) is a rare, autosomal dominant hereditary disorder—where mutations in the he cases ,97. Germed cells ,98,99. Sed cells , Li–Fraued cells .TP53 gene occur in 14% to 56% of cases. The most common types of cancer found were sarcoma (32.7%), brain tumor—including choroid plexus (9.3%), breast cancer (31.2%), or adrenocortical carcinoma (9%), bone tumors were rare and found in 0.7% of the studied group. In another large study, including 474 families, germline alteration of TP53 was found in 82 families (17%) [TP53 gene. Deletions removing the entire TP53 locus, the promoter, and exons 1–10 were also observed [In a study including 525 families diagnosed with Li-Fraumeni (at least a three-generation pedigree in the family history), according to various criteria classification, mutations in the es (17%) . Most muobserved .TP53 gene in a group of 765 patients. Among missense mutations, the most frequent alteration was the common exonic variant P72R. Other more frequently observed mutations were P47S, Y107H, R273H, R248Q [wt p53 [TP53 R172H (corresponds to human R175H) in mouse p53 null fibroblasts or heterozygous OS cell lines results in overexpression of Pla2g16 protein. Increased level of Pla2g16 leads to a more aggressive and metastatic cell phenotype [wt p53, lung metastasis was still observed [NGS sequencing of tumor samples revealed 32 variants of the H, R248Q . P47S is [wt p53 . Mutatio [wt p53 . The mut [wt p53 . These t [wt p53 . The act [wt p53 . Expresshenotype ,110. Hethenotype . In a moobserved .TP53 gene found in LFS patients by Birch et al. were localized in codons E180K, R175H, Y220C, D245G, and R248Q [FOXM1 expression, and inhibiting AMPK and FOXO3a activation [Mutations in the nd R248Q . The Y22nd R248Q . In yeasnd R248Q . This mund R248Q . In silind R248Q . Mutatiotivation . Tumor ctivation . Molecultivation .TP53 mutations were only detected in 20% of OSs, but currently, this frequency is believed to be much higher—from 47–90%. Moreover, numerous structural alterations involve this gene, especially the translocation of the first intron [TP53 mutations had poorer overall survival. Lorenz et al. [MDM2 amplifications were also observed. Somatic TP53 mutations are an unfavorable prognostic marker for 2-year survival in OS patients [Initially, t intron . Patientz et al.  found thpatients .TP53 gene were detected. In one case, an MDM2 amplification was found, and as MDM2 is a negative regulator of the p53 protein, this would lead to down-regulation of the protein. They extended their sequencing analysis to 32 further OS samples and found that 50% had rearrangements of the TP53 gene, 38% had missense  or nonsense mutations (codons 169 and 211), 6% a TP53 gene deletion and again 3% (1 case) had an MDM2 gene amplification. Two fusions of the TP53 gene with ASIC2 or SFSWAP genes were also observed. The rearrangements involving intron 1 of TP53 have not been found in other neoplasms, thus appearing specific for OS [Chen et al.  performe codons 1 and 211,c for OS . Expressc for OS . Amifostc for OS . Some stc for OS ,124. Argc for OS . R337H ic for OS ,127 and c for OS ,129. ThiTP53 exons were sequenced; mutations were found in 9.5% of young patients but none in patients aged 30 years or older. A more extensive paper by this group confirmed these findings, though a TP53 germline mutation was also found in one 39-year-old patient. Frequent mutations were: P47S, Y107H, R273H, R248Q, and P36, R213 in the exonic splice site [Mirabello et al.  analyzedice site . The firTP53 gene mutations were deletions, missense type (A159N), STOP codon (192), and splice site mutation (307). The authors maintain these alterations probably lead to degradation of changed TP53 transcript or result in expression of a truncated form of the TP53 gene.Rare mutations were reported by Bousquet et al. , and sevWGS data from 25 OS tumors collected from patients of both sexes in the age range between 8–79 years revealed mainly rearrangements in intron 1 and intron 9. The final transcripts lose essential parts of the protein, so these aberrations result in loss-of-function. Identified point somatic missense mutations were C238F, I255T, R273L, C275Y, D281E. Based on yeast assays or structure predictions, these mutations were classified as loss of protein function. The loss-of-function type was confirmed by Jordan et al.  for mutaTP53 intron 1 led to the fusion of the promoter to various genes, and showed by transcriptomic analyses that this led to the activation of numerous genes, also ones belonging to the tumor protein p53 pathway, and the rearrangement leads to reactivation of many of these genes. The interesting findings of that paper are that this rearrangement is an early event in OS genesis, and it is present in all OS cells, thus could be a potential target for future therapies.Saba et al.  analyzedTP53 gene—P72R and R248W were found in human osteosarcoma cancer stem cell line 3AB-OS. The authors classified these alterations as gain-of-function. Cells bearing these mutations proliferate rapidly [TP53 were found. These transcripts consisted of the first exon of TP53 joined with exons of VAV1, DDX39B, SAT2, EMR1, PPRAD genes. A series of truncated transcripts of TP53 were also observed. In the IOR/SARG cell line, the stop codon at position 205 results in the loss of a part of the DNA—binding and C-terminal domains [Two mutations in the  rapidly . In othe domains .Trp53 (mouse homolog of human TP53) knockout  mutant mice develop tumors at three to six months of age [Trp53 is inactivated by Cre-loxP-mediated recombination. Additionally, mesenchymal sarcoma stem cells (Sca-1low) have been isolated from these animals, but these mice are deficient in p53 and pRb, making detailed analyses difficult [The development of pre-clinical systems is crucial to enable basic research on the role of p53 in OS and provide a platform for testing the potential therapeutic approaches. Several animal models mimicking dysregulation of p53 were already proposed . We lists of age . They arifficult .TP53 mutations would be modeled more accurately rather by heterozygous than homozygous animal models [Trp53 mouse models, which require LOH before tumorigenesis occurs, develop a spectrum of tumors that is more similar to humans. While homozygotes develop lymphomas, the most frequent tumors in heterozygotes were OSs and soft tissue sarcomas [It was postulated that the human Li-Fraumeni syndrome characterized by heterozygous l models . Indeed,sarcomas . In thissarcomas .Trp53 missense mutations was investigated in transgenic mice with knock-in alleles that mimic the hot spot mutations found in human TP53. p53 R172H and p53 R270H mutations were introduced to the mouse p53 corresponding to amino acids 175 and 273 in human p53. These are most often mutated in human carcinomas [The role of rcinomas . The speTrp53−/− mice, these knock-in models display a higher degree of heterogeneity in the spectrum of tumor types with more frequent carcinomas (than sarcomas). On the other hand, homozygous Trp53−/− mice are highly prone to develop T-cell lymphomas and selected sarcomas [Trp53 in mice results in thymic lymphoma as the dominant tumor type, in humans, these lymphomas are less frequently TP53 mutated/dependent [Compared with sarcomas . Althougependent .Tp53-deficient Dark Agouti rat [Several p53-deficient animal models were also developed in rats. One example is the outi rat . In thisouti rat .tm1(EGFP-Pac)Qly/Rrrc (F344-Tp53) strain) develops predominantly meningeal sarcomas and OSs [Another rat model, obtained in the Fischer-344 (F344) rat . In one of them, mutation p53I166T was induced by exposure of the embryos to gamma irradiation [Tp53 knockout was also generated [Another recently proposed model was developed in zebrafish , the mutated protein will affect the structure of the whole tetramer, which will not function properly [Though many papers are devoted to this subject, the p53 protein is not considered an easy target for cancer therapy. The problems are caused by the numerous different mutations, their varied effects on the protein, and the gain-of-function that some of them convey, and also even if the wild-type protein is present  is overexpressed in OSs, and its knockdown decreases the oncogenic properties of OS cell lines. Moreover, a small molecule inhibitor of the protein affects the viability of OS cells, and Samsa et al. have developed an animal model which shows that JAB1 oncogenesis depends on TP53. It is suspected that targeting JAB1-associated miRNAs can be used for clinical intervention in OS.The way to treat OS by acting on p53 could be indirect by acting on its interactors. An example is shown by Samsa et al. . JAB1  are established regulators of tumor suppression, and their expression is transcriptionally controlled by p53 ,157,158.wt p53 protein-coding gene into four sarcoma lines. They found that the cell lines became sensitive to cisplatin and doxorubicin, though these were not OS lines. The crucial problem in OSs and therapy aimed at TP53 is that in many OSs, the rearrangements lead to the activation of numerous genes. This is to some extent due to rearrangements involving the TP53 gene promoter, it is not clear whether attempts to reactivate or introduce a normal copy of the gene would be successful [TP53 and/or its interactors; however, they rarely refer to OS.Ganjavi et al.  used adeccessful . NumerouTP53 mutations, which may not be relevant for osteosarcoma [For tumors with decreased p53 levels, therapy could act on its interactors, which regulate p53 activity—MDM2 can affect p53 localization and promote its degradation; moreover, it can block its ability to transactivate by binding to the N-terminal domain of the p53 protein . MDMX alosarcoma .TP53 gene. Moreover, as TP53 mutations are rarely recurrent, and there are many different ones, this restoration would have to be tested for numerous mutations to see how effective it would be.The literature on potential treatments of various types of cancer through acting on the p53 protein is abundant. It is essentially based either on stabilizing the wild-type form by acting on its destabilizers—mainly MDM2  . The forOne of the molecules which could restore wild-type activity is PRIMA-1 1-azabicyclo3-octan-3-one) (PRIMA is an abbreviation for p53 reactivation and induction of massive apoptosis) and its methylated form PRIMA-1MET . Both coTP53 mutations is that there are so many different ones. Still, some attempts to target