Psoralea corylifolia Linn. (BGZ) is a commonly used traditional Chinese medicine (TCM) for the treatment of kidney-yang deficiency syndrome (Yangsyn) with good curative effect and security. However, BGZ was also reported to induce liver injury in recent years. According to TCM theory, taking BGZ may induce a series of adverse reactions in patients with kidney-yin deficiency syndrome (Yinsyn), which suggests that BGZ-induced liver damage may be related to its unreasonable clinical use.syn and Yinsyn rat models and identify the corresponding characteristic biomarkers.Liver injury caused by TCM is a rare but potentially serious adverse drug reaction, and the identification of predisposed individuals for drug-induced liver injury (DILI) remains challenging. The study aimed to investigate the differential responses to BGZ in Yangsyn and Yinsyn were induced by hydrocortisone and thyroxine + reserpine respectively. Body weight, organ index, serum biochemistry, and Hematoxylin and Eosin (HE) staining were used to evaluate the liver toxicity effect of BGZ on rats with Yangsyn and Yinsyn. Transcriptomics and metabonomics were used to screen the representative biomarkers ) changed by BGZ in Yangsyn and Yinsyn rats, respectively.The corresponding animal models of Yangsyn and Yinsyn rats, respectively, and the results also were confirmed by the pathological changes of liver tissue. The results showed that 102 DEGs and 27 metabolites were significantly regulated related to BGZ’s protective effect on Yangsyn, which is mainly associated with the glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate, and coenzyme A (CoA) biosynthesis pathways. While 28 DEGs and 31 metabolites, related to the pathway of pantothenate and CoA biosynthesis, were significantly regulated for the BGZ-induced liver injury in Yinsyn. Furthermore, 4 DEGs , solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), out at first homolog (Oaf)) and 4 metabolites  in the Yangsyn group and 1 DEG [galectin 5 ] and 1 metabolite (5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate) in Yinsyn group were significantly correlated to the ALT and AST levels of BGZ treated and untreated groups (receiver operating characteristic (ROC) ≥ 0.9).The level changes of liver organ index, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), suggested that BGZ has liver-protective and liver-damaging effects on Yangsyn and Yangsyn are the predisposed syndromes for BGZ to exert liver damage and liver protection respectively, which are mainly related to the regulation of amino acid metabolism, lipid metabolism, energy metabolism, and metabolism of cofactors and vitamins. The results further suggest that attention should be paid to the selection of predisposed populations when using drugs related to the regulation of energy metabolism, and the Yinsyn/Yangsyn animal models based on the theory of TCM syndromes may be a feasible method for identifying the susceptible population to receive TCM.YinThe online version contains supplementary material available at 10.1186/s13020-023-00793-x. Polygonum multiflorum Thunb.[Psoralea corylifolia Linn (BGZ) [Drug-induced liver injury (DILI) refers to one of the most common and serious adverse drug reactions induced by various prescription or non-prescription chemical drugs, biological agents, traditional Chinese medicine (TCM), natural medicine, health care products, dietary supplements and their metabolites and even excipients, which can lead to acute liver failure or even death . Due to um Thunb. and Psornn (BGZ) , which hPolygonum multiflorum Thunb., Green Tea, and Kampo products , etc. are closely related to HLA-B*35:01 [HLA-B*57:01 [DILI usually involves two factors, the “drug” and the “host” . For dru-B*35:01 –12, whil-B*57:01 –17. Scretherapy with syndrome differentiation” of TCM, both of which are based on identifying drug-susceptible populations for rational medication use. However, for TCM theory, in addition to the correct understanding of medicine, it is also necessary to correctly identify TCM syndromes that summarize the pathological changes of the body at a certain stage of disease development based on TCM theory. TCM treatment requires “therapy with syndrome differentiation”. Symptomatic treatment can result in a better therapeutic effect, otherwise, it can aggravate the disease process and induce new diseases. Therefore, it may be feasible to explore the predisposition of individuals to TCM liver injury based on TCM syndrome theory which may provide a reference for other similar studies on the hepatotoxicity of TCM.Actually, the identification of susceptible genes or populations is similar to the “Psoralea corylifolia Linn, is widely used in Asia and has been used for many years in China to treat symptoms such as impotence, nocturnal emission, enuresis, frequent urination, cold aching in the lower back and knees, kidney deficiency, and premature ejaculation [BGZ, the dried mature fruit of the leguminous plant culation . BGZ hasculation . Its actculation , 21. In culation , 22–24. culation .syn) and kidney-yin deficiency syndrome (Yinsyn) according to TCM diagnosis [Lei Gong Concocting (Paozhi) Theory” also recorded during the Northern and Southern Dynasties in China that people with “asthenic yin causing excessive pyrexia” should avoid taking BGZ. Thus, the BGZ-induced DILI may be related to its inappropriate symptomatic treatment. However, whether it has a causal relationship with the imbalance of Yangsyn and Yinsyn in the body remains unclear.Although some studies have confirmed that BGZ and some of its components have hepatotoxicity , 26, 27,iagnosis –31. The syn generally have low body temperature, loose stools, and less drinking and urine, while patients with Yinsyn generally have high body temperature, dry stool, polydipsia, and polyuria. In addition, some studies have found that the expression of cAMP in patients with Yangsyn decreased [syn increased [syn and Yinsyn, respectively. The changes in behavioral signs and cAMP levels in the above animal models are similar to the clinical patients with the corresponding syndromes and have been generally recognized and adopted by experts in the related fields of TCM. With a good description of the changes in the metabolic characteristics of endogenous metabolites and the differential expression changes of a series of functional genes in organisms, metabolomics and transcriptomics have been successfully applied to screen various diseases and their metabolic or gene profile changes in drug intervention [syn and Yinsyn animal models to evaluate whether the liver damage induced by BGZ is related to its non-symptomatic use and the possible susceptible individual characteristics.According to the diagnostic criteria of TCM syndromes , patientecreased , 34, whincreased . Accordincreased  or thyroncreased  in rats rvention , 39. Thi+-K+-ATPase tests were purchased from Jiancheng Biological Technology, Co., Ltd. . Cyclic adenosine monophosphate (cAMP) was purchased from Mlbio Biotechnology Co., Ltd. .Hydrocortisone succinate sodium was provided by Tianjin Biochemical Pharmaceutical Co., Ltd. . Thyroxine and reserpine were provided by Shanghai MACKLIN Technology Co., Ltd. . Alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Scr), lactate dehydrogenase (LDH), and NaBGZ was purchased from the Anhui Puren Herbal Pieces Co., Ltd.  and authenticated by Professor Xiaohe Xiao of the Institute of Hepatology, Fifth Medical Center, PLA General Hospital . In addition, BGZ was crushed and filtered through 200 mesh sieves and then suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) for administration.Male Sprague–Dawley rats (180–200 g) were obtained from the SPF Biotechnology Co. Ltd. , and housed in the Laboratory Animal Center of the Fifth Medical Center, Chinese PLA General Hospital . All rats were raised under specific pathogen-free conditions under a 12 h light/dark cycle, with free access to adequate food and water. All animals were fed adaptively for 1 week before starting the experiments.syn), kidney-yang deficiency syndrome-treated with BGZ group (Yangsyn+BGZ), kidney-yin deficiency syndrome group (Yinsyn); kidney-yin deficiency syndrome-treated with BGZ group (Yinsyn+BGZ). Rats in the BGZ-related treatment groups were consecutively administered intragastrically with BGZ suspension for 21 consecutive days, while the CON, Yangsyn, and Yinsyn groups received the same volume of 0.50% CMC-Na solution for the same amount of time. The changes in body weight were recorded before the end of the experiment. From the 8th day of administration, the Yangsyn and Yangsyn+BGZ group rats were given 25 mg/kg hydrocortisone subcutaneously once a day for 14 consecutive days to prepare the Yangsyn model [syn and Yinsyn+BGZ group rats were given thyroxine (16 mg/mL) and reserpine (1 mg/mL) at the dosage of 0.5 mL/100 g via gastric perfusion once a day for 7 consecutive days to prepare the Yinsyn model [The rats were randomly divided into six separate groups (N = 8) as follows: Control group (CON), BGZ group (BGZ), kidney-yang deficiency syndrome group  pathological staining analysis.After centrifugation , serum biochemistry of ALT, AST, Scr, LDH, Na260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RNA integrity number (RIN) ≥ 6.5, 28 S:18 S ≥ 1.0, > 1 µg) was used to construct the sequencing library. RNA-seq transcriptome libraries were prepared using a TruSeqTMRNA sample preparation kit from Illumina  and sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer under standard protocols. All samples had a Q30  of > 91%. Sequence readers were trimmed and quality controlled using SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters and aligned to the reference genome through the orientation mode using hierarchical indexing for spliced alignment of transcripts 2 (HISAT2) [http://deweylab.biostat.wisc.edu/rsem/) [Padjust < 0.05 (DESeq2) [http://www.geneontology.org) functional enrichment and Kyoto Encyclopedia of Genes and Genomes  pathway analysis carried out by Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do) were performed to determine