specific mutations have been described, including a carbazole derivative molecule PhiKan083 which stabilizes the eighth most common mutation Y220C [Moreover, two compounds with potential activity to reactivate mutant p53, APR-246, a methylated structural analog of PRIMA-1, and COTI-2, a third-generation thiosemicarbazone, have been shown to work in pre-clinical models . These ton Y220C .Different strategies are required to obtain stable p53 for the relatively rare nonsense mutations; this would require drugs, such as aminoglycosides, which promote translational readthrough, which would lead to a normal protein. Another approach would be to target NMD (nonsense-mediated decay) of nonsense-codon-containing mRNAs. At least one such inhibitor, NMD114, has been shown to improve the stability of the mRNA for p53 .TP53 in OS cell lines, affecting their oncogenic properties. Similar results were obtained with NSC59984, which accelerates mutant p53 degradation. Both strategies decreased the expression of anti-apoptotic proteins survivin and Bcl-2 in the two tested OS cell lines; however, as mentioned above, many OSs do not carry mutated p53, but rearrangements involving its promoter, and Tang et al. only tested two OS cell lines [There are practically no data on targeting mutated p53 in OS. Still, some interesting results were recently obtained by Tang et al. , who usell lines .TP53 gene is also an important target for gene therapies [TP53 was reported [The herapies . Reductireported ,11,12. ITP53 gene is delivered into cancer cells by an adenovirus vector, and as a result, p53 protein is expressed. Gendicine is being used in China to treat patients with other advanced or unresectable tumors, including advanced lung cancer, liver cancer, gynecological tumors, and soft tissue sarcomas. Multimodal therapy with Gendicine and chemotherapy, radiotherapy, or other conventional treatment regimens demonstrated significantly higher response rates and progression-free survival than standard therapies alone [Gendicine™ (rAd-p53) is the first gene-drug—recombinant human p53 adenovirus. In 2003, the China Food and Drug Administration (CFDA) approved it to treat head and neck squamous cell carcinoma when used in combination with radiotherapy. The recombinant human es alone . The useAt this point, alternative molecules that have been proven useful for redirecting adenoviral attachment and entry may be of potential interest in targeting OS, including fibroblast growth factor receptor, folate receptor, transferrin receptor, and vascular endothelial growth factor receptor . TargetiMDM2 amplification [MDM2 status does not correlate with Nutlin-3a induced apoptosis [GADD45A, which, when not hypermethylated, was associated with the apoptotic response following Nutlin-3a treatment [Pharmacological targeting of p53 is another approach used in the development of novel therapeutic strategies. There were few ways for p53 pharmacological modulation . The firfication . Interespoptosis . An altereatment .Since the development of Nutlin-3a, new generations of MDM2 inhibitors have been identified. MI-219 is an MDM2 inhibitor with higher affinity and selectivity than Nutlin-3a ; howeverMDM2 gene amplification and MDM2 protein overexpression [Another small molecule MDM2 inhibitor, RG7112, showed antitumor activity in a panel of different solid tumor cell lines with the strongest response in OS cells . Moreovepression .TP53-wild-type tumors, and 7 had concurrent MDM2-amplification. Other MDM2 antagonists included idasanutlin (RG7388), AMG-232 (KRT-232), APG-115, BI-907828, CGM097, siremadlin (HDM201), and milademetan (DS-3032b); however, they have not yet been studied in OS [APG-115, another small molecule MDM2 inhibitor, was tested in a phase I study of 21 patients with liposarcomas and other solid tumors, including one with OS. In the study, one partial response and 12 stable diseases were achieved . Among ted in OS .Another approach to target p53 function with pharmacological intervention is restoring wild-type p53 activity by promoting proper folding of mutant p53. Since the development of the first p53-reactivating compound, CP-31398, several mutant p53-targeting substances were identified, including PRIMA-1 and its methylated analog APR-246, PK083, PK11007, NSC19630, NSC319726, and others ,182. Deswt p53 function can be achieved by targeting specific p53 mutations. The Y220C mutation was identified as the eighth most common TP53 mutation [wt p53 conformation. Suppression of other p53 mutants, including R175H and R273H, can be induced using P53R3, which has shown p53-dependent inhibition of glioma cell proliferation [Moreover, reactivation of the mutation . Small-mmutation ,188, resferation . SCH5290feration . The comApart from restoring wild-type p53 activity, depletion of mutant p53 was studied as a possible approach to target p53 function in cancer . CholestA group of compounds that act through mutant p53 destabilization are heat shock protein 90 (Hsp90) inhibitors. Hsp90 causes the accumulation of mutant p53 by inactivating MDM2 and CHIP ,194. TreAnother way to target p53 is treatment with substances that exclusively inhibit pathways involved in the proliferation of p53-mutated cells, including Chk1, polo-like kinase 1 (Plk1), and wee1 kinase (Wee1) . AZD7762Several Plk1 inhibitors are currently investigated in clinical trials in various cancer types, but none in OS patients . Wee1 iswt p53 pathway via the activation of p73. At effective doses, it preferentially induces the death of tumor cells, stimulates p73 activity, and targets mutant p53 for degradation. This compound is nongenotoxic; thus, its clinical application could be possible. In contrast to p53, p73, a transcription factor with high structural and sequence homology top53 and has similar functions, is rarely deleted or mutated in human cancer. In mammalian cells under stress conditions, p73 is activated by complex signaling pathways that induce apoptosis and increase chemosensitivity. Many chemotherapeutic agents, such as camptothecin, etoposide, and cisplatin, up-regulate p73 expression. Mutant p53 inhibits p73 activation by binding to p73.NSC59984 is a small molecular compound that destabilizes mutant p53 and restores the TP53 using CRISPR-Cas 9 increases the cytotoxic effect of doxorubicin in MDR osteosarcoma cells. Improvement of the anti-cancer activity of doxorubicin was observed after NSC59984 treatment in MDR osteosarcoma cells in vitro. Thus, a potential application of NSC59984 would be to reduce chemoresistance [Additionally, NSC59984 induces mutant p53 protein degradation via MDM2-mediated ubiquitination and proteasomal degradation. Stabilization of mutant p53 results from its inability to interact with MDM2, an E3 ubiquitin ligase. It is unclear how mutant p53 and MDM2 are phosphorylated by signaling pathways stimulated by NSC59984 in mutant p53-expressing cancer cells. According to Fan Tang et al. (2019), NSC59984 strongly inhibits the expression of mutant p53 R156P in osteosarcomas. NSC59984 treatment results in mutant p53 degradation by MDM2-mediated ubiquitination and proteasomal degradation and restores TP53 pathway signaling via p73 activation. Administration of NSC59984 up-regulated the expression of p21, a potent CDK inhibitor that leads to cell-cycle arrest ,208. Fansistance .Association between p53 and IGF-1R receptors is not fully understood but is expected to impact OS cell viability. IGF-1 is considered one of the main factors in osteosarcoma pathogenesis, so modulating the IGF-IR pathway is an attractive anticancer treatment strategy in p53 mutated OS. PPP  inhibits IGF-IR expression in osteosarcoma cell lines. PPP was shown to be active against osteosarcoma cell lines resistant to conventional chemotherapy with doxorubicin, paclitaxel, and vincristine. PPP is also active against primary tumor cells from osteosarcoma patients ,209,210.After the introduction of doxorubicin, methotrexate, and cisplatin in the late 1970s, the 5-year overall survival of OS patients and therapeutic outcomes have remained essentially unchanged . CurrentTP53 gene, the wild-type TP53 gene has become an important target for novel cancer gene therapy [TP53 gene encodes an important transcription factor (p53) with a tumor suppressor role. Mutated TP53 seems to play a critical role in maintaining cell proliferation and growth in OSs. In response to cellular stress, it activates various pathways that induce cell-cycle arrest, apoptosis, cell senescence, DNA repair, or changes in metabolism. Moreover, p53 plays a regulatory role in ontogenesis, myogenesis, angiogenesis [GLUT1 and GLUT4 transporters genes) [Because a significant fraction of human cancers has mutations in the  therapy . The TP5ogenesis . p53 is ogenesis ,28,29, iogenesis , regulats genes) , or modus genes) ,60,216. s genes) ,106.TP53 gene may exert three possible effects: deprivation of the ‘guardian’ p53 function, overcontrolling its co-expressed wild-type form (the dominant-negative effect), or gain-of-function [TP53 gene frequently occur in Li–Fraumeni syndrome. Patients suffering from LFS develop sarcomas most frequently (32.7%). TP53 mutations are also commonly detected in OS specimens; current studies estimate that these mutations are present in 47–90% of cases. Importantly, somatic TP53 mutations are an unfavorable prognostic marker in OS patients [The presence of a mutation in the function . Howeverpatients .TP53-targeted gene therapy approaches are also conducted in academic centers outside China.The p53 protein is considered a challenging target for cancer therapy. In particular, the challenges are associated with numerous different mutations and their varied effects on the p53 protein. Even in the presence of the wild-type protein, the mutated protein may affect the structure of the whole tetramer . CurrentAnother approach is pharmacological p53 modulation. Several agents are in an early phase of development. One example could be the pharmacological inhibition of the principal ubiquitin ligase for p53, MDM2, with Nutlin-3a (RG7112)  or SAR40