significant GO terms and pathways associated with the DEGs [Padjust < 0.05 were considered potential target pathways.Total RNA was extracted from liver tissues using Trizol reagent  according to the manufacturer’s protocol, and genomic DNA was removed using DNase I (TaKara). RNA quality was assessed with a Bioanalyzer 2100 (Agilent) and measured with a NanoDrop 2000 spectrophotometer. Only a high-quality RNA sample (OD(HISAT2) . Messengu/rsem/) . Differe(DESeq2) . Furtherthe DEGs . Pathway−△△Ct method 2−∆∆Ct [Liver tissue RNA was extracted by the Tissue RNA Purification Kit Plus  and reverse-transcribed into cDNA using the Fast All-in-One RT Kit  according to the manufacturer’s instructions. The qPCR of aldehyde dehydrogenase 1 family member B1 (Aldh1b1), galectin 5 , solute carrier family 25 member 25 (Slc25a25), Pim-3 proto-oncogene, serine/threonine kinase (Pim3), and out at first (Oaf) were quantified by the SYBR Green PCR master mix  with the QuantStudio 6 Flex PCR System . The amplification parameters were set according to the standard protocol. Primer sequences used in this study are shown in Additional file d 2−∆∆Ct .The liver was homogenized with normal saline at 1:1 (1 g:1 mL) using a homogenizer. The 300 uL homogenized sample and 900 uL methanol were mixed and vortexed for 30 s. The supernatant was centrifuged at 12,000 r/min for 10 min and concentrated to dry using a vacuum centrifugal concentrator. Then 100 uL methanol was added for redissolving and centrifuged at 13,000 r/min for 10 min. Finally, a 4 uL supernatant was taken out for UPLC-QTOF/MS  detection.P < 0.05 were selected as potential metabolites. Online metabolic databases including the Human Metabolome Database (HMDB) (http://www.hmdb.ca/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/) were combined with exact masses and secondary ion mass spectrometry of the metabolites acquired through Progenesis QI to identify the differential metabolites.Metabolic profiling analysis of the biofluids was performed using the Waters Xevo G2-XS QTOF/MS . An analytical Acquity UPLC HSS T3 C18 column (temperature 30 °C) was injected with 4 µL aliquots of each sample. For positive electrospray ionization source (ESI+) and negative electrospray ionization source (ESI-) analysis, samples were isolated using a 30 min linear gradient of solvent A (water spiked with 0.1% formic acid) and solvent B (acetonitrile spiked with 0.1% formic acid) as mobile phases. The flow rate was fixed at 0.30 mL/min. For each sample, 10 µL was drawn as a quality control sample to ensure that the system was stable and the analyses were repeatable. Every 20th sample was injected with the control sample and subsequently analyzed. Masslynx software  and Progenesis QI  were used for identifying the original mass spectral data and normalizing the total ion intensity of each chromatogram to acquire a data matrix containing the m/z value, retention time (RT), and normalized peak area. SIMCA-P 14.1 software  was used for principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). The PCA score chart was used to show the natural interrelation of observation results. Variable importance in the projection (VIP) ≥ 1 and https://js.cytoscape.org/) is a visualization tool for exploring biomedical networks composed of compounds, genes, and other types of interactions [http://MetScape.ncibi.org) is a plugin of Cytoscape that allows users to construct and analyze gene and compound networks, and identify and visualize enriched pathways changes in expression profiling data and metabolite data [syn/Yinsyn and corresponding BGZ-treated groups were imported into Metscape to obtain a global understanding of gene and metabolic changes to assess the underlying mechanisms of Yinsyn/Yangsyn-associated differential responses to BGZ in rat models, respectively.Cytoscape 3.8.0 . All results are expressed as mean ± SD. A one-way analysis of variance (ANOVA) was used to statistically analyze the multiple-group analysis. The student’s t-test was used to statistically analyze the receiver operating characteristic (ROC) curve analysis. The significance threshold was set at P < 0.05) and accompanied with the significantly increased fecal water content (P < 0.01) in the Yangsyn group, while the levels of anal temperature, water intake, urine volume, and cAMP were all significantly increased  and accompanied with the significantly decreased fecal water content (P < 0.05) in the Yinsyn group. The change trends of behavioral characteristics and cAMP in the above-mentioned Yangsyn/Yinsyn model rats were similar to the definition of Yangsyn/Yinsyn in the diagnostic criteria of TCM syndromes. Under the intervention of BGZ, the levels of anal temperature, urine volume, and cAMP were all significantly increased  and accompanied by the significantly decreased fecal water content (P < 0.05) in the Yangsyn+BGZ group compared with the Yangsyn group, while the levels of weight, water intake, urine volume, and cAMP were all significantly decreased  and accompanied with the significantly increased anal temperature and fecal water content (both P < 0.05) in the Yinsyn+BGZ group compared with the Yinsyn group.As shown in Fig. P < 0.001) and accompanied by the significantly increased spleen index (P < 0.05) in the Yangsyn group, but the organ indexes of the liver, kidney, adrenal gland, spleen, lung, and heart in the Yinsyn group were all significantly increased . Under the intervention of BGZ, the spleen index in the Yangsyn+BGZ group was significantly increased (P < 0.05) compared with the Yangsyn group, while all organ indexes (except for kidney and heart) in the Yinsyn+BGZ group were significantly increased compared with the Yinsyn group .As shown in Fig. syn group increased prominently, while BGZ significantly reversed the phenomena . Though the AST level in the Yinsyn group was significantly increased (P < 0.01), there was no obvious change in the levels of AST between the Yinsyn+BGZ group and the Yinsyn group, while the ALT level was significantly increased after administration of BGZ in the Yinsyn+BGZ group (P < 0.001), illustrating that there was a certain risk of liver injury in rats with Yinsyn after administration of BGZ. As for kidney function . Combined with the changes in the kidney organ index, it revealed that BGZ may have no obvious renal toxicity in rats with Yangsyn or Yinsyn. As shown in Fig. syn group exhibited hepatocyte focal necrosis, loss of central vein intima, and inflammatory cell infiltration in portal vein areas, while the above symptoms were alleviated in the Yangsyn+BGZ group. The liver samples from the Yinsyn group exhibited slight inflammatory infiltration in the portal area but no evident hepatocyte injury, while the hepatocyte focal necrosis and inflammatory cell infiltration were aggravated in the Yinsyn+BGZ group. Combined with the levels of ALT, AST, liver index, and liver pathological examination, it was suggested that BGZ has a preferable liver protection effect on rats with Yangsyn and a certain risk of liver injury effect on rats with Yinsyn.Serum ALT, AST, and Scr, which are well-recognized markers of various types of liver and kidney damage, were used for the analysis. As for liver function Fig. A–B, therion Fig. C, no sigsyn and Yangsyn rats, liver gene expression profiles were obtained from the CON, BGZ, Yangsyn, Yangsyn+BGZ, Yinsyn, and Yinsyn+BGZ groups using RNA-Seq analysis. As shown in Fig. syn group, and 508 up-regulated DEGs along with 847 down-regulated DEGs were found in the Yinsyn group. Compared to the Yangsyn group, the number of up-regulated and down-regulated DEGs decreased to 59 and 43 by BGZ treatment, respectively  and DEGs changed using BGZ in Yinats Fig. , the ressyn+BGZ group and Yangsyn group, and 10 DEGs had better discrimination between the Yinsyn+BGZ group and Yinsyn group  of the ROC curves ≥ 0.9 and P < 0.05) , while the levels of Aldh1b1 and Lgals5 were both decreased (both P < 0.05) and accompanied with the significantly increased Slc25a25 and Pim3 (both P < 0.05) in the Yinsyn group. Under BGZ intervention, the levels of Aldh1b1, Slc25a25, Pim3, Oaf, and Lgals5 were all significantly decreased in the Yangsyn+BGZ group compared with the Yangsyn group , while only Lgals5 was significantly increased in rats with Yinsyn+BGZ compared with the Yinsyn group (P < 0.05).To further explore the diagnostic efficacy of DEGs, a ROC curve analysis was performed using GraphPad Prism software (version 8.01). The results revealed that 22 DEGs could be better discriminated between the Yangoup Fig. A, revealsyn Fig. B–E. Vennoup Fig. F, revealsyn Fig. G. RT-qPCsyn+BGZ group compared with the Yangsyn group, such as GO:0009991 (response to extracellular stimulus), GO:0032922 (circadian regulation of gene expression), GO:0031667 (response to nutrient levels), and GO:0009892 (negative regulation of metabolic process), while 6 GOs including GO:0019825 (oxygen binding), GO:0020037 (heme-binding), GO:0046906 (tetrapyrrole binding), GO:0005833 (hemoglobin complex), GO:0005344 (oxygen carrier activity), and GO:0042743 (hydrogen peroxide metabolic process) were significantly changed in the Yinsyn+BGZ group compared with the Yinsyn group. However, there is no intersection in the GO terms of regulating liver function in rats with Yangsyn/Yinsyn by BGZ, Fig. Padjust < 0.05, respectively.GO functional enrichment analysis found that 95 GOs , carbohydrate metabolism , lipid metabolism (arachidonic acid metabolism), metabolism of cofactors and vitamins (porphyrin and chlorophyll metabolism), were significantly changed in the Yinsyn+BGZ group compared with the Yinsyn group. However, the KEGG pathway of BGZ regulating liver function in rats with Yangsyn/Yinsyn has only one intersection (tryptophan metabolism). Figure Padjust < 0.05, respectively.KEGG functional enrichment analysis found it mainly involves 15 pathways, including amino acid metabolism , carbohydrate metabolism , lipid metabolism (glycerolipid metabolism), were significantly changed in the Yangsyn, and Yinsyn groups can be well distinguished, suggesting that the metabolic information between the Yangsyn and Yinsyn groups has been changed. OPLS-DA analysis found that the comparison between the Yangsyn group and the Yangsyn+BGZ groups  and differential metabolites Figs. , 20 metasyn+BGZ group and Yangsyn group, and 5 metabolites had better discrimination between the Yinsyn+BGZ group and Yinsyn group  of the ROC curves ≥ 0.9 and P < 0.05). Venn analysis showed the ROC results of metabolites and their correlation with the levels of ALT and AST among the comparison of the Yangsyn+BGZ group and Yangsyn group . The schematic diagram of the disturbed metabolic pathways changed by the treatment of BGZ for Yangsyn and Yinsyn rats is presented in Fig. P value of the metabolic pathway. The details for the value of P are shown in Additional file syn mainly involved glycerophospholipid metabolism, arachidonic acid metabolism, pantothenate and coenzyme A (CoA) biosynthesis. The metabolites of BGZ regulating liver function in rats with Yinsyn mainly involve pantothenate and the CoA biosynthesis, glycerophospholipid metabolism. To compare the differences in metabolic profiles for BGZ treatment on Yinsyn and Yangsyn rats, a network map was constructed based on the identified metabolites and enriched metabolic pathways. As shown in Fig. To further understand the metabolic disorder of susceptible syndromes, the KEGG identification code of differential metabolites was used for pathway analysis via MetaboAnalyst 5.0  but increased in the Yinsyn group (P < 0.01) while compared with the CON group. In addition, the levels of LDH were significantly increased in both model groups (both P < 0.01). Under BGZ intervention, the levels of Na+-K+-ATPase in the Yangsyn+BGZ group and Yinsyn+BGZ group were both significantly increased when compared with corresponding model rats, respectively (both P < 0.05), while the levels of LDH in the Yangsyn+BGZ group and Yinsyn+BGZ group showed the opposite trends of change when compared with corresponding model rats, respectively (both P < 0.05).In the Venn analysis of the metabolite enrichment pathways and DEGs enrichment pathways, it was found that the pathways of BGZ regulating liver function in rats with Yinism Fig. A, while ism Fig. B. Figure and LDH , 50. As syn and Yangsyn are two different predisposed individual states of hepatotoxicity or hepatoprotection caused by BGZ. From the perspective of the gene expression profile, BGZ mostly does not affect normal rats, but it has a mild or greater impact on the change numbers of DEGs in Yinsyn (28 DEGs) and Yangsyn (102 DEGs) rats, respectively. From the perspective of endogenous metabolites, Yinsyn and Yangsyn rats can be well distinguished on the PCA scatter plot, and the OPLS-DA plot can further better distinguish Yinsyn, Yangsyn, and their corresponding BGZ-treated groups, suggesting that BGZ exerts different metabolic perturbation patterns for different models.Individual variability in liver injury following drug ingestion is a major challenge for DILI research and clinical prevention . In the syn and Yangsyn rats, which is consistent with the phenomenon of abnormal liver function [syn rats was inhibited by BGZ, indicating that Slc25a25 may be a hepatoprotective gene for BGZ in Yangsyn rats, and the way to aggravate liver injury in rats with Yinsyn may not by regulating Slc25a25. Pim3 is a liver growth-stimulating factor with serine/threonine kinase activity and is involved in gp130-mediated induction of cell proliferation and protection of apoptosis downstream of signal transducer and activator of transcription 3 (STAT3) [syn rats with almost no change in Yinsyn rats. The above results indicated that Pim3 may be a marker gene for BGZ to exert a hepatoprotective effect on Yangsyn rats. In addition, BGZ may also have a potential therapeutic effect on tumor patients with Yangsyn, and the anti-tumor effect of BGZ has been confirmed [Among the DEGs with ROC ≥ 0.9 and significant correlation with ALT and AST, Slc25a25 may function as an ATP-Mg/Pi carrier to mediate the transport of Mg-ATP in exchange for phosphate. It is also likely responsible for the net uptake or efflux of adenine nucleotides into or from the mitochondria and is highly expressive in acute liver failure or induced by exogenous thyroxine , 52. Thi (STAT3) , 54. Pim (STAT3) . Therefo (STAT3) , 57. Pim (STAT3) , and the (STAT3) , which c (STAT3) . This stonfirmed , 62.Among the metabolites with ROC ≥ 0.90 and significant correlation with ALT and AST, glycerophospholipids  are the most abundant phospholipids in the body. In addition to constituting biofilm, they are also one of the components of bile and membrane surface-active substances and participate in protein recognition and signal transduction through the cell membrane . Hepatocsyn and Yangsyn both showed opposite trends, suggesting that regulating glycerophospholipid metabolism may be another way in which BGZ exerts liver damage and protection in Yinsyn and Yangsyn rats, respectively.In the glycerolipid metabolic pathway, phosphatidate can activate hepatic interleukin 6 (IL-6) signaling through inter-organ crosstalk and alleviate acetaminophen-induced liver injury in mice . Phosphasyn+BGZ group under the intervention of BGZ, while the relative expressions of 12(S)-HETE and PGE2 were significantly decreased in the Yangsyn+BGZ group, suggesting that the regulation of arachidonic acid metabolism may be another way in which BGZ regulates the effects of liver protection or liver damage in rats with Yangsyn and Yinsyn, respectively.In the arachidonic acid metabolic pathway, 12(S)-HETE, as an important inflammatory marker, can affect the inflammatory process by stimulating the release of cytokines such as tumor necrosis factor alpha-like (TNF-α) and IL-6. Blocking the production of 12(S)-HETE can inhibit ischemia-reperfusion-induced liver dysfunction, inflammation, and cell death . As a reIn the metabolic pathway of porphyrin and chlorophyll, porphyrins mediate related to tissue and cell damage by inducing protein oxidation and aggregation , and thesyn rats accompanied by the inhibition expressions of bilirubin and FMN in Yinsyn rats, suggesting that the regulation of porphyrin and chlorophyll metabolism and tyrosine metabolism via Blvrb, may be a mechanism by which BGZ exerts hepatoprotective or liver damage effects on Yangsyn and Yinsyn rats, respectively.Blvrb has also been shown to catalyze the reduction of flavin mononucleotide , flavin adenine dinucleotide (FAD), and riboflavin , and ribsyn group and the Yinsyn group, the levels of 3-Hydroxy-L-kynurenine were significantly decreased and increased under the intervention of BGZ, respectively, suggesting the kynurenine metabolism pathway in regulating tryptophan metabolism may be another mechanism by which BGZ exerts hepatoprotective or liver damage effects on Yangsyn and Yinsyn rats.In the metabolic pathway of tryptophan, tryptophan is involved in immune regulation, neural function, and intestinal homeostasis through the kynurenine pathway for metabolism . For exaGlycine, serine and threonine metabolism are directly or indirectly involved in the above metabolic pathways  by providing raw materials –107. Glysyn and (or) Yangsyn, were almost all related to energy metabolism, amino acid metabolism, and lipid metabolism, and the disorder of the above pathways has also been reported in Yinsyn and Yangsyn [+-K+-ATPase, two indexes directly reflecting energy level, were significantly correlated with cAMP expression [+-K+-ATPase, in rats with Yinsyn and Yangsyn, respectively. It is suggested that the cAMP signaling pathway may be one of the mechanisms by which BGZ regulates energy metabolism to affect rats with Yinsyn or Yangsyn. As for the different intervention effects of BGZ on the different syndromes, the interaction of endogenous metabolites and genes may be the main reason, which is worthy of further study in the future.The above-discussed pathways changed by BGZ in Yin Yangsyn , 113. Th Yangsyn . cAMP, t Yangsyn , can cro Yangsyn –118. LDHpression , 120. Copression , and thesyn rats but also has a potential risk of inducing liver injury in Yinsyn rats. The mechanism is mainly reflected in BGZ having different regulatory effects on amino acid metabolism, energy metabolism, lipid metabolism, and metabolism of cofactors and vitamins in the above two different syndromes. In response to the seemingly contradictory results that BGZ has both liver-damaging and liver-protective effects in previous reports [In conclusion, this study found that BGZ has a double-edged sword-like effect that not only exerts a good hepatoprotective effect on Yang reports , 19, thi Additional file 1:Table S1 Primer sequences of target genes. Table S2 DEGs and correspondingfold changes (FC) regulated by BGZ in the treatment of Yangsyn inrats. Table S3 DEGs and correspondingfold changes regulated by BGZ in the treatment of Yinsyn in rats. Table S4 The relationship between the relativeabundance ofDEGsand the levels of serum biochemistry (ALT and AST). Table S5 GO functional enrichmentpathways analysis of DEGs regulated by BGZ in Yangsyn+BGZ ratscompared with Yangsyn rats (Padjust < 0.05). Table S6 The relationship between the relativeabundance of metabolites and the levels of serum biochemistry(ALT and AST). Table S7 Metabolic pathways of BGZin the treatment of Yinsyn in rats. Table S8 Metabolic pathways of BGZin the treatment of Yangsyn in rats. Figure S1 ROC analysis of DEGs. A the DEGs between the Yangsyn+BGZ group and the Yangsyngroup; B the DEGs between the Yinsyn+BGZ group and the Yinsyngroup. Figure S2A The 100-permutationtest for the Yangsyn+BGZ group and Yangsyn group in ESI-mode; B The 100-permutation test for Yinsyn+BGZ group and Yinsyngroup in ESI- mode; C The 100-permutation test for Yangsyn+BGZ groupand Yangsyn group in ESI+ mode; D The 100-permutation test for Yinsyn+BGZgroup and Yinsyn group in ESI+ mode. Figure S3 Secondaryfragment ions characteristic maps of identified metabolites. Figure S4 ROC analysis ofmetabolites. A the metabolites between the Yangsyn+BGZ group and theYangsyn group; B the metabolites between the Yinsyn+BGZgroup and the Yinsyn group.