Gossypium hirsutum) is widely planted around the world for its natural fiber, and producing high-quality fiber is essential for the textile industry. CCRI70 is a hybrid cotton plant harboring superior yield and fiber quality, whose recombinant inbred line (RIL) population was developed from two upland cotton varieties (sGK156 and 901-001) and were used here to investigate the source of high-quality related alleles. Based on the material of the whole population, a high-density genetic map was constructed using specific locus-amplified fragment sequencing (SLAF-seq). It contained 24,425 single nucleotide polymorphism (SNP) markers, spanning a distance of 4,850.47 centimorgans (cM) over 26 chromosomes with an average marker interval of 0.20 cM. In evaluating three fiber quality traits in nine environments to detect multiple environments stable quantitative trait loci (QTLs), we found 289 QTLs, of which 36 of them were stable QTLs and 18 were novel. Based on the transcriptome analysis for two parents and two RILs, 24,941 unique differentially expressed genes (DEGs) were identified, 473 of which were promising genes. For the fiber strength (FS) QTLs, 320 DEGs were identified, suggesting that pectin synthesis, phenylpropanoid biosynthesis, and plant hormone signaling pathways could influence FS, and several transcription factors may regulate fiber development, such as GAE6, C4H, OMT1, AFR18, EIN3, bZIP44, and GAI. Notably, the marker D13_56413025 in qFS-chr18-4 provides a potential basis for enhancing fiber quality of upland cotton via marker-assisted breeding and gene cloning of important fiber quality traits.Upland cotton ( Gossypium hirsutum L., 2n = 52) is a widely planted cash crop providing natural fiber. Due to its excellent environmental adaptability and yield, among the four cultivated tetraploid species, it is G. hirsutum that contributes almost 95% of the harvested cotton production (Upland cotton (oduction . Given tG. raimondii (G. arboreum (G. barbadense (G. hirsutum (via marker-assisted selection (MAS)  .Based on an assessment of previous research, fiber development could be classified into four stages: initiation [−3 to 3 days postanthesis (DPA)], elongation (3–23 DPA), secondary wall biosynthesis (20–40 DPA), and maturity (40–50 DPA) . Initiat8:9 RIL population was used to evaluate fiber quality performance in nine environments and to construct a genetic linkage map. The whole-genome-based high-density genetic map contained 24,425 single nucleotide polymorphism (SNP) markers spanning a distance of 4,850.47 cM. Meanwhile, 289 QTLs for the three fiber quality traits and seven QTL clusters were identified. Accompanying the RNA-Seq analysis done for the whole process of fiber development, differentially expressed genes (DEGs) in those obtained QTLs and clusters were also identified, which provides new and timely insights into the genetic basis of fiber development and fiber quality traits.The hybrid variety “CCRI70”, a nationally approved variety in China released in 2008, is capable of good yield and has high fiber quality, whose recombinant inbred line (RIL) population consisting of 250 individuals was developed to investigate the source of high-quality related alleles for further upland cotton breeding . In this8:9 RILs were developed from the hybrid cotton variety CCRI70 whose parents are “sGK156” (P1) and “901-001” (P2); this population was developed from 2011 onward at the experimental farm of the Institute of Cotton Research, Chinese Academy of Agricultural Sciences (CAAS), in Anyang, Henan Province. The CCRI70 (F1) was obtained and planted in Sanya, where the F2 seeds were harvested. Shuttle breeding was applied to further develop the population during 2012–2016 between Anyang and Sanya. Full details of the procedure used to generate the RIL populations were reported already  and MBZ70-236 (L2), known to differ in their fiber quality performance were designated for RNA sequencing. Concerning the above four types of plant materials, P2 and L1 performed high-fiber quality, and P1 and L2 showed low-fiber quality in terms of their FL and FS. The day of anthesis was marked as 0 DPA, and flowers were marked by hanging labels with the flowering date recorded. Cotton bolls were collected in the morning at 5, 10, 15, 20, 25, and 30 DPA with three biological replicates, with at least five bolls collected in each replicate. The fiber samples were collected using sterilized medical scalpel and tweezers. Then all the samples were frozen in liquid nitrogen and stored at −805:7 and F5:8 families, reported previously  by using an HVI1000  with the HVICC Calibration at the Cotton Quality Supervision, Inspection, and Testing Center, Ministry of Agriculture, Anyang, China . The desin silico simulation, SLAF libraries for 250 RILs were constructed by using two endonucleases in combination, HaeIII and SspI , to digest the genomic DNA . According to the pilot experiment and the preexperiment omic DNA . The detG. hirsutum reference genome   , and QTL = 0.05) . Places  = 0.05) , in whic = 0.05) , those g1 was searched for using the physical positions of the border markers of QTLs   , and theTotal RNA from each sample was extracted following the manufacturer’s protocol with the RNAprep Pure Plant Kit . The quantification and purity of the RNA were respectively assessed by a NanoDrop 2000 spectrophotometer  and a NanoPhotometer spectrophotometer . The RNA integrity was confirmed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . Next, according to the manufacturer’s recommendations, 2 μg RNA per sample was used for the transcriptome library construction, implemented with the Illumina TruSeq™ RNA Sample Preparation Kit . In total, 72 libraries were separately sequenced using Illumina Novaseq 6000  for the two RILs and the two parents at six developing stages  with three biological replicates (4 × 6 × 3 = 72).N ≥ 10%), and low-quality reads , the clean data set was finally generated. Meanwhile, the GC percentage and Q30 were calculated to evaluate the quality of the clean data. HISAT2 v2.1.0  vis-à-vis the inferior trait group  at six stages, and also from horizontal comparisons within each group between the different developmental stages.Based on the gene count number of each sample, the DESeq2 package for R was run to identify the DEGs , for whi3 based on Kyoto Encyclopedia of Genes and Genomes (KEGG) were all employed  was performed and WGCNA R package was used to identify modules and hub (or highly correlated) genes that were strongly associated with the fiber development stages . Those DThe phenotypic data for the CCRI70 RIL population were selected and evaluated in nine environments during 2015 and 2016 . The ranAfter library construction, sequencing, and filtering, 1.1 TB of clean data was obtained, for which the Q30 bases were 94.76% and its mapping ratio was 99.51%. Specifically, 82.01 GB per each parent and 3.72 GB per each RIL were obtained; comparing both to the upland cotton reference genome (2.14 GB), indicating that the sequencing depth of our study was 35.41-fold for each parent and 1.61-fold per progeny, respectively .A total of 1,943,288 SNPs were detected in sGK-156 whereas 2,073,924 SNPs were identified in 901-001, whose homozygosity SNP markers amounted to 8,11,619 and 8,82,936, respectively. After filtering these markers, 24,425 SNP markers were retained for the genetic map’s construction.t) contained 14,220 SNP markers and 2564.93 cM and the D subgenome (Dt) contained 10,205 SNP markers and 2285.54 cM  were stable QTLs and mainly distributed on chromosomes 6, 14, 16, 18, and 24  were stable QTLs and mainly distributed on chromosome 5, 6, 10, 14, 24, and 25  were stable QTLs  were identified on seven chromosomes  , the QTLs for FS and FL traits shared the same direction of additive effects. Besides, additive effects from qCl-chr17-1 were in opposing directions for FS and FM, and likewise for qCl-chr25-1 with respect to the FL and FM traits. These results suggested that the clusters could improve FS and FL simultaneously yet decrease the FM.According to the overlapping of confidence intervals of stable QTLs for the different traits, seven QTL clusters ( and 25) , of whicComparing these stable QTLs with earlier published work for CCRI70  and otheCRA002982 and CRA004731). The average number of clean reads, GC content, and Q30 value were, respectively, 38.34 million, 44.92%, and 94.93% for each sample, implying that the quality of the RNA-Seq data was reliable. After filtering out four low-correlation samples, the FPKM values were calculated for the biological replicates, and their percentages in the categories of 0.5 ≤ FPKM < 5, 5 ≤ FPKM ≤ 100, and FPKM > 100, respectively were 23.50, 12.24, and 0.78% on average  were significantly differentially expressed between two groups among all six stages. Meanwhile, 13 and 16 genes were expressed differently in five and four stages, respectively , with the narrow confidence intervals, and another four QTLs  more than 20 MB in size on the physical map. Among those 1,476 unique promising genes, 1,005 of them were for the FS trait, 412 were for the FL trait, and 241 were for the FM trait  contained no more than 20 such genes. The qFS-chr18-1 and qFL-chr24-1, which could be detected in most environments, contained 77 and 20 promising genes, respectively. Among the seven QTL clusters where 182 promising genes were identified, qCl-chr10-1 had the most genes, with 54, whereas two clusters (qCl-chr17-1 and qCl-chr24-1) had less than 20 each , of which 246 were highly expressed . Comparing these promising genes with DEGs found revealed that 473 were expressed differentially during the process of fiber development, consisting of 320, 120, and 83 DEGs for FS, FL, and FM, respectively . ApplyinWhen deriving a genetic map, it has often been constructed with restriction fragment length polymorphism (RFLP) markers, SSR markers, and SNP markers with gene chip and sequencing technologies . The SNPqFL-chr11-2 and qFM-chr02-1 were specifically detected in the Yellow River basin, whereas qFM-chr17-2 was only identified in the Xinjiang Autonomous Region, and hence it might be environment-specific QTLs. Meanwhile, the fact that qFS-chr18-1, qFL-chr24-1, and qFM-chr03-2 were detected in at least six environments is strong evidence suggesting that genetic factors may play a dominant role in fiber quality traits.Fiber quality traits are quantitative plant traits that are sensitive to the environment and other factors; so detecting QTLs in multiple environments was necessary to be done here, especially as it could improve accuracy. Some QTLs could be detected only in specific environments, whereas others that may be detected in multiple environments and across generations are considered as stable QTLs. In our study, the planting locations’ selection in experimental design took into account both geographic and