Within the wide scope of interstitial lung diseases (ILDs), familial pulmonary fibrosis (FPF) is being increasingly recognized as a specific entity, with earlier onset, faster progression, and suboptimal responses to immunosuppression. FPF is linked to heritable pathogenic variants in telomere-related genes (TRGs), surfactant-related genes (SRGs), telomere shortening (TS), and early cellular senescence. Telomere abnormalities have also been identified in some sporadic cases of fibrotic ILD. Air pollution and other environmental exposures carry additive risk to genetic predisposition in pulmonary fibrosis. We provide a perspective on how these features impact on screening strategies for relatives of FPF patients, interstitial lung abnormalities, ILD multi-disciplinary team (MDT) discussion, and disparities and barriers to genomic testing. We also describe our experience with establishing a familial interstitial pneumonia (FIP) clinic and provide guidance on how to identify patients with telomere dysfunction who would benefit most from genomic testing. The most common fibrotic ILD is idiopathic pulmonary fibrosis (IPF) . BesidesThe terminology to refer to cases of ILD with a familial predisposition can be confusing. While initial reports used the term “familial IPF,” this would not account for the heterogeneity of ILD diagnoses in affected family members  . A commo2.2.1.TTAGGG) and associated proteins thought to maintain genomic integrity during repeated cell divisions. Critical TS can lead to diverse clinical syndromes  , 15, 16.2.2.TERT, TERC, RTEL1, PARN, DKC1, TINF2, NOP10, NHP2, ACD, NAF1, ZCCHC8, RPA, POT1 (TERT (telomerase reverse transcriptase) and TERC (telomerase RNA component) are components of the telomere complex which is a group of proteins and RNA that drive the addition of telomeric repeats (TTAGGG) to the ends of chromosomes and were the first TRGs identified in FPF , macrocytosis (24–41%) or thrombocytopenia (8–54%) may be associated to ILD  can also be found in 5–27% TRG mutation patients and early hair greying is noted in 15–40%   include F cases <% 5). AB. ABSFTPCn adults . In chiln adults .MUC5B rs35705950 minor allele promoter variant and low penetrance, genotyping of the variant is not currently part of FPF gene investigations in most countries  was shown to be associated with both FIP and sporadic IPF . It show disease .In FPF, there is overall a 50% chance of inheriting a deleterious allele in first-degree relatives   compared2.4.TERT) favor the development of fibrosis following infectious or noxious challenge. Immune processes can also be affected by cellular senescence (such as T cell dysfunction and shifting to Th17 responses leading to immune dysregulation and propensity to autoimmune responses) and environmental exposures can accelerate this process  air pollution , NOx = 3.08 (2.21–4.27), PM2.5 = 3.65 (2.6–5.13), and PM10 = 3.23 (2.32–4.5)  .2.5 can lead to dose-dependent epigenomic modifications, altered telomerase activity and TS   decline in IPF patients  , 47. Res FBXO32) . Increashylation . Increaspatients . Increasortality .4.4.1.p = 0.12) (Progressive pulmonary fibrosis (PPF) is the dominant phenotype seen in familial cases. There is evidence to suggest that FPF is worse, faster progressing and has higher mortality than sporadic fibrotic ILD, although data is incomplete and at times contradictory . In a sm = 0.12) . The ave = 0.12) . The mea = 0.12) , 50. In  = 0.12) . Regardl = 0.12) .4.2.As genomic tests can be expensive or only reimbursed when certain criteria are met, it is recommended to pre-screen patients based on family history and clinical features of TS prior to testing , 2 5, 5, 55, 52.Appropriate counselling and a tactful approach are necessary because genomic testing can be seen as opening a “Pandora’s box.” It often leads to major changes in patients’ lives and those of their relatives. In our experience with the Manchester FIP clinic, most patients are extremely interested in genomic tests if the wider context is clearly explained, and a pathway exists to deal with possible results. Importantly, testing provides an answer to the question “Why did I get ILD?” which leads to better acceptance of the ILD diagnosis and treatment requirements. Many patients are also very interested in research projects.4.3.In our experience, familial and sporadic cases with telomere dysfunction frequently have atypical ILD presentations, and this can delay referral to an ILD specialist center, accurate diagnosis, and timely treatment.As FPF cases may progress faster than sporadic ones, more frequent follow-up with lung function monitoring and early lung transplant referral should be considered as part of the MDT discussion, ideally involving transplant specialists and genetic counsellors. Transplant outcomes between FPF and sporadic PF were comparable , 55, theAntifibrotics are just as useful in FPF as in sporadic fibrotic ILDs (IPF and PPF). Immunosuppression can increase the risk of complications and efficacy may be limited in patients who require such treatments  . It woul4.4.Interstitial lung abnormalities (ILAs) on chest CT scanning are a common finding in relatives of FPF patients . They arPrevious studies have demonstrated a prevalence of ILAs on lung cancer screening of 4–20% . Recent Interstitial lung abnormalities are associated with radiological progression and mortality . Having 4.5.There is no clear pathway or optimal age for screening relatives of FPF patients. As there is a 1–3-year delay between symptom onset and diagnosis for FPF patients, this represents an opportunity for early diagnosis .It is recommended that all first-degree relatives of FPF patients should have a screening chest high-resolution CT (HRCT) scan especially if they have non-resolving respiratory symptoms (chronic cough and dyspnea) . The preLung function is important for screening and follow-up, but simple spirometry may not identify early cases since FVC < 80% predicted in asymptomatic relatives is rare. However, relatives with ILAs have a lower diffusing capacity of the lung for carbon monoxide (DLCO) than those with normal HRCT , 69. AnnAdditionally, due to the high prevalence of other TS features, a full blood count (FBC) and liver function testing should also be performed in first-degree relatives .Certain relatives may be more open to screening, while others would prefer to wait and clinicians should abide by their preferences, to not cause undue anxiety. Eligible relatives may also be offered genomic testing, usually via local/regional Clinical Genetics services who follow-up families of the proband.To date, there is no consensus on the frequency and duration of clinical, functional, and radiological evaluations of first-degree relatives of FPF patients. Management could be customized through a risk assessment, considering resources available in each country/national health system.5.R = −0.28, p < 0.0001). It appears that sex-adjusted LTL below median is uniformly associated with chronological age and increased risk of mortality in all racial groups although in the Asian population it was not statistically significant  (Although patients with PF were found to have disproportionately short leukocyte telomere length (LTL), there was very little genetic variability in cohorts (to account for ethnic and socio-economic differences around the world). LTL decreased with age, more so in PF patients compared to controls (5–8.23]) . Patient5–8.23]) , 72. Fur5–8.23]) .The availability of genomic testing, telomere length measurement and whole genome sequencing varies greatly around the world, making it difficult to standardize global recommendations. At the time of this writing, a 25-gene panel test for FPF is available in the UK, but telomere length measurement is not reimbursed by the health service . Local l6.In our opinion, actively eliciting a family history of ILD and other rare conditions, as well as asking about TS features , 2 couldThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.SS and PR-O contributed equally to the conceptualization and literature search. JC contributed to summarizing the key points from the references. SS and PR-O structured the manuscript. SS, JC, and PR-O provided comments and contributed equally to finalizing the draft manuscript for submission. 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.


To assess the diagnostic performance of high-resolution contrast-enhanced MRI (hrMRI) with three-dimensional (3D) fast spin echo (FSE) sequence by comparison with conventional contrast-enhanced MRI (cMRI) and dynamic contrast-enhanced MRI (dMRI) with 2D FSE sequence for identifying pituitary microadenomas.κ analysis.This single-institutional retrospective study included 69 consecutive patients with Cushing’s syndrome who underwent preoperative pituitary MRI, including cMRI, dMRI, and hrMRI, between January 2016 to December 2020. Reference standards were established by using all available imaging, clinical, surgical, and pathological resources. The diagnostic performance of cMRI, dMRI, and hrMRI for identifying pituitary microadenomas was independently evaluated by two experienced neuroradiologists. The area under the receiver operating characteristics curves (AUCs) were compared between protocols for each reader by using the DeLong test to assess the diagnostic performance for identifying pituitary microadenomas. The inter-observer agreement was assessed by using the p ≤ .002) and dMRI  for identifying pituitary microadenomas. The sensitivity and specificity of hrMRI were 90–93% and 100%, respectively. There were 78% (18/23) to 82% (14/17) of the patients, who were misdiagnosed on cMRI and dMRI and correctly diagnosed on hrMRI. The inter-observer agreement for identifying pituitary microadenomas was moderate on cMRI (κ = 0.50), moderate on dMRI (κ = 0.57), and almost perfect on hrMRI (κ = 0.91), respectively.The diagnostic performance of hrMRI  was higher than cMRI (AUC, 0.74–0.75; The hrMRI showed higher diagnostic performance than cMRI and dMRI for identifying pituitary microadenomas in patients with Cushing’s syndrome.The diagnostic performance of hrMRI was higher than cMRI and dMRI for identifying pituitary microadenomas in Cushing’s syndrome.• About 80% of patients, who were misdiagnosed on cMRI and dMRI, were correctly diagnosed on hrMRI.• The inter-observer agreement for identifying pituitary microadenomas was almost perfect on hrMRI.• The online version contains supplementary material available at 10.1007/s00330-023-09585-1. Cushing’s syndrome, caused by excessive exposure to glucocorticoids, is associated with considerable morbidity and increased mortality . CushingHowever, there are considerable challenges in identifying ACTH-secreting pituitary adenomas. This is because about 90% of the tumors are microadenomas (less than 10 mm in size) and the median diameter at surgery is about 5 mm . ConventThe aim of our study was to assess the diagnostic performance of hrMRI with 3D FSE sequence by comparison with cMRI and dMRI with 2D FSE sequence for identifying ACTH-secreting pituitary microadenomas in patients with Cushing’s syndrome.This single-institutional retrospective study was approved by the Institutional Review Board of our hospital. The study was conducted in accordance with the Helsinki Declaration. The informed consent was waived due to the retrospective nature of the study.n = 97), patients with ectopic ACTH syndrome who underwent pituitary exploration (n = 2), and patients with macroadenomas (n = 5) or lack of pathology (n = 13) were excluded from the study. Finally, 69 patients with ACTH-dependent Cushing’s syndrome were included in the current study  using an 8-channel head coil. The MRI protocol included coronal T2-weighted imaging, coronal T1-weighted imaging, and sagittal T1-weighted imaging before contrast injection. After contrast injection of gadopentetate dimeglumine (Gd-DTPA) at 0.05 mmol/kg (0.1 mL/kg) with a flow rate of 2 mL/s followed by a 10-mL saline solution flush, dMRI and cMRI with 2D FSE sequence were obtained first, and hrMRI with 3D FSE sequence using variable flip angle technique was performed immediately afterward. Detailed acquisition parameters are presented in Table Image interpretation was independently conducted by two experienced neuroradiologists , who were blinded to patient information. The evaluation order of cMRI, dMRI, and hrMRI sequences was randomized. The identification of pituitary microadenomas on images was scored based on a three-point scale . Scores of 1 or 2 represented the identification of the lesion. Reference standards were established by using all available imaging, clinical, surgical, and pathological resources, with a multidisciplinary team approach.Two readers  were asked to assess the image quality of cMRI, dMRI, and hrMRI. Before exposure to images used in the current study, these readers underwent a training session to make sure that they were comparable to the experienced neuroradiologists in terms of image quality assessment. Images were presented in a random order. Image quality was assessed by using a 5-point Likert scale , includi2 region of interest was placed in the background, and noise was defined as the standard deviation of the signal intensity of the background [The mean signal intensity of pituitary microadenomas, pituitary gland, and noise on cMRI, dMRI, and hrMRI was measured using an operator-defined region of interest. For noise, a 10-mmckground . For pitckground . Supplemκ analysis was conducted to assess the inter-observer agreement for identifying pituitary microadenomas. The κ value was interpreted as follows: below 0.20, slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; greater than 0.80, almost perfect agreement.The U test was used to evaluate the difference in image quality scores and the Wilcoxon signed-rank test was used to evaluate SNR and CNR measurements between MR protocols. A p value of less than 0.05 was considered statistically significant. Statistical analysis was performed using MedCalc Statistical Software  and SPSS Statistics .To assess the diagnostic performance of cMRI, dMRI, and hrMRI for identifying pituitary microadenomas, the receiver operating characteristic curves were plotted and the area under curves (AUCs) were compared between MR protocols for each reader by using the DeLong test. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated. The Mann–Whitney A total of 69 patients  with ACTH-dependent Cushing’s syndrome were included in the study and their clinical characteristics are shown in Table κ statistic between two readers was moderate on cMRI (κ = 0.50), moderate on dMRI (κ = 0.57), and almost perfect on hrMRI (κ = 0.91), respectively.The inter-observer agreement for identifying pituitary microadenomas by p = 0.002), dMRI , and combined cMRI and dMRI . For reader 2, the diagnostic performance of hrMRI  was higher than that of cMRI , dMRI , and combined cMRI and dMRI .The diagnostic performance for identifying pituitary microadenomas on cMRI, dMRI, hrMRI, and combined cMRI and dMRI is summarized in Table For reader 1, 23 of the 69 patients (33%) were misdiagnosed on both cMRI and dMRI, but 18 of the 23 misdiagnosed patients (78%) were correctly diagnosed on hrMRI. For reader 2, 17 of the 69 patients (25%) were misdiagnosed on both cMRI and dMRI, but 14 of the 17 misdiagnosed patients (82%) were correctly diagnosed on hrMRI.Figure p < 0.001 for all) and dMRI .Image quality scores of cMRI, dMRI, and hrMRI are presented in Table p < 0.001), but higher than that on dMRI . The CNR on hrMRI  was higher than that on cMRI  and dMRI .The SNR and CNR measurements are shown in Table p ≤ 0.002) and dMRI  for identifying pituitary microadenomas. To our knowledge, there are no previous studies specifically evaluating the identification of pituitary microadenomas on hrMRI with 3D FSE sequence by comparison with cMRI and dMRI in patients with ACTH-dependent Cushing’s syndrome, and this is the largest study conducted in ACTH-secreting microadenomas with a sensitivity of more than 90%.The identification of pituitary microadenomas is considerably challenging but critical in patients with ACTH-dependent Cushing’s syndrome. Our study demonstrated that hrMRI with 3D FSE sequence had higher diagnostic performance  than cMRI , which was moderate on cMRI (κ = 0.50) and dMRI (κ = 0.57). Therefore, hrMRI using the 3D FSE sequence is a potential alternative that can significantly improve the identification of pituitary microadenomas.The identification of pituitary adenomas on preoperative MRI in patients with ACTH-dependent Cushing’s syndrome could help the differential diagnosis of Cushing’s syndrome and aids surgical resection of lesions. It should be noted that most of the pituitary adenomas in patients with Cushing’s disease are microadenomas , 6. In oLimitations of the study included its retrospective nature and the relatively small sample size in patients with ectopic ACTH syndrome as negative controls. The bias may be introduced in the patient inclusion process. Only those patients who underwent all the cMRI, dMRI, and hrMRI scans were included. In fact, some patients will bypass hrMRI when obvious pituitary adenomas were detected on cMRI and dMRI. These patients were not included in the current study because of lack of hrMRI findings. Given the situation, the sensitivity of identifying pituitary adenomas will be higher with the enrollment of these patients. Besides, the timing of the sequence acquisition after contrast injection is essential  and biasIn conclusion, hrMRI with 3D FSE sequence showed higher diagnostic performance than cMRI and dMRI for identifying pituitary microadenomas in patients with Cushing’s syndrome.Supplementary file1 (PDF 242 kb)Below is the link to the electronic supplementary material.


Citrullus lanatus L.) under Zn stress, one of the most important horticultural crops, remains largely unknown. In this study, we revealed that 24-epibrassinolide (EBR), a bioactive BR enhanced Zn tolerance in watermelon plants, which was related to the EBR-induced increase in the fresh weight, chlorophyll content, and net photosynthetic rate (Pn) and decrease in the content of hydrogen peroxide (H2O2), malondialdehyde (MDA), and Zn in watermelon leaves. Through RNA deep sequencing (RNA-seq), 350 different expressed genes (DEG) were found to be involved in the response to Zn stress after EBR treatment, including 175 up-regulated DEGs and 175 down-regulated DEGs. The up-regulated DEGs were significantly enriched in ‘phenylpropanoid biosynthesis’ pathway (map00940) using KEGG enrichment analysis. The gene expression levels of PAL, 4CL, CCR, and CCoAOMT, key genes involved in phenylpropanoid pathway, were significantly induced after EBR treatment. In addition, compared with Zn stress alone, EBR treatment significantly promoted the activities of PAL, 4CL, and POD by 30.90%, 20.69%, and 47.28%, respectively, and increased the content of total phenolic compounds, total flavonoids, and lignin by 23.02%, 40.37%, and 29.26%, respectively. The present research indicates that EBR plays an active role in strengthening Zn tolerance, thus providing new insights into the mechanism of BRs enhancing heavy metal tolerance.Zinc (Zn) is an important element in plants, but over-accumulation of Zn is harmful. The phytohormone brassinosteroids (BRs) play a key role in regulating plant growth, development, and response to stress. However, the role of BRs in watermelon ( Zinc (Zn), an essential biological element, participates in plant physiological metabolism as well as in the process of protein synthesis and transport2. However, high concentration of Zn will cause serious environmental pollution, and excessive accumulation of Zn in plants causes serious toxicity to plants3. Excessive Zn can cause the imbalance of nutrient and water absorption of plants, change the ultrastructure of chloroplasts to inhibit plant photosynthesis, destroy the membrane system and macromolecular substances of plant cells through accumulation of reactive oxygen species (ROS), and then seriously damage the growth, development, metabolism, and other plant physiological processes, causing plant death4. For example, high concentration of Zn in M. sativa produces a large amount of ROS, resulting in oxidative stress and cell damage, which seriously interferes M. sativa growth and quality5. Therefore, it has become a research hotspot in the field of environmental science to control Zn pollution, study the mechanism of plant tolerance to Zn and reduce the excessive absorption of Zn by plants.Heavy metals (HMs) pollution is a major environmental problem around the world. HMs are transported to the aboveground part through roots and accumulated in the edible part of crops, and enter human or animal bodies through the food chain, threatening human life and health7. The regulation of exogenous BRs on HMs tolerance has attracted more and more attention8. Previous studies have shown that BRs can eliminate ROS produced by Zn stress by combining with membrane proteins, thereby alleviating the damage effect of Zn on plants. BRs can also affect the absorption of Zn by plants by stabilizing the electrical properties of cell membranes and enzyme activities, thus reducing Zn toxicity9. For example, Exogenous EBR spraying can improve eggplant seedlings tolerance to Zn stress through enhancement of antioxidant enzyme activity, osmotic substance accumulation, and hormone metabolism balance10. Exogenous EBR further increases the levels of antioxidant enzymes, thus effectively eliminating ROS, alleviating the toxicity of Zn to soybean seedlings11. Recently, EBR application to M. sativa under Zn stress could reduce Zn accumulation, promote the response of antioxidant defense system, and actively regulate the mechanism of heavy metal detoxification5.Brassinolides (BRs) is a kind of natural plant hormone widely existing in plants. It has been identified as the sixth kind of plant hormone and plays a crucial role in plant growth and development, as well as biotic and abiotic stresses13. Phenylpropane metabolism pathway plays an antioxidant role directly or indirectly in plant tolerance to heavy metal stress, and can improve plant absorption of heavy metal ions and stress tolerance14. In recent years, some studies have shown that BRs can improve plant stress resistance by regulating phenylpropane metabolism. Seed priming with EBR changes the growth and phenylpropanoid pathway of flax to water deficit15. Leaf application of EBR alleviates rice salt stress by up regulating secondary metabolites produced via the phenylalanine biosynthesis pathway16. EBR plays an active role in Colletotrichum fructicola resistance through the induction of lignin synthesis in Camellia sinensis17. Up to now, there are few reports on the research of heavy metal resistance mediated by BRs through the regulation of phenylpropane metabolism pathway, let alone Zn stress.Phenylpropane metabolic pathway is an important secondary metabolic pathway in plants, and its metabolites phenolics, flavonoids, and lignin are closely related to plant stress resistance18, which reveal the signal transduction pathways involved in Zn stress tolerance in some plants, such as wheat19, barley20, and Arabidopsis21. However, the specific gene expression profile of plant Zn tolerance induced by BR is still unclear. Watermelon (Citrullus lanatus) is one of the most important horticultural crops. However, so far, the possibility of using EBR to improve Zn tolerance of watermelon has not been studied, let alone the regulatory mechanism. Considering the increase of Zn content in horticultural soil and the stress improvement characteristics of BRs, the molecular mechanism of EBR regulating the response of watermelon to Zn stress were studied in the current study. 