temporal replicates. For example, Anyang and Linqing are located in the Yellow River basin while Alaer, Kuerle, and Shihezi are in the Xinjiang Autonomous Region. Meanwhile, Alaer, Anyang, and Linqing had been arranged for 2 years. It was our goal to detect the stable QTLs of upland cotton across multiple environments. Notably, of the36 stable QTLs, 1) was the first nationally approved hybrid cotton capable of a high yield of superior fiber quality. The goal of this study was to detect QTLs and identify promising genes related to key fiber quality traits, which could be used to reveal the parental source of fiber quality related alleles. Compared with previous QTL studies of fiber quality, few fiber quality related QTLs, especially FS-related QTLs, were identified on chr18 (D13) , making it worthy of further study and providing information to better understand the underlying genetic mechanisms, as well as a reference for improving the upland cotton fiber quality.Among the 36 stable QTLs found in this study, 18 had been identified before , but the other 18 were novel . Together, they provide a sound basis for further functional characterization and upland cotton marker-assisted breeding programs. Our work differs from previous research, in that the CCRI70 RIL population is a breeding population developed from two upland cotton cultivars, which themselves are breeding material rather than germplasm material. Moreover, CCRI70 (F18 (D13) . However-chr18-4 , 901-001-chr18-4 ; four le-chr18-4 , which s-chr18-4  and rean-chr18-4  for mark1 cN/tex . The marThe promising genes identification was confirmed by the CI of stable QTLs, whose dynamics of expression are of significance for functional characterization. We identified 24,941 unique DEGs through the comparative transcriptome analysis  and 1,47GH_A06G0729, GH_D02G0231, GH_D11G1746) were enriched in the amino sugar and nucleotide sugar metabolism pathway. Among them, GH_D02G0231 was located in qFS-chr14-1 and identified as UDP-D-glucuronate 4-epimerase 6 (GAE6). UDP-d-galacturonate is necessary for pectin’s synthesis, in that the latter is synthesized from UDP-d-glucuronate by GAE6  were enriched in the phenylpropanoid biosynthesis pathway and annotated as aldehyde dehydrogenase 2C4, CINNAMATE 4-HYDROXYLASE (C4H), O-METHYLTRANSFERASE 1 (OMT1), and AMP-dependent synthetase and ligase family protein. Interestingly, GH_D10G2039, GH_D13G2625, and GH_D13G1855 each exhibited high expression levels during fiber developmental stages and were identified in qFS-chr20-1, qFS-chr18-1, and qFS-chr18-4, respectively. All currently known building blocks of the lignin polymer are produced by, or derived from the general phenylpropanoid pathway  were enriched in the plant hormone signal transduction pathway. GH_D07G1483 was enriched into auxin-signaling pathway as AUXIN RESPONSE FACTOR 18 (ARF18) and located in qCl-chr16-1, a genome region that influences both the FS and FL of upland cotton. Through phylogenetic analysis, ARF18 was classified into the class containing ARF1, ARF2, ARF9, and ARF11  ETHYLENE INSENSITIVE 3 (EIN3), which is considered the key transcriptional regulator of the ethylene response in plants , which could activate JA signaling  signaling pathways, respectively. EIN3 could regulate ETHYLENE RESPONSE FACTOR 1 (ERF1), and GbERF1-like reportedly activates the JA/ET signaling pathway and lignin synthesis , whose mutants were shown to reduce GA responses. GAI is involved in regulating stem elongation via the GA signaling pathway  belonging to the TCP family class I group  yet the other 18 were novel ; they provide a robust basis for understanding the distribution and the sources of high fiber quality-related alleles in cotton plants. Notably, qFS-chr18-4 and marker D13_56413025 could serve as important references for upland cotton maker-assisted selection breeding. Transcriptome sequencing for 72 libraries of the two parents and two RILs during fiber development with three biological repeats was performed, this aiming to understand the potential candidate genes expression patterns and DEGs  in superior and inferior fiber quality groups during the process of fiber development. Based on this transcriptome analysis, 24,941 unique DEGs were identified via vertical and horizontal comparisons, from which 473 of the 1,476 promising genes were identified as DEGs during fiber development. Among 320 differentially expressed promising genes for FS, 10 significant promising genes with high expression levels were enriched in the pathways of pectin synthesis, phenylpropanoid biosynthesis, and plant hormone signaling; these, as well as several others, annotated as transcription factors may jointly impact FS and regulate fiber development. Altogether, our results provide a fresh basis for improving cotton fiber quality via marker-assisted breeding that could benefit the textile industry.To investigate the parental source of high-quality related alleles, we constructed a genetic map of the hybrid CCRI70 RIL population by using SLAF-seq, this containing 24,425 SNP markers spanning a distance of 4850.47 cM. To detect stable QTL across multiple environments, we evaluated three key fiber quality traits in nine environments at six locations in 2015 and 2016. This revealed 289 QTLs, 36 of these were stable QTLs , of which 15 were located on Ahttps://ngdc.cncb.ac.cn/, (CRA002982 and CRA00473).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: XJ: conceptualization, formal analysis, validation, software, and writing—original draft. JG: resources. JZ, HC, ZK and JP: investigation. ZZ: software and methodology. YS: methodology. JL, QG, and SZ: visualization. AL and WG: data curation. XD and SF: project administration. JC: formal analysis. TJ and RW: software. QC: investigation and writing—review and editing. SW: writing—review and editing. HS and YY: conceptualization, writing—review and editing, supervision, and funding acquisition. All authors contributed to the article and approved the submitted version.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.


Droogmansia munamensis, is typically native to the DRC flora; 117 species are growing in the DRC and neighboring countries; 31 species are either introduced from other regions, and 64 are not specified. Alongside the treatment of Diabetes, about 78.13% of plants have multiple therapeutic uses, depending on the study sites. Experimental studies explored the antidiabetic activity of 133 plants, mainly in mice, rats, guinea pigs, and rabbits. Several chemical classes of antidiabetic compounds isolated from 67 plant species have been documented. Rare phase II clinical trials have been conducted. Critical issues included poor quality methodological protocols, author name incorrectly written (16.16%) or absent (14.25%) or confused with a synonym (4.69%), family name revised (17.26%) or missing (1.10%), voucher number not available 336(92.05%), ecological information not reported (49.59%). Most plant species have been identified and authenticated (89.32%). Hundreds of plants are used to treat Diabetes by traditional healers in DRC. However, most plants are not exclusively native to the local flora and have multiple therapeutic uses. The analysis showed the scarcity or absence of high-quality, in-depth pharmacological studies. There is a need to conduct further studies of locally specific species to fill the gap before their introduction into the national pharmacopeia.Several studies have been conducted and published on medicinal plants used to manage Diabetes Mellitus worldwide. It is of great interest to review available studies from a country or a region to resort to similarities/discrepancies and data quality. Here, we examined data related to ethnopharmacology and bioactivity of antidiabetic plants used in the Democratic Republic of Congo. Data were extracted from Google Scholar, Medline/PubMed, Scopus, ScienceDirect, the Wiley Online Library, Web of Science, and other documents focusing on ethnopharmacology, pharmacology, and phytochemistry antidiabetic plants used in the Democratic Republic of Congo from 2005 to September 2021. The Kew Botanic Royal Garden and Plants of the World Online web databases were consulted to verify the taxonomic information. CAMARADES checklist was used to assess the quality of animal studies and Jadad scores for clinical trials. In total, 213 plant species belonging to 72 botanical families were reported. Only one plant,  Most African traditional healers who detain ancestral heritages are illiterate, and their knowledge transmitted verbally from generation to generation is at risk of disappearing. To minimize such risk, the World Health Organization (WHO) recommends scientists carry out ethnopharmacological and experimental studies to record folk knowledge, create databases, and validate scientifically traditional claims from the perspective of developing improved medications . WHO estOne of the most explored diseases is diabetes mellitus (DM). Over 800 plant species showing hypoglycemic activities can be essential sources for discovering and developing new types of antidiabetic molecules . The magRecent data show about 1.7 million people suffer from DM in the Democratic Republic of Congo (DRC), ranking fourth in the top ten countries by diabetes cases in Africa . Like otThis review aimed to describe what is known hitherto about ethnopharmacological, pharmacological, and clinical studies embracing medicinal plants used to manage DM in the traditional medicine of the DRC, to highlight which plants are native or introduced, how they are formulated and used, what valid experimental studies have been conducted in preclinical and clinical phases. A critical analysis is made to assess the quality of studies carried inside DRC and resort similarities/discrepancies with studies conducted outside.n = 24), preclinical bioactivity (n = 9); and one clinical trial (n = 1) studies. One paper includes both an in vivo study and an Ethnobotanical survey.The review was an Internet search on Google Scholar, Medline, PubMed, Scopus, ScienceDirect, the Wiley Online Library, Web of Science, and other documents focusing on ethnopharmacology, pharmacology phytochemistry of antidiabetic plants used in the Democratic Republic of Congo from 2005 to September 2021. The review was conducted following Preferred Regulatory Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines 2009. A total of 34 studies were included. Ethnopharmacological/Ethnobotanical/Ethnomedicinal ; Relative Frequency of Citation : index, where FC is the number of informants who mentioned the use of the species and N, is the total number of informants  where U is the number of usable reports for a given plant species cited by each informant and n is the total number of informants interviewed for a given plant (Relative Importance Index (RII) of each plant species was calculated based on the normalized number of pharmacological properties attributed to it and the normalized number of body systems (BS) it affects  and Allium cepa in 5 zones. The vernacular names were linked or not to ethnic dialects. Swahili is the most reported language 48(12.87%), followed by Kongo 46(12.33%), Luba 36(9.65%), and Bemba 32(8.58%). In most cases, the vernacular name is not specified 47(12.60%) or not reported 8(2.14%). The formulations prepared consisted more often of decoction 173(60.49%), maceration 31(10.84%), and infusion 29(10.14%). The leaf is the most used part 122(39.23%), followed by roots 73(23.47%), and stem bark 43(13.83%).As shown in Albizia adianthifolia, Azanza garckeana, Gladiolus klattianus, Panda oleosa, Raphia gentiliana, Rauvolfia caffra, and Vitex madiensis; five studied in DRC. It also reported the only plant species native exclusively from DRC.Only seven plants presented in Panda oleosa. Endpoints consisted mainly of mortality, pathophysiological syndromes, and microscopic examination of the pancreas and other vital organs pathological changes. The sub-chronic evaluation focused on assessing biochemical, hematological, and histopathological markers after a relatively long period (14 days and sometimes 90 days). The level of exposure to different organs, including the fetus, liver, kidney, heart, etc., of different doses of plant extracts was also determined. Thus, most plant extracts produce a toxic effect in specific organs or systems at high doses.Many toxicological studies have been carried out in animals (rodents) using plant extracts. Some studies have been undertaken in mice, guinea pigs, and rabbits to explore the acute toxicity of Raphia gentiliana fruit extract was given to 25 males and 20 females, aged 18–50 years old, with normal blood sugar levels (R. gentiliana as food (0.14 g/kg), while fifteen were introduced to the glucose solution (0.07 g/kg) (standard). The glycemia was measured by spectrophotometry, and the triangle surface area ratio method was used to calculate the glycemic and load index. The observed values of glycemic index and load were −3.1% and −1.36%. The approach followed by the authors did not comply with any clinical trial requirements, and instead, their behavior went like traditional healers themselves.Data from the present study showed the lack of local clinical trials of antidiabetic plants used to manage Diabetes in the DRC. Of seven native herbals, only r levels . Thirty Panda oleosa, Physalis peruviana, and Vernonia amygdalina.Some studies have been undertaken to explore the chemical composition of The analysis presented in The errors in plant authors included entirely different authors, spelling mistakes, inappropriate use of the period, improper use of bracket, and incomplete author name.Droogmansia munamensis was the only species exclusively native to DRC flora (“Haut-Katanga”). Concerning the data quality, the author names of plant species were correctly written in 69.59% of cases, not correctly registered in 16.16%, or absent in 14.25%. Furthermore, 17.26% of plants had family names changed, and 81.64% not changed. In few cases (n = 10), the main plant was confused with its synonym. For example, Antidesma metacarpus (A. membranaceum), Annona senegalensis (A. arenaria), Cassia alata , Chenopodium ambrosioides (Dysphania ambrosioides), Citrus x aurantium (C. sinensis), Hibiscus esculentus (Abelmoschus esculentus), Mitragyna stipulosa , Nauclea latifolia , Solanum americanum (S. nigrum), and Solanum gilo (S. aethiopicum and S. subsessile). Most species (89.32%) from different sites were identified and authenticated in other herbariums or laboratories of ecology, but only a few (7.95%) had a voucher number published. This situation implicates the responsibilities of publishers and reviewers. http://powo.science.kew.org) database and http://plantsoftheworldonline.org via the Royal Botanic Garden Kew database.The origin of plants was specified in 69.95% and not in 30.05% of species. However, 54.93% of plants with known origin were native to Africa, 14.55% species were introduced, and The country is home to different ethnic groups, making it one of the most diverse countries globally, with more than 200 other ethnic groups speaking an estimated 213 native languages. Sometimes referred to as the Baluba, the Luba people are the largest ethnic group. The community is native to the Kasai, Maniema, and Katanga regions. The Mongo people comprise several smaller constituent groups, including the Mbole, Ekonda, Boyela, Bolia, and Nkutu. The Kongo ethnic group is native to DRC and Angola, speaking Kongo alongside Lingala, Kyanzi, and Kintandu. The Mangbetu ethnicity is concentrated within the Orientale Province (Kisangani). The Zande people reside in the tropical rainforest and the savanna and speak nearly five dialects of the Azande language. The Pygmies are considered to have been some of the earliest peoples to inhabit the Congo River Basin. Their short stature characterizes them, they are mainly hunters and gatherers, and they occupy the rainforest. The plants are distributed within tropical and subtropical ecological regions, flooded grasslands, moist broad-leaf forests, savannas, and mangroves.Swahili is the most reported language 48(12.87%), followed by Kongo 46(12.33%), Luba 36(9.65%), Bemba 32(8.58%), Tshiluba 29(7.77%), Mashi 26(6.97%), French 21(5.63%), and Lingala 14(3.75%). After le French, which is the official language, there are four regionally distributed national languages, including Ciluba (Tshiluba), Kongo, Lingala, and Swahili, among 213 native languages identified in DRC. Those four languages are used in out-group communication, in lower primary school years , cultural and religious purposes, etc. .Fabaceae was the most representative family, consistent with other studies that showed this family is commonly found in tropical rain and dry forests in the Americans and Africa . Around The leaf was the most used part 122(39.23%), followed by roots 73(23.47%), and stem bark 43(13.83%). According to , the freCatharanthus roseus was found at almost all locations (except site R4) and Allium cepa at five sites. However, A. sativum, Cassia alata, C. occidentalis, Mangifera indica, Persea americana, and Vernonia amygdalina were quoted at four locations.Out of 213 plants listed, 103(33.66%) were found at site R5 and 85(27.78%) at R6. The majority of plants had local vernacular names, except in few cases where the author did not mention the names. For instance, Albizia adianthifolia was the most reported antidiabetic with eight citations representing an RFC of 0.0063, followed by Catharanthus roseus 7(RFC = 0.0055). However, Allium cepa, Annona senegalensis, and Cassia occidentalis were reported six times (RFC = 0.0047), followed by Mangifera indica, Morinda morindoides, Phaseolus lunatus, and Vernonia amygdalina with five citations (RFC = 0.0039).Studies undertook outside DRC confirmed the use of the majority of plants cited as antidiabetic remedies. Elaeis guineensis was endorsed by 28 uses; the (UV score of 0.0221 was the highest compared to Ocimum gratissimum (0.0150), Antidesma membranaceum (0.0142), Jatropha curcas (0.0126), Bridelia ferruginea (0.0118), and Quassia africana (0.0118). Also, Balanites aegyptiaca, which is employed to treat nine body systems, showed the highest Relative Importance Index (32.5%), compared to Vitis vinifera (28.7%), Zingiber officinale (27.1%), Solanum seretii (24.9%), Thomandersia hensii (24.9%), Lippia multiflora (23.2%) and Stachytarpheta indica (23.2%). The use-value indicators , Fidelity Level of Citation (FL), Family Use Value, Importance Consensus Factor (ICF), etc. It should be noted that some rare studies make an effort to investigate these parameters, although the information on the number of respondents remains a challenge. One of the weaknesses of ethnopharmacological surveys is that the respondents are often the healers themselves and not or seldom the users. The questionnaires do not scrutinize evidence on the number of people treated and outcomes.Different strategies and pathways are used to determine the mechanism of antidiabetic agents, as shown in in vivo reported study models. Streptozotocin presents many advantages over alloxan, including its longer half-life, more productive, stable, and selective to islet beta cells, less toxic, and causing less mortality in animal models . Enzymes are a frequent pharmacological target for establishing the mechanism of action of new drugs. Upon in vitro studies, alpha-glucosidase activity inhibition was the most common investigation (45.3%), followed by inhibition of PTP1B (13.8%), alpha-amylase (9.7%), DPP-4 (1.4%), and 11ß-HSD1 activity (1.0%). Additionally, in cell lines studies, glucose uptake (28.0%) was to be the most commonly used, followed by glucose uptake regulation markers such as GLUT4 translocation and expression levels (9.7%) and PPAR (9.6%)  and alloxan (24.64%) represented 60.19% of all l models . The majR (9.6%) . Some exAzadirachta indica aqueous leaf extract (400 mg/kg bw) improved levels of BG, serum insulin, lipid profile, insulin signaling molecules, and GLUT4 proteins in the tissue of high-fat fructose-induced type-2 diabetic male rat after 30 days of treatment, compared to the control group. In Goto-Kakizaki rats, the acetone extract of Syzygium cumini seed was a potent inhibitor of alpha-glucosidase hydrolysis of maltose compared to untreated control animals  significantly reduced glucose levels in a dose-dependent manner in rabbits under OGTT conditions  reduced hypoglycemia to 43% compared to 55% obtained with glibenclamide . Overall, it is not easy to define an exact upper cut-off dose. In most cases, an oral dose range of 100–200 mg/kg body weight for plant extracts in vivo investigations should be considered the upper limit . Experiments on Albizia adianthifolia, Azanza garckeana, Gladiolus klattianus, and Rauvolfia caffra extracts used the highest dose (500 mg/kg bw) calculated from the human patients of 60 kg treated with 750 ml of plant extract . Par the