0.05 µM EBR could effectively alleviate Zn stress in watermelon. The gene expression pattern regulated by EBR under Zn stress was studied by RNA-seq and the different expressed genes (DEG) was significantly enriched in ‘phenylpropanoid biosynthesis. In addition, the important enzymes and secondary metabolites involved in the phenylpropanoid biosynthesis pathway have also been proved to be related to EBR application. The results of this study will provide new insights into the mechanism of BR enhancing Zn tolerance in plants.In the past decade, RNA-sequencing (RNA-seq) technique, a high-throughput method, has been used to investigate the global expression proﬁlesC. lanatus L.) variety 8424 was from Anhui Fengsheng Agricultural Technology Co., Ltd. Watermelon seeds were sterilized, washed, placed on filter paper, and incubated at 25°C. After germination, the seedlings were sown in pots (diameter of 8 cm), which was filled with sterilized sand and vermiculite (3:1). Growth conditions: temperature 25 ± 2°C, relative humidity 70 ± 5%, photoperiod 12/12 h, photosynthetic photon flux density 500 μmol m−2 s−1. After 10 days, plants were irrigated with 1/2 Hoagland’s nutrient solution containing Zn or not.Watermelon (4·7 H2O) concentration was selected based on preliminary experiment, using 1.0, 2.5, 5.0, or 10.0 mM of Zn. It was found that plant growth was inhibited at 5 mM Zn but not completely inhibited (IC50) (data not shown). The EBR concentrations were selected using 0.025, 0.05, 0.10, 0.20, or 0.50 μM EBR and 0.05 μM EBR were selected.The Zn , or subjected to 5.0 mM Zn with different concentration of EBR . Seedlings were sprayed with EBR one day in advance and were watered with the nutrient solution containing Zn every 3 days. After 10 days of Zn/EBR application, the third leaves were frozen in liquid NThe 0.1 g fresh leaf samples were homogenized in 10 ml of 80% frozen acetone at 4°C at 10，000 g centrifuge for 10 min. The absorbances of chlorophyll a and b in the supernatant at 663, 645, and 480 nm were recorded.−2 s−1, leaf temperature is 25°C, CO2 concentration is 400 µmol mol−1, and relative humidity is 75%.The Li-6400 portable photosynthetic instrument  was used to measure Pn of the third true leaf of watermelon, and the instrument automatically recorded the net photosynthetic rate. The measured light intensity is 1,000 µmol m−1 dry weight. H2O2 and MDA content were measured according to the method of Wu et al.22.For content measurement of Zn, the dried material was weighed, ground to a powder, and digested with a 1:3 mixture of HCl:HNO3. The digests were then dissolved in ultrapure water. Then, the digested sample were analyzed using a ﬂame-atomic absorption spectrometer  and the content was expressed as mg gTM2000 platform. Three biological replicates were performed. Each sample got more than 6 Gb of data. After sequencing, the raw data was filtered to obtain high-quality clean data. The ﬁltered clean reads were aligned to the reference genome (http://www.watermelondb.cn) using HISAT2 software23. Differentially expressed genes (DEGs) were analyzed using the DESeq2 package with |log2 Fold change| ≥ 1 and p-value<0.05. The fold change indicates the ratio of the expression amount between two groups.Total RNA from watermelon leaves of Zn+EBR and Zn treatments was isolated. After the RNA sample was qualified, the enrichment of mRNA and the construction of cDNA library were carried out. After the library inspection was qualified, the RNA-seq was carried out using an Illumina HiSeqhttps://www.kegg.jp/kegg/)24. Using R (v3.6.2) to make KEGG enrichment scatter diagram for the first 20 most significant pathways. Gene ontology (GO) enrichment of DEGs was annotated into GO database, enriching and analyzing the BP, MF, and CC functions, respectively, using Fisher exact test. GO and KEGG terms with corrected p-value<0.05 were considered significantly enriched.BLASTALL (v2.2.26) software was used to carry out annotations of GO, KEGG, COG, KOG, NR, Swissprot, Pfam databases to analyze the DEG functions. KEGG pathway enrichment was analyzed using the KEGG database (http://www.watermelondb.cn), as shown in Supplementary Table S1. β-Actin gene was used as a reference for normalizing the data.Total RNA from watermelon leaves of Zn+EBR and Zn treatments was isolated and the SuperscriptIII first-strand synthesis system  was used for cDNA synthesis according to the manufacturer’s protocol. Use SYBR Premium Ex Taq  to conduct RT-qPCR in real-time PCR system. The selected specific primers for DEG were designed according to the Watermelon Genome Database , 4-coumaric ligase (4CL), and peroxidase (POD)25. Mainly, the activities of PAL, 4CL, POD and CAD were determined by ELISA kit  following the protocol.Plant material was homogenized with 50 mM sodium borate buffer (pH 8.0) supplying 1 mM ethylenediaminetetraacetic acid disodium salt (EDTA) and 2% polyvinyl pyrrolidone (PVP), and then immediately centrifuged at 12,000 g and 4°C for 20 min. The supernatant fraction was frozen in liquid nitrogen and placed at−80°C for determining enzyme activities of PAL, 4CL, and POD according to the method of Shao et al.26, the phenol content was estimated by Folin – Ciocalteu reagent, and the phenol content was expressed as mg gallic acid equivalent (GAE) g−1 of sample. The flavonoid was determined by sodium nitrite aluminum nitrate colorimetry with sophorin as the standard sample. The flavonoid content was expressed as mg g−1 sample. The lignin content was determined by spectrophotometry according to the method of Xu et al.27. Acetyl lignin is produced after the phenol hydroxyl in lignin is acetylated. The product has a characteristic absorption peak at 280 nm. The lignin content was measured on the basis of the changing absorbance.According to the method of Alam et al.P value≤0.05) were used to analyze the differences. SPSS version 20.0  was used for statistical analysis.Each treatment has three biological replicates. The data is the average ± SD (Standard Deviation) of the replicates displayed by the vertical error bar. One-way analysis of variance (ANOVA) and the Least Significance Difference (LSD) test  was used. The growth of watermelon was obviously inhibited under Zn treatment alone . CompareEBR-induced watermelon tolerance to Zn stress is accompanied by a decrease in Zn accumulation . CompareThe Chl content and Pn in watermelon seedlings under Zn treatment alone decreased significantly compared with the control . The app2O2 and MDA in leaves . These reads were, thus, mapped to the watermelon reference genome (http://www.watermelondb.cn). The DEGs analysis was conducted with the screening threshold was: | log2FC| ≥1 and q-adjust<0.05. We identified 350 DEGs between Zn+EBR and Zn treatments  were sequenced using the Illumina HiSeqeatments .Figure ZAT10 (ClG42_02g0071700), RMA1 (ClG42_03g0060400), POD (ClG42_10g0201300), CCR (ClG42_09g0033200), PAL (ClG42_04g0096300), PPO (ClG42_03g0058500), VIT1 (ClG42_01g0066400), PYL4 (ClG42_04g0073000), BKI1 (ClG42_05g0217200), and PepA (ClG42_02g0149200). The results showed that the expression level of all DEGs was consistent with the transcriptomic data and the correlation coefficient (R2 = 0.9126) was observed . A total of 175 up-regulated DEGs were signiﬁcantly enriched in four functional terms including ‘response to oxidative stress (GO:0006979)’, ‘extracellular region (GO:0005576)’, ‘cell wall (GO:0005618)’, and ‘endoplasmic reticulum lumen (GO:0005788)’. For down-regulated DEGs, metabolic process (GO:0008152) and ‘protein kinase inhibitor activity (GO:0004860)’ were signiﬁcantly enriched.To check the DEG-related pathways, they were searched using the KEGG pathway database. KEGG enrichment indicated that the up-regulated DEGs were significantly associated with ‘phenylpropanoid biosynthesis (map00940)’, ‘protein processing in endoplasmic reticulum (map04141)’, and ‘biosynthesis of secondary metabolites – unclassified (map00999)’ .Figure DEGs related to oxidative stress. The biological process of up-regulated DEGs was significantly enriched to ‘response to oxidative stress, such as common antioxidant enzyme genes peroxidase  and polyphenol oxidase (ClG42_03g0058500) (Table S4), suggesting that EBR-alleviated Zn stress was related to EBR-induced antioxidant protection to alleviate oxidative damage, which was consistent with the physiological data , 4-coumarate-CoA ligase , cinnamoyl-CoA reductase , and caffeate O-methyltransferase   of up-regulated DEGs was significantly enriched in extracellular region and cell wall (Table S4), such as ClG42_05g0027500 encoding pectinesterase 2 and ClG42_10g0192200, ClG42_09g0179200, ClG42_08g0082000, and ClG42_07g0166400 encoding extensin-like protein enriched in external encapsulating structure and cell wall organization. Besides, ClG42_01g0049000 is a lignin-forming anionic peroxidase. PAL, 4CL, CCR, and COMT are involved in lignin biosynthetic process.DEGs related to protein processing in the endoplasmic reticulum (ER). There were 12 DEGs involved in protein processing in the ER . Among them, the 11 differentially expressed HSPs and a E3 ubiquitin-protein ligase RMA1 genes were up-regulated. The DEGs were all enriched in the degradation of ubiquitin – proteasome-dependent process of ER-associated degradation (ERAD). The results indicate that ERAD is involved in EBR mediated Zn stress tolerance.Due to the significant enrichment in the phenylpropanoid biosynthesis pathway . Therefo1. Here, Zn stress treatment significantly inhibited the growth of watermelon, and also significantly inhibited the accumulation of Zn, chlorophyll content, and photosynthetic rate of watermelon  on plants, many methods have been adopted, including foliar application of different chemicals/phytohormones31–35. BRs act as multidimensional regulators of plant responses under different environmental stresses, and epibrassinolide (EBR) is a synthetic brassinolide analog, which has been widely used in production7. In this study, the addition of EBR could significantly promote the increase of the biomass of watermelon seedlings under Zn stress , which further demonstrated the mechanism of EBR mediated oxidative stress response to alleviate Zn damage.Cell membrane is an important structure for plant cells to isolate protoplasts from the external environment, which can control the material exchange and signal transmission between cells and the external environmentsed by Zn , indicat42. On the one hand, ROS neutralization is achieved through the complex antioxidant system developed by plant cells. Stress tolerance of major metabolites is mainly through classical antioxidant enzyme system and non-enzyme system42. Here, under Zn stress exogenous EBR treatment could significantly increase the expression level of three peroxidase and one polyphenol oxidase genes (Table S4) and have a high activity of POD  acts as a quencher of chloroplast photooxidation. In addition, due to the vacuole position of PPO substrate, PPO may synthesize specific metabolites and react to environmental factors44. It is also believed that the polymerization of polyphenols by peroxidase increases after heavy metal absorption and detoxification, which is the reason for heavy metal binding in the skin gland of Nymphaea epidermal45. Alam et al.26 found that providing EBR to soybean plants under NaCl stress led to the enhancement of peroxidase activities coupled with the reduction in H2O2 and MDA content. These results indicated that exogenous EBR with appropriate concentration can stimulate and induce the antioxidant enzyme protection system, at least including the gene expression and activity of POD, accelerate the clearance of ROS, reduce ROS accumulation in cells, and reduce membrane lipid peroxidation damage.The main way for plant systems to overcome the oxidative stress is to use primary metabolites and/or secondary metabolitesy of POD . Peroxid42. These compounds are important metabolic intermediates of phenylpropane metabolic pathway. In the present study, KEGG enrichment revealed that the up-regulated DEGs were significantly associated with ‘phenylpropanoid biosynthesis (map00940)’, and ‘biosynthesis of secondary metabolites-unclassified (map00999)’ .It is speculated that there are two mechanisms for phenylpropanoid pathway to participate in EBR-mediated Zn tolerance: one is that EBR further activates phenylpropyl biosynthesis pathway, leading to the generation of various phenolic compounds, which may eliminate harmful ROS; the other is to block Zn transport in plants. The rich metabolites produced by phenylpropane biosynthesis pathway coupled with high antioxidant capacity give mulberry higher salt tolerance2O2 and MDA in watermelon leaves. The comparative transcriptome data and physiological analysis revealed that the increased H2O2 scavenging and activated phenylpropanoid biosynthesis pathway contributing to the mitigation of Zn toxicity by EBR. As far as we know, this is the first study to clarify the mechanism of BR reducing Zn stress through phenylpropanoid biosynthesis pathway. In conclusion, these results enhance our understanding of the mechanism of exogenous EBR enhancing Zn tolerance and the heavy metal responsive genes identified after EBR application, providing a target for future molecular breeding.53The present study showed that exogenous EBR enhanced Zn tolerance in watermelon seedlings with higher levels of chlorophyll and Pn and lower accumulation of HClick here for additional data file.


ObjectiveThe main purpose of this study was to determine the end treatment response (ETR) and sustained viral response (SVR) in hepatitis C virus (HCV) patients receiving sofosbuvir and daclatasvir daily for 12 weeks.MethodsThis is a prospective open-label interventional study conducted from March 2018 to December 2020 in the outpatient departments of Abbasi Shaheed Hospital and Lyari General Hospital, Karachi. Patients with chronic infection of HCV, confirmed with ribonucleic acid (RNA) polymerase chain reaction (PCR)  were invited to participate in the study. All patients with positive HCV antibodies were evaluated clinically, with laboratory, and imaging assessment earlier to treatment. Statistical analysis was performed using SPSS version 20.0 .ResultsA total of 1043 patients participated in the study with a female predominance, 699 (67%) females. A majority (67.9%) of the study participants were aged between 15 and 45 years. After treatment of 12 weeks with sofosbuvir and daclatasvir 1039 (99.9%) patients achieved SVR while 1038 (99.6%) achieved an end treatment response. There was no significant association found between changes in alanine aminotransferase (ALT) levels, gender, and age among study participants.ConclusionSofosbuvir and daclatasvir are found to be extremely effective for patients with hepatitis C in Pakistan. However, additional investigation including a larger sample size and involving a multicenter setting is recommended. Viral infections (hepatitis A to E), alcohol intake, toxins, and metabolic dysfunction can result in hepatitis (inflammation of liver parenchyma) . InfectiApproximately 58 million people have chronic HCV infection worldwide, with around 1.5 million new infections each year . AccordiNon-pegylated and pegylated interferons (PegIFN) therapy over the first two decades of chronic HCV therapy, as well as ribavirin, resulted in a decline in effectiveness and was ineffectively tolerated. During the period 2001-2011, depending upon the HCV genotype, PegIFN with ribavirin was the standard therapy for treatment. In genotype 1, the percentage of people who had a durable virological response ranged from 40% to 50% after 24 weeks or 48 weeks of combination treatment . Even thFor patients older than 12 years of age, the WHO suggests therapy with pan-genotypic direct-acting antivirals (DAAs). Most people with HCV infection may be cured with DAAs, and treatment time is brief (about 12-24 weeks), depending on whether cirrhosis is present or not .After endorsement by Food and Drug Administration in 2013, sofosbuvir a non-structural protein 5B (NS5B) inhibitor was the driving DAAs taken after daclatasvir which is an NS5A inhibitor . By loweCombination therapy of interferon and ribavirin has been used in Pakistan for a long time. For this reason, much research is being done on this topic ,12. In rThe present study was an interventional study carried out in the Department of Medicine of Abbasi Shaheed Hospital and Liyari General Hospital between 2018 and 2020. A favorable ethical opinion was obtained (KMDC/2018) from Abbasi Shaheed Hospital and Karachi Medical and Dental College Research Ethics Board before the commencement of the study. Written informed consent was obtained from the study participants. All eligible participants had their identifiable data protected by assigning each participant a unique identifier (ID), which was only linked to their medical record number. The master list contained the study-assigned participant number and participant name only. De-identified data were entered during data collection. This database was stored with the principal investigator on encrypted and password-protected jumpdrives, to which only they had access. All other members of the research team were only able to view the study ID and the relevant information that is associated with it. A total of 1129 patients were contacted for prospective clinical research with chronic liver infection and compensated chronic liver diseases (CLD), which was confirmed with RNA polymerase chain reaction (PCR)  . Frequencies and percentages were calculated for sex, ETR, and SVR rate. Serum glutamic pyruvic transaminase (SGPT) was expressed as mean, standard deviation, and 95% confidence interval (CI). Chi-square tests and logistic regression analysis were used to assess the association between the ETR and SVR with sex, and age, and independent t-tests were used to compare the mean of SGPT between ETR and SVR groups.A total of 1043 patients agreed to participate in the study. Overall, female predominance was noted in 699 (67%) females  between mean serum glutamic pyruvic transaminase in SVR who achieved (56.29±47.03) and not achieved (56.41±47.06). Similarly, No significant difference was observed  between mean SGPT in SVR who achieved (56.41±47.06) and not achieved (45.33±2.89). Logistic regression and chi-square test results show no significant association between sex and SVR (p=0.669). Similarly, no significant association was observed between sex and ETR (p=0.598)  and sustained virological response in 248 (95.8%) patients when given a combination of sofosbuvir plus daclatasvir in genotype 3 patients . AnotherA study conducted by Butt et al. showed ETR was achieved in 196 98%) and SVR in 186 (93%) chronic hepatitis C (naïve) patients, respectively, who received sofosbuvir and daclatasvir . Another% and SVRSeveral factors, including bioequivalence of generics vs branded medications, compliance, study population, and probable underlying drug resistance, may be responsible for this disparity in outcomes . ResearcKwo et al. also showed that sofosbuvir/daclatasvir therapy, with or without ribavirin, had high SVR12 rates (84%) and was usually well-tolerated in this patient population . FurtherIn this study, ALT remained the same for patients who attained ETR and SVR but an increase in ALT levels was observed who did not achieve ETR and SVR from 45.33±2.89 to 61.00±0 but the association was insignificant, but a study done by El Raziky et al. and El Kassas showed a decrease in ALT levels with significant association ,23.In this study, 344 (100%) male and 695 (99.9%) female patients achieved SVR, while 343 (99.7%) males and 695 (99.6%) females achieved ETR, however, there was no significant association was observed between gender and status of SVR and ETR. The results are similar to the study by El Raziky et al.  and MushIn Pakistan, mainly from Punjab province, there have been various studies, reporting the efficacy of sofosbuvir-based therapies in HCV genotype 3-infected patients. It has been found that sofosbuvir-based dual or triple therapy has so far shown to be very effective in genotype 3 patients, with SVR after 24 weeks of treatment found to be 82.2-99.34% . HoweverIn this study, even with our effective study design, the study sample size was restricted to only the two tertiary care hospitals in the city of Karachi perhaps excluding a substantial population in other infirmaries and catchment areas. In the future, randomized control trials for longer follow-ups or a higher dose of treatment are needed before recommending it in our population due to its unseemly effects, and financial and observance-related implications.Daclatasvir and sofosbuvir is a highly efficient combination in individuals with chronic hepatitis C genotype 3 infection in Pakistan, with SVR12 of about 99.6% in genotype 3 patients. These non-proprietary medicines will be critical in eradicating hepatitis C infection in the underdeveloped globe. However, further investigation with a larger sample size, and longer follow-up involving a multicenter site is required.


Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women. This study was designed to investigate the associations of vitamin D receptor (VDR) gene variants with PCOS risk and the severity of the disease phenotype among Egyptian women.Man allelic discrimination real-time polymerase chain reaction.In this study, 185 women with PCOS and 207 fertile women as controls were recruited. Cases were divided into phenotype groups based on their clinical and paraclinical features. Clinical and laboratory data were measured in the patient and control groups. All individuals were genotyped for nine single-nucleotide polymorphisms (SNPs) located across the VDR gene using TaqP ≤ 0.001) higher body mass index (BMI) (22.77 ± 2.5) than controls (21.68 ± 1.85 kg/m2). Women with PCOS had significantly higher anti-Mullerian hormone, prolactin, luteinizing hormone (LH), LH/follicle-stimulating hormone (FSH), free testosterone, total testosterone, and dehydroepiandrosterone sulfate levels than the control group (P ≤ 0.001). The level of FSH was significantly lower in women with PCOS than in the control group (P ≤ 0.001). Analysis of the VDR rs4516035, rs2107301, rs1544410 (BsmI), and rs731236 (TaqI) SNPs showed a significant association with PCOS phenotype A. Furthermore, rs2228570 (FokI), rs3782905, rs7975232 (ApaI), and rs739837 SNPs showed a significant association with PCOS phenotype C. Furthermore, rs11568820 SNP showed a significant association with PCOS phenotype D (P < 0.05).Women with PCOS were significantly (The findings of this study indicate that variations in the VDR gene were associated with an increased risk of PCOS in Egyptian women. Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder that affects 6-12% of women of reproductive age worldwide . PCOS woA significant potential gene for PCOS is the vitamin D receptor (VDR) gene, also known as the calcitriol receptor or NR1I1 . It is aApproximately, 200 single-nucleotide polymorphisms (SNPs) of the VDR gene were found to be involved . VitaminThis study was designed to examine the association of the nine functionally most relevant VDR SNPs rs11568820 (C/T), rs4516035 (C/T), rs3782905 (C/G), rs2228570 (FokI) (C/T), rs2107301 (C/T), rs1544410 (BsmI) (G/A), rs7975232 (ApaI) (A/C), rs731236 (TaqI) (T/C), and rs739837 (T/G) with PCOS risk and the severity of the disease phenotype among Egyptian women.This case-control study included a convenience sample of 392 women  recruited at the Obstetrics and Gynecology Department, Mansoura University Hospital, Egypt. Laboratory workup was performed at the Molecular Genetic Unit in Endemic Hepatogastroenterology and Infectious Diseases, Faculty of Medicine, Mansoura University, during the period from January 2014 to November 2016.The subjects were classified according to the Rotterdam consensus . PCOS was diagnosed according to the Rotterdam criteria [2 (m2), were reported to exclude obese women. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (TT), and free testosterone (FT) were detected by chemiluminescence immunization  with intra- and inter-assay variation coefficients of 10% during the early follicular phase of the cycle. Several recognized causes of androgen excess and ovulatory dysfunction were excluded. Serum cholesterol and triglycerides (TG) were evaluated in the fasting blood samples using precipitation and enzymatic methods.All participants were females of reproductive age who had not been pregnant and had not received hormone treatment for at least three months prior to the study. Age and body mass index (BMI), which is determined by dividing weight (kg) by heightTM2000/2000c, Thermo-Fisher Scientific, CA, USA).All individuals had their genomic DNA extracted from peripheral blood using a commercial Qiagen DNA isolation kit , following the manufacturer’s instructions. A 2% ethidium bromide-stained agarose gel was used to determine the integrity of the DNA using a NanoDrop spectrophotometer  was used to evaluate the fluorescence data files from each plate [The following 20 µL were used for the reaction: 10.0 µL Universal Master Mix II by TaqMan (2×), 1.0 µL Assay Mix for SNP Genotyping (20×), 1.0 µL template for DNA, and 8.0 µL of RNase free water. A thermal cycler was used to carry out real-time PCR genotyping . The subsequent cycles were utilized: the first step in denaturation at 95ºC for 10 min, then the denaturation stage for 40 cycles at 95ºC for 15 s, and the stage of annealing/extension at 60ºC for 1 min. Taqch plate . To confP < 0.05.Statistical analysis was performed on the data using the SPSS software program . Numbers and percentages were used to convey categorical data The categorical data were compared using the chi-square or Monte Carlo test if required. The Shapiro test was used to determine whether quantitative data were normal. The mean and SD were utilized as variables with a normal distribution. More than two groups were compared using one-way ANOVA with Bonferroni post hoc multiple comparisons. The non-parametric variables were described using the median and interquartile ranges. In contrast to the Mann-Whitney test, which was used to compare just two groups, the Kruskal-Wallis test was used to compare several groups. Each allele’s total genotyping was scored and totaled in each group. Allele carriage is the proportion of people who carry at least one variation of a certain allele. A given allele’s odds ratio (OR) and 95% confidence interval (CI) were computed, compared to the case in which the target allele is not carried, using the program Med Calc . Each SNP’s allele carriage variables were compared using chi-square and Fisher’s exact tests. The difference was thought to be significant if 2 vs. 21.68 ± 1.85 kg/m2) than the control group.The clinical characteristics of the PCOS and fertile groups and the PCOS phenotype groups, including PCOS phenotype, age, pre-pregnancy BMI, gravity, parity, and primary and secondary infertility data, are shown in Table P ≤ 0.001). Furthermore, women with PCOS had significantly higher levels of cholesterol, TG, and HbA1c than the controls (P ≤ 0.001). The level of FSH was significantly lower in women with PCOS than in the controls (P ≤ 0.001).Laboratory data of the fertile and PCOS groups and the PCOS phenotype groups, including anti-Mullerian hormone (AMH), prolactin, LH, FSH, LH/FSH, FT, TT, dehydroepiandrosterone sulfate (DHEA-s), cholesterol, TG, and HbA1c levels, are shown in Table The different genotypes of PCOS phenotypes and the fertile group of the VDR gene SNPs are shown in Table P > 0.05). The allele carriages and frequencies of the sub-phenotypes of PCOS are also shown in Table Furthermore, the minor allele frequencies of rs1544410 (BsmI) and rs7975232 (ApaI) located in intron 9 were 0.15 and 0.21 in the fertile group, whereas those in the PCOS group were 0.30 and 0.32, respectively. Meanwhile, the minor allele frequency of rs731236 (TaqI) located in exon 10 was 0.12 in the fertile group, whereas that in the PCOS group was 0.32. Finally, rs739837 SNP located in the UTR region was genotyped. The minor allele frequency was 0.13 in the fertile group, whereas that in the PCOS group was 0.28. The differences in minor allele carriage of different SNPs were analyzed in the PCOS and PCOS phenotype groups and compared with those in the fertile group using the autosomal dominant model. The total minor allele frequency ranged from 0.18 to 0.30, and the global minor allele frequency ranged from 0.18 to 0.49. The distribution of allele carriages was tested for equilibrium using the Hardy–Weinberg equation. The allele carriages of all SNPs in all groups were balanced (P < 0.0001) . The association of the T allele carriage of rs4516035 located in the promoter region was also higher among the PCOS group and phenotypes A and C than that in the control group.The risk of minor allele carriage of different SNPs was compared in different phenotypes and compared with that in the fertile group using a dominant model, which means that at least one copy of a minor allele is associated with the disease Table . The car01) Fig. . The samAlong the transcriptional region, a significant association of the T allele carriage of rs2228570 (FokI) located in the initial codon was observed among the PCOS group and phenotypes C and D compared with that in the control group. Furthermore, there was a significant association between the G allele carriage of rs3782905, which is located in intron 3, and the T allele carriage of rs2107301, which is located in intron 4, among the PCOS group and all PCOS phenotype groups. In intron 9, there was a significant association of the A allele carriage of rs1544410 (BsmI) among the PCOS group and all PCOS phenotype groups and the C allele carriage of rs7975232 (ApaI) among the PCOS group and phenotypes C and D only. Furthermore, there was a significant association between the C allele carriage of rs731236 (TaqI) located in exon 10 among the PCOS group and all PCOS phenotype groups. Finally, there was a significant association between the G allele carriage of rs739837 located in the UTR region among the PCOS group and all PCOS phenotype groups.P, probability < 0.05. The highlighted cells indicate the highest significant risk.SNP, single-nucleotide polymorphism; PCOS, polycystic ovary syndrome; OR, odds ratio; CI, confidence interval; A popular genetic approach for examining relationships between candidate genes and dichotomous disease features is the unrelated case-control design. The probed SNP may be in high linkage disequilibrium with a causal variant or the SNP itself may be a common variant that significantly affects the trait if the association is significant. Population stratification within unrelated case-control populations, however, is a potential issue that should be taken into account in studies that integrate data. A family-based case-control design is an alternate strategy to prevent spurious association , 19. HowPatients with PCOS present with alterations in sex hormone production and cholesterol and TG levels. The results showed a significant association of PCOS with BMI, AMH, prolactin, LH, FSH, FT, TT, and DHEA-s levels. When the hormone levels were compared, the level of LH was significantly higher in the PCOS group than in the control group, whereas FSH levels were significantly lower. However, the LH/FSH ratio was significantly higher in the PCOS group than in the control group. These results agree with those of many studies , 22.PCOS is a multigenic disease with several causes, where several genes interact with one another as well as with environmental conditions, impacting the syndrome’s development and symptoms. Numerous biological systems rely on vitamin D and It uses VDR to moderate its activities. VDR is found in a variety of tissues, including pancreatic beta cells, skin, parathyroid, pituitary gland, or reproductive tissue, participating in the regulation of several endocrines, metabolic or reproductive functions . SNPs inThe transcription factor rs11568820 controls the transcription of the VDR gene, mRNA, and protein level , 26. YamStatistics from a prior meta-analysis showed that VDR ApaI rs7975232) and VDR BsmI (rs1544410) polymorphisms are linked to PCOS susceptibility in the Asian population . Mahmoud and VDR In conclusion, this study suggests that VDR gene polymorphisms can be a good candidate for PCOS development among the Egyptian population. Further case-control studies on various ethnic populations with a larger sample size are needed to verify the current conclusions in the future.