way, differences in doses that normalize interspecies variation should be taken into account .Of the 213 plant species listed, 134(62.91%) underwent experimental studies in animals or Temperature and humidity were not reported. The effeteness of the treatment was based on the capacity of the extract to reduce baseline glycemia (hypoglycemia effect) or the capacity to reduce induced hyperglycemia; this varied between 25 and 75%, compared to reference drugs (glibenclamide and metformin). According to , over 8550 reported for Cola nitida, Sida acuta, Ficus sycomorus, Moringa oleifera, Panda oleosa, Alchornea cordifolia, Morinda lucida, Physalis peruviana, Musanga cecropioides, Vitis vinifera, Erythrina abyssinica, Persea americana, Jatropha curcas, Momordica charantia, and Rauwolfia caffra. Almost all plants but Jatropha curcas are relatively non-toxic (LD50 > 500 mg/kg).For acute toxicity, 50 was observed at 8,600 mg/kg bw in rodents with an oral administration of Alchornea cordifolia. The bark extract of Panda oleosa was practically non-toxic in guinea pigs with an LD50 of approximately 7,892 mg/kg bw; no signs of intoxication were observed with oral doses less than 2,000 mg/kg. However, at doses above 6,000 mg/kg, poor mobility, poor appetite, anuria, and death have been noted in animals  . Extracts of Aframomum melegueta (286–345 mg/kg bw) and Artemisia annua (300 mg/kg bw) produced a toxic effect on the development of fetus by discontinuation of first trimester pregnancies in rats , including losing appetite, slow movement, depression, less aggression, and lying at the corners of the cage  are considered the most toxic. Their consumption is not recommended in pregnancy and lactation because they are harmful to the fetus and newborn  and Mishra et al. (2018) are contradictory. The first group did not report any toxicological signs in biochemical, hematological, and morphological markers, while the second group noticed some changes.The highest value of LD animals . The adm female) . Despite in rats . Except  in rats . Aloe-emn organs . A daily in rats . Leaf mel weight . Bulbs’ the cage . Erythrint doses . Some co newborn . A methaney rats . SometimRaphia gentiliana fruit extract was given to 25 males and 20 females, aged 18–50 years old, with normal blood sugar levels  have been validated by clinical evidence. These are Allium cepa  improved glycemic indices, total antioxidant capacity, malondialdehyde, C-reactive protein, serum paraoxonase, dietary intake, and physical activity, measured at the beginning and end of the study, and after 12 h fasting compared to control groups. A randomized, placebo-controlled, parallel-group study with 42 treated patients treated with leaf hydroethanolic extract (500 mg/8 h for 3 months) and 44 as placebo groups showed that the Salvia officinalis leaves lowered fasting glucose and HbA1c the baseline at the endpoint with no adverse effects reported. A clinical trial on a juice extract from the fruit of Morinda cordifolia (2 ml/kg bw once a day) in patients with DMT2, after 90 days of treatment, presented a significant reduction of morning BG in several cases, an improvement of hyperglycemia status. In a prospective, randomized, double-blind, placebo-controlled clinical investigation, the administration of Terminalia chebula  in 60 diabetic patients significantly improved the endothelial function (reflection index) compared to placebo . In an 8-weeks randomized controlled clinical trial study of the effect of Trigonella foenum-graecum intake seed in 50 patients with T2DM, the plant significantly reduced fasting blood glucose. It improved some liver and kidney function compared with control interventions.For example, a double-blind, placebo-controlled, randomized clinical trial conducted on 20–60 year-old DMT2 patients who did not receive insulin showed that 3 months supplementation of 3 g of ginger  combined with conventional oral antihyperglycemic drugs was conducted in 46 treated patients vs 46 placebo groups. At the endpoint, the extract significantly lowered the blood levels of fasting glucose, 2 h postprandial glucose, and HbA1c, without significant effects on other hepatic or cardiovascular parameters, vs the placebo. All considered these results demonstrated that nettle is safe and may have a beneficial effect on glycemic control in patients with advanced DMT2 needing insulin therapy. Vernonia amygdalina elicited a significant reduction in BG levels at the most postprandial time points and area-under-curve.A randomized, double-blind, placebo-controlled clinical trial of A. cepa, A. sativum, B. aegyptiaca, E. guineensis, M. charantia, R. vomitoria, S. officinalis, U. dioica, and Z. officinale presented excellent quality (Jadad score ≥ 3). In addicore ≥ 3).Various second metabolites have been identified and isolated, as shown in Allium cepa contains ferulic acid, alliin  Czern has cinnamic acid  significantly inhibited amylase-catalyzed starch digestion and played roles in hyperglycemia management by increasing hepatic glycolysis and glycogen concentration and lowering hepatic gluconeogenesis. Furthermore, hesperidin, naringin, and nobiletin reduced hepatic gluconeogenesis and improved insulin sensitivity in animal models benzyl]}thiocarbamate, reduced blood glucose levels in STZ-induced diabetic mice and promoted the glucose consumption of IR cells .Isothiocyanates inhibited gluconeogenesis and hepatic glucose-6-phosphatase (G6P) expression in hepatoma cells and improved glucose tolerance and insulin signaling sensitivity .Galactomannan showed significant dose-related hypoglycaemic and antihyperglycaemic effects; the obtained results were better than glibenclamide used as reference .O-glycoside enhanced glucose transport through proximal and distal marker modulation involved in glucose uptake and its transformation into glycogen  significantly increased eostasis . Hirsutreostasis .According to A stearoyl glucoside of ursolic acid (urs-12-en-3β-ol-28-oic acid 3β-D-glucopyranosyl-4′- octadecanoate) demonstrated an antidiabetic property by lowering sugar blood in rats from the 8th day to the 21st day of the experiment .in vitro. In mice, it alleviated insulin resistance, glucose tolerance by reducing fasting blood glucose levels and blood lipid (triglyceride and cholesterol) (esterol) .O-β-D-glucuronopyranoside enzymes, quercetin-3-O-glucoside, avicularin, castalagin, and 2,3-hexahydroxydiphenoyl-(α/β)-D-(4)C1-glucopyranose showed inhibition capacity of sucrase  are associated with a beneficial influence on Diabetes and its complications through many mechanisms. The most well-known are: stimulation of insulin secretion, improvement of pancreatic β-cell functionality, inhibition of hepatic gluconeogenesis, enhanced glucose uptake, increased insulin signaling pathway, delay of carbohydrate digestion and glucose absorption, and inhibition of protein glycation  ellagic acid showed significant alpha-glucosidase inhibitory efficacy with IC50 values of 2.9 and 6.4 mM, respectively -[8]-Gingerol was found to be the most potent on glucose uptake and increase in the surface distribution of GLUT4 protein on the L6 myotube plasma membrane .ρ-Coumaric acid exhibited higher inhibition activity against alpha-glucosidase (98.8%) than acarbose (62.5%). However, acarbose showed the most potent inhibition against alpha-amylase (98.6 vs 66.8%) (s 66.8%) .Luteolin showed significant alpha-glucosidase and alpha-amylase inhibitory activities . ChebulaN-trans-feruloyl tyramine, N-trans-p-coumaroyl tyramine, and N-cis-p-coumaroyl tyramine (Phenylmethyl cinnamates) showed inhibitory activity against alpha-glucosidase -5b,19-epox y-19,25-dimethoxycucurbita-6,23-dien-3b-ol, cucurbitane exhibited significant inhibitory activity on alpha-glucosidase with ICectively . Oral adectively .in vitro and stimulated insulin secretion at both basal and stimulatory glucose concentrations in INS-1 832/13 cells, and enhanced acute glucose-stimulated insulin secretion cultured β-cells , is an ingredient of essential oils of many great different herbs; it can increase free insulin-stimulated glucose transporter by activation of adenosine monophosphate-activated protein kinase .α-amyrin acetate (a pentacyclic triterpenoid) lowered the blood glucose profile in STZ-induced diabetic rats and db/db mice at 50 mg kg1 dose level . PahlavaCucurbitane-type compounds -dien-19-al, charantal, charantoside XI, and 25ξ-isopropenylchole-5, 6-ene-3-O-D-glucopyranoside), demonstrated an alpha-amylase and alpha-glucosidase inhibitory activities ranging from 56 to 79% .Two monoterpenes -2-hydroxymethyl-6,6-dimethylbicyclo[3.1.1]heptane-2,3- diol, and sobrerol significantly increased glucose uptake in 3T3-L1 adipocytes . On the Multiple investigations have been carried out on natural products, mainly plants used to treat Diabetes Mellitus worldwide. In DRC, a country with a high ecological, cultural and human diversity, traditional medicine through plants occupies an important place in the health system. Several ethnopharmacological and ethnobotanical studies have been conducted previously in this perspective, and various plant species have been identified. Contrary to the previous review, the present review assessed the quality of studies carried inside DRC and resorted similarities/discrepancies with studies conducted outside. The findings confirm the high diversity of the flora and the various ethnic groups in DRC. Most of the plants claimed as antidiabetic and used by traditional healers in the DRC are not specifically native to DRC. One hundred thirty-four native and introduced species have been experimentally validated by various pharmacological, toxicological, and phytochemical researches. Many plants are safe at doses < 500 mg/kg, but long-term use may trigger sub-chronic toxicity. Exclusively conducted in DRC, preclinical and clinical studies of some plant species demonstrated poor protocol quality. Locally specific species deserve in-depth investigations to meet scientific requirements for their introduction studies into the national pharmacopeia. Although a few plants reduced blood sugar levels, clinical data and antidiabetic studies of the isolated compounds remain limited to allow the availability and accessibility of standardized phytomedicines to Congolese. This review constitutes a primary database for further experimental studies, especially for unstudied species in the perspective of safe and efficient use of easily accessible natural resources.


Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans. Our data show that a HFD (45% energy fat versus 16% energy fat in an AIN-93 diet (AIN)) promotes azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation in a mouse cancer model. However, the underlying metabolic basis remains to be determined. In the present study, we hypothesize that AOM treatment results in different plasma metabolomic responses in diet-induced obese mice. An untargeted metabolomic analysis was performed on the plasma samples by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found that 53 of 144 identified metabolites were different between the 4 groups of mice , and sparse partial least-squares discriminant analysis showed a separation between the HFD and HFD + AOM groups but not the AIN and AIN + AOM groups. Moreover, the concentrations of dihydrocholesterol and cholesterol were inversely associated with AOM-induced colonic ACF formation. Functional pathway analyses indicated that diets and AOM-induced colonic ACF modulated five metabolic pathways. Collectively, in addition to differential plasma metabolomic responses, AOM treatment decreases dihydrocholesterol and cholesterol levels and alters the composition of plasma metabolome to a greater extent in mice fed a HFD compared to the AIN. Consumption of a high-fat diet (HFD) links obesity to colon cancer in humans ,2. The gWe recently reported that in a mouse model, a HFD promotes colonic aberrant crypt foci  formation accompanied by increased systemic levels of proinflammatory cytokines . We therThe plasma samples were collected at the end of the study (Week 14) . We idenTo examine the diet-AOM effect on metabolome, sparse partial least-squares discriminant analysis (sPLS-DA) was used to visualize the metabolite group separation and individual sample variability A. We fouThe 10 major metabolites influencing separation along Component 1 were (1) beta-sitosterol (>120% increase), dihydrocholesterol (>89% increase), alpha-tocopherol (>94% increase), methanol phosphate (>18% increase), cholesterol (>32% increase), arachidonic acid (>6% increase), and nicotinamide (>58% increase), which were the highest concentrations in the HFD group compared to the other three groups; (2) the concentrations of citric acid, isocitric acid and myristic acid were decreased by (>23%) in the HFD group compared to the other three groups B.The 10 major metabolites influencing separation along Component 2 were: (1) oxoproline (>29% increase), oleic acid (>72% increase), 3-ureidopropionate (>67% increase), 2-aminobutyric acid (>58% increase), ornithine (>65% increase), and uracil (>25% increase) were the highest concentration in the HFD + AOM group compared to the otherthree3 groups; (2) palmitoleic acid (>30% decrease), alpha-ketoglutarate (>28% decrease), malic acid (>40% decrease), and citrulline (>30% decrease) exhibited a lower concentration in the HFD and HFD + AOM groups compared with the AIN or AIN + AOM group C.To visualize the effect of diet and AOM interaction on the abundance of these 53 metabolites, a two-way ANOVA (including interaction tests) was performed. In These 25 interactive metabolites included (1) the concentrations of alpha-tocopherol, dihydrocholesterol and nicotinamide were increased (> 58%) in the HFD group compared to the other 3 groups; in contrast, the concentrations of citric acid and myristic acid were decreased at least 23% in the HFD group compared to the other 3 groups . These 5cis-aconitic acid, citric acid, pyruvic acid, fumaric acid; (2) arginine biosynthesis: citrulline, aspartic acid, ornithine, oxoglutaric acid, fumaric acid; (3) aminoacyl-tRNA biosynthesis: phenylalanine, glycine, aspartic acid, serine, methionine, lysine, threonine, tyrosine; (4) alanine, aspartate, and glutamate metabolism: aspartic acid, citric acid, fumaric acid, pyruvic acid, succinic acid, oxoglutaric acid; and (5) glyoxylate and dicarboxylate metabolism: cis-aconitic acid, citric acid, serine, glycine, isocitric acid, pyruvic acid  ,26,27. IDihydrocholesterol biosynthesis occurs through two major pathways: (1) unabsorbed cholesterol in the colon is biohydrogenated to dihydrocholesterol via bacterial enzymes ,32, and l-tRNA biosynthesis are two pathways, which are involved in endothelial cell migration and angiogenesis in tumorigenesis [Further functional analysis demonstrates that 53 plasma metabolites were involved in five major metabolic pathways , Table 2igenesis ,39. Howeigenesis ,41, a chIn this report, although our data on differential plasma metabolomic responses are interesting, there are a few limitations. For example, our plasma metabolomic data show only a disease-associated relationship but not a causal relationship, and these data are from a single-time-point experiment. Because the cause of colon cancer is involved multiple metabolic pathways (collective effects), it is currently difficult for us to experimentally validate one or a few disease-causal metabolites. However, in the future, we will be able to shed some light on disease-causal metabolites if more metabolomic data/studies  are available.TM ventilated cages within a pathogen-free facility that maintained a 12 h light/dark cycle and a temperature of 22 ± 1 °C. Mice were given free access to food and deionized water. This study was approved by the Animal Care and Use Committee of the Grand Forks Human Nutrition Research Center , and animals were maintained in accordance with NIH guidelines for the care and use of laboratory animals. The study design, diet composition, and preparation have been previously reported [n = 25/group). On Week 3, within a given diet group, mice received either weekly intraperitoneal injection of the colon carcinogen, AOM  (n = 15/group), at a concentration of 8 mg/kg body weight [n = 10/group) for 4 weeks. At the termination of the experiment, mice were fasted for 6 h and then euthanized with a mixture of ketamine and xylazine (100 mg/kg body weight). Plasma samples were collected at the end of the study (Week 14) and stored at −80 °C for metabolomic analysis.Three- to four-week-old male C57BL/6 mice  were individually housed in Plexiglasy weight  or phospMetabolomic analysis was performed at the West Coast Metabolomics Center  ,15. PlasBioinformatic analysis: to avoid a skewed distribution, obtained (peak intensity) data were normalized by Log10 transformation with an autoscaling method which is highly recommended for most peak intensity data ,45. In aOur data demonstrated that AOM, a commonly used carcinogen in a chemical-induced colon cancer model, altered the plasma metabolome to a greater extent in mice fed the HFD compared to the AIN. These plasma metabolites were involved in five major metabolic pathways included (1) citrate cycle, (2) Arg biosynthesis, (3) aminoacyl-tRNA biosynthesis, (4) Ala, Asp, and Glu metabolism, and (5) glyoxylate and dicarboxylate metabolism. Our newly identified (inverse) association between plasma dihydrocholesterol levels and AOM-induced colonic ACF formation in a mouse model warrants future exploration as one of the unique plasma “multiple analytes” at the quest of human colon cancer biomarker search.


The global prevalence of nonalcoholic fatty liver disease (NAFLD) or metabolic associated fatty liver disease (MAFLD), as it is now known, has gradually increased. NAFLD is a disease with a spectrum of stages ranging from simple fatty liver (steatosis) to a severe form of steatosis, nonalcoholic steatohepatitis (NASH), which could progress to irreversible liver injury (fibrosis) and organ failure, and in some cases hepatocellular carcinoma (HCC). Although a liver biopsy remains the gold standard for accurate detection of this condition, it is unsuitable for clinical screening due to a higher risk of death. There is thus an increased need to find alternative techniques or tools for accurate diagnosis. Early detection for NASH matters for patients because NASH is the marker for severe disease progression. This review summarizes the current noninvasive tools for NAFLD diagnosis and their performance. We also discussed potential and newer alternative tools for diagnosing NAFLD. Nonalcoholic fatty liver disease (NAFLD), now known as metabolic associated fatty liver disease (MAFLD), is a common liver disease that affects 25% of the population worldwide ,2. Even Currently, the gold standard assessment for NASH and fibrosis is a histological assessment of the liver (liver biopsy) . HoweverUnderstanding the pathogenesis of NAFLD is crucial for identifying the important molecular biomarkers crucial for accurate diagnosis. NAFLD is a multifactorial disease associated with unhealthy lifestyles and diets, metabolic dysregulation, genetics, oxidative stress, and altered gut–liver axis, all of which might influence disease development and progression. Detailed pathogenesis and molecular mechanisms have been described before .Currently, the new proposed name for NAFLD is MAFLD. In the early 1980s, the term NAFLD was applied to patients with liver histological characteristics similar to those found in cases of alcohol-associated liver disease (ADL) yet the patients did not have heavy alcohol consumption . Later, Dysregulation of the metabolic features drives early NAFLD/MAFLD disease development and steatosis. Overnutrition, insulin resistance, and obesity contribute multiple insults that modulate excess hepatic lipid accumulation . Among tSCD1), fatty acid synthase (FASN), and the elongation of long-chain fatty acids family member 6 (ELOVL6) . M. M177].  (P4HA1) . Moreove (P4HA1) . Since t (P4HA1) , high se (P4HA1) . Thus, aHnf4a), forkhead box transcription factors (Foxa2 and Foxo1), Ppara, and the signal transducer and activator of transcription 3 (Stat3) [Ppara and sirtuin 1 (Sirt1) expression [SREBF1 and -2 genes [ABCA1) expression, HDL production, and circulating levels [Besides miR-122, other miRNAs also regulate steatosis and lipid metabolism. One such is the miR-21 that is higher in liver tissue and circulating plasma of NAFLD individuals and animal models ,133. Inh (Stat3) ,135. Ano (Stat3) ,140. Simpression ,140. Onepression . This mipression  and mammpression . The miR-2 genes . SREBF1 -2 genes . Therefog levels , suggestg levels , confirmEREG), activated leukocyte cell adhesion molecule (ALCAM), and moesin (MSN), and these molecules are involved in epithelial-to-mesenchymal transitions (EMT) [Zeb2) expression. This Zeb2 is a well-known EMT modulator [Another liver-specific miRNA is miR-192, mainly implicated in fibrosis via the TGFB/SMAD pathway . Followins (EMT) . A studyodulator , criticaodulator  and coul2 [These circulatory miRNAs are used in a diagnostic panel to identify and diagnose NAFLD. A panel of five serum miRNAs, miR-122, miR-34a, miR-375, miR-192, and miR-21, was specific enough to differentiate NAFLD individuals from drug-induced liver injury cases . A syste2 . Another2 . AlthougSimilar to miRNAs, long noncoding RNAs (lncRNAs) also do not produce protein products, and they could regulate multiple processes in NAFLD . LncRNAsH19), which is one of the essential lncRNAs in hepatic steatosis. In a study of H19 knockout mice, H19 lncRNA acts as a scaffold to facilitate the interaction between the polypyrimidine tract binding protein 1 (Ptbp1l) and Srebf1, thus activating downstream lipogenesis genes [H19 lncRNA acts as a sponge to miR-130a to upregulate peroxisome proliferator-activated receptor gamma (Pparg) expression, concomitant with hepatic steatosis [Pparg, Srebf1, Scd1, Acc1, and Fasn [H19 and miR-130a to regulate Srebf1 could be the primary driver for hepatic steatosis. Another lncRNA is the steroid receptor RNA activator (SRA). This lncRNA negatively regulates the expression of Atgl and promotes hepatic steatosis via the suppression of fork-head box protein O1 (Foxo1) and Pparg which promotes the transactivation of the Atgl enzyme. Silencing SRA expression in the mice restored Atgl expression, prevented FA oxidation, and relieved hepatic steatosis [SRA, the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) lncRNA is an inflammatory lncRNA associated with diabetic conditions [MALAT1 expression was higher, and this upregulation caused an increase of Srebf1 mRNA and protein expression and promoted hepatic steatosis [MALAT1 interacts with the Srebf1 protein to stabilize it, thus leading to lipid lipogenesis gene activations [lncSHGL, also known as B4GALT1-AS1, the human homolog) is a protective lncRNA for hepatic steatosis [B4GALT1-AS1/Hnrnpa1/Cam axis could be used as a therapeutic target for individuals with NAFLD and T2D.A few lncRNAs regulate hepatic steatosis . One sucis genes . In anotteatosis . The miRand Fasn . Therefoteatosis . Besidesnditions . In animteatosis . Furtherivations . In contteatosis . This lnteatosis . This CAteatosis ,194. ThiLFAR1), and this lncRNA expression was higher in the animal model of fibrosis [LFAR1 acts as a scaffold to allow for the binding of the Smadfamily protein complex, Smad2/Smad3, to the receptor of Tgfb1, in turn activating the downstream fibrosis-related genes in TGFB signaling [HOTAIR). This lncRNA expression is higher in the animal and cell model of fibrosis [HOTAIR acts as a sponge to miR-148b, a known negative regulator of the DNA methyltransferase 1 (DNMT1) [DNMT1 expression leads to hypermethylation on the promoter region of another lncRNA, maternally expressed 3 (MEG3), and suppressed this lncRNA expression. Furthermore, HOTAIR also acts as a scaffold at the MEG3 promoter region and recruits the polycomb repressive complex 2 (PRC2) to initiate H3K27 methylation, further suppressing MEG3 gene transcription [MEG3 is anti-fibrotic lncRNA which increases tumor suppressor p53 (TP53) expression to initiate HSCs apoptosis [LRP6), thus alleviating lipid accumulation [APTR), a recently recognized cell cycle and proliferation regulator [APTR was observed in animal models and human patients with fibrosis [APTR expression prevented an accumulation of extracellular matrix protein (ECM) and HSCs activation [Some lncRNAs regulate hepatic fibrosis. One such is liver-specific lncRNA, liver fibrosis-associated lncRNA 1  [TGFB2/TGFB2-OT1 in a panel with Fibroscan was also able to identify fibrosis in individuals with similar AUROC. For NASH identification, the serum expression of lncRNA RP11-128N14.5 has similar diagnostic potential with serum AST level; despite that, this lncRNA expression was higher in NASH individuals [RP11-128N14.5 expression with the clinical markers did not improve the diagnostic performance. Therefore, the possibility of identifying novel NASH-specific lncRNAs as biomarkers for early diagnosis is still open, and it is still to be determined whether these lncRNAs could be used in primary care settings.The lncRNAs are also incorporated into panels for diagnosing NAFLD and NASH. Although the data are still limited, one study investigated the diagnostic potential of serum expression of is study , Lnc-SPA of 100% . Similaris study , the exp: 0.891) . In the : 0.891) , this raividuals . FurtherCircular RNAs (circRNAs) are large ncRNAs that do not encode for functional proteins and are the products of the transcription process, though circRNAs are derived from the back-splicing events ,200,201.circRNA_0046366 [circRNA_0046367 [circRNA_021412 that acts as a sponge to miR-1972. This inhibition of miR-1972 causes an increase of Lipin 1 (LPIN1) expression, and LPIN1 is a co-activator of PPARA [circRNA_021412, circRNA_0046366, and circRNA_0046367 to suppress the miRNAs that control PPARA signaling may be an alternative therapeutic target to reduce hepatic steatosis.Although the information about circRNAs in NAFLD is scarce, there are some circRNAs that are known to regulate hepatic steatosis. Two circRNAs act as a sponge to miR-34a: _0046366  and circ_0046367 . Both ci_0046367 ,170. Anoof PPARA . TherefocircRNA_002581, as a central modulator in NASH [Slc1a5, Plp2, and Cpeb1), validated with real-time PCR. All of these three genes are involved in NAFLD [In NASH, an experimental study of a NASH animal model identified one circular RNA,  in NASH . This ciin NAFLD .circUBE2K from the parental gene of ubiquitin-conjugating enzyme E2 K (UBE2K). In a human HSC cell line study, circUBE2K was found to act as a sponge to miR-149-5p and increase TGFB2 expression to initiate the expression of fibrosis genes (ACTA2 and COL1A1) [circRNA_0074410, which acts as a sponge to miR-9-5p [TGFB receptors and suppress TGFB signaling [circPWWP2A also regulates TGFB signaling, as this circRNA is a sponge to miR-203, thus removing the suppression of follistatin-like 1 (FSTL1) expression, and therefore, FSTL1 could interact with SMAD proteins to facilitate TGFB signaling [circTUBD1 [circRSF1 [TLR4) [RAC1) [circRNA_0067835, which acts as a sponge to miR-155 and induces FOXO3A suppression [circRNA_0067835 caused a reduction of FOXO3A due to higher miR-155 expression and inhibition of HSC proliferation via the suppression of the PI3K/AKT pathway [Some circRNAs regulate fibrosis. One such is  COL1A1) . AnothermiR-9-5p . AlthougmiR-9-5p , a previignaling . circPWWignaling . Two cirircTUBD1  and circcircRSF1 . A loss 1 [TLR4)  and Ras-) [RAC1) . Other cpression . Knockdo pathway .circ_0007874, or cMTO1, which acts as a sponge to miR-17-5p and increases SMAD7 expression, a negative regulator of TGFB signaling [SMAD7 expression is circPSD3, which acts as a sponge to miR-92b-3p [cMTO1 showed that cMTO1 could act as a sponge to miR-181b-5p [PTEN), a negative regulator of the PI3K/AKT pathway [cMTO1, circFBXW4 acts as a sponge to miR-181b-5p, and this suppression leads to higher expression of SREBF1 and inhibition of the PI3K/AKT pathway [There are several circRNAs that negatively regulate fibrosis. An example is ignaling . AnotherR-92b-3p . Besides-181b-5p . The sup pathway . Similar pathway ,165. AltInter-cellular communication is not limited to direct contact in cell-to-cell interaction, there being adjacent communication via the secretion of information-bearing membrane lipid vesicles known as extracellular vesicles (EVs) . GeneralROCK1) and subsequently enriches the TRAIL receptor on EVs to initiate macrophage activation [Pparg [Although the research on EVs in NAFLD is still new, the findings show that EVs could play significant roles in NAFLD progression, as summarized previously ,204. Fortivation . Anothertivation . Mechanitivation . EVs alsn [Pparg . Anothern [Pparg .Although EVs are detectable in circulating biofluids, their origins are essential to determine the specificity of the disease. A recent study of a mouse model of NAFLD showed that hepatic EVs could be isolated using nanoscale flow cytometry by detecting the surface markers of asialoglycoprotein receptor 1 (ASGR1) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) on the EVs . In thisAnother potential area for developing a diagnostic tool for NAFLD and NASH is the metabolomics of NAFLD. Metabolomics is a study of all metabolites, the small molecules, intermediates, and products of cell metabolism ,212. A rAlmost all current tools and technologies for diagnosing NAFLD could diagnose advanced fibrosis and cirrhosis well, though gaps remain with respect to the identification of good markers for NASH and early fibrosis. Liver biopsy remains the gold-standard method to assess NASH; however, the development of noninvasive tools to limit or avoid the usage of liver biopsy has become a research priority. Therefore, understanding and identification of biomarkers specific to different stages of NAFLD, notably NASH, are of great importance. Alternative biomarkers, such as circulating noncoding RNAs, genetic markers, and extracellular vesicles, show significant potential. Therefore, these most promising new biomarkers should be further developed and validated in various populations.