Objective:To investigate the effect of the key glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) on the biological activity of hemangioma-derived endothelial cells (HemECs) .Methods:Totally, 4 proliferating infantile hemangioma (IH) tissues and 4 involuting IH tissues were collected. Primary HemECs were isolated from the proliferating IH tissues, and human umbilical vein endothelial cells (HUVECs) served as controls. Immunohistochemical study and Western blot analysis were performed to determine the expression of PFKFB3 in the IH tissues and HemECs, respectively. Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of PFK15 (a specific inhibitor of PFKFB3) at concentrations of 0 - 10 μmol/L on the proliferation of HemECs, and HemECs treated without PFKFB3 served as the control group. Some in vitro cultured HemECs were treated with 5 μmol/L PFK15, and served as a PFK15 intervention group, while HemECs treated without PFK15 served as a control group; then, the migratory ability of HemECs was assessed by Transwell assay, and the apoptosis level of HemECs was detected by flow cytometry. Comparisons between groups were performed by using t test or analysis of variance. Results:Immunohistochemical study showed that the positive rate of PFKFB3 was significantly higher in the proliferating IH tissues (74.34% ± 5.26%) than in the involuting IH tissues (41.46% ± 2.99%, t = 9.40, P < 0.001). Western blot analysis showed that the relative expression level of PFKFB3 was also significantly higher in HemECs (0.73 ± 0.05) than in HUVECs (0.45 ± 0.04, t = 8.50, P < 0.001). CCK8 assay revealed significantly decreased proliferative activity of HemECs in the 0.625-, 1.25-, 2.5-, 5-, and 10-μmol/L PFK15 groups compared with the control group (all P < 0.01). Compared with the control group, the PFK15 intervention group showed significantly decreased number of migratory HemECs (297 ± 15 vs. 422 ± 8, t = 12.59, P < 0.001), but significantly increased apoptosis rates of HemECs (6.69% ± 0.64% vs. 0.34% ± 0.07%, t = 17.07, P < 0.001) . Conclusion:The key glycolytic enzyme PFKFB3 was highly expressed in the proliferating IH tissues and HemECs, and the PFKFB3 inhibitor PFK15 could suppress the proliferation, migration, and increase the apoptosis of HemECs.

Objective:To analyze demographic and clinical characteristics of infantile hemangioma (IH) , and to explore related risk factors for IH.Methods:A multicenter case-control study was conducted. IH patients (case group) and healthy children (control group) were collected from West China Hospital of Sichuan University, West China Second University Hospital of Sichuan University and Yulin Community Central Hospital of Chengdu from October 2018 to December 2020. The data on patients′ demographic characteristics, and risk factors during their mothers′ pre-pregnancy, pregnancy and perinatal period were collected and retrospectively analyzed. Univariate and multivariate analyses were performed using binary logistic regression.Results:A total of 1 479 patients with IH and 1 086 healthy children were included in this study. There were 456 males and 1 023 females in the case group, with the age being 3.74 ± 2.82 months, and there were 359 males and 727 females in the control group, with the age being 3.95 ± 2.77 months. There was no significant difference in the gender ratio, age, ethnic composition, birth weight or birth height between the case group and control group (all P > 0.05) . IH lesions mostly affected the head and face (564 cases, 38.1%) , followed by the trunk (449 cases, 30.6%) and limbs (356 cases, 24.1%) . At the visit, 1 109 (75.0%) patients presented with proliferating IH, 1 059 (71.6%) with superficial IH, and 1 306 (88.3%) with focal IH. The IH lesion area ranged from 0.01 to 168.00 (6.24 ± 12.91) cm 2, and the segmental IH area ranged from 7.50 to 168.00 (32.17 ± 26.94) cm 2. Univariate logistic regression analysis showed some factors influencing the occurrence of IH (all P < 0.05) , including pre-pregnancy factors (delivery history and miscarriage history) , pregnancy factors (fetal distress, cord entanglement, history of threatened abortion, placenta previa, oligohydramnios, gestational hypothyroidism, gestational anemia, history of progesterone supplementation, history of thyroxine drug use, history of uterus myomas) , and perinatal factors (including fetal position, gestational weeks, premature rupture of membranes and preterm premature rupture of membranes) . Multivariate binary logistic regression adjusted analysis showed that fetal breech presentation, preterm birth, cord entanglement and history of thyroxine drug use during pregnancy did not influence the occurrence of IH (all P > 0.05) ; the delivery history was the strongest independent risk factor for IH (adjusted OR = 5.624, 95% CI: 4.275 to 7.398, P < 0.001) , and gestational hypothyroidism and history of uterus myomas were protective factors for IH. Conclusions:In this study, the average age of IH patients at visit was 4 months, skin lesions mostly occurred on the head and face, and most were superficial and focal in the proliferative stage. The occurrence and development of IH may be associated with placental diseases, hypoxia, maternal hormone levels during pregnancy, etc.

Objective:To investigate the effect of the glucose transporter 1 (Glut-1) inhibitor resveratrol on the activity of infantile hemangioma (IH) -derived endothelial cells (HemEC) .Methods:IH tissues were collected from 4 cases of proliferating IH and 4 cases of involuting IH, and immunohistochemical study was performed to determine the Glut-1 expression. Primary HemEC were extracted from 4 proliferating IH tissues, real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of Glut-1 in HemEC and human umbilical vein endothelial cells (HUVEC) , respectively. HemEC were cultured in vitro and treated with 0 (control group) , 50, 100, 200, 400 and 800 μmol/L resveratrol for 24 hours, respectively. Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative ability of HemEC in the above groups, and the 50% inhibitory concentration (IC50) was calculated. The migratory ability and apoptosis level of HemEC were assessed by Transwell assay and flow cytometry, respectively. Intergroup comparisons were performed using t test or analysis of variance, and multiple comparisons were performed using least significant difference- t test. Results:Immunohistochemical study showed that Glut-1 was expressed in vascular endothelial cells derived from both proliferating and involuting IH tissues, and the Glut-1 expression was abundant in the proliferating IH but markedly decreased in the involuting IH tissues. The mRNA and protein expression levels of Glut-1 were significantly higher in HemEC (1.793 ± 0.041, 1.959 ± 0.144, respectively) than in HUVEC (0.820 ± 0.073, 0.648 ± 0.046, t = 16.35, 12.28, respectively, both P < 0.001) . After the treatment with Glut-1 inhibitor resveratrol at different concentrations, the proliferative ability of HemEC significantly differed among the control group, 50-, 100-, 200-, 400- and 800-μmol/L resveratrol groups ( F = 1 043.00, P < 0.001) , and was significantly lower in all the resveratrol groups than in the control group (all P < 0.05) . The IC50 of resveratrol was calculated to be 150 μmol/L by using GraphPad Prism 8 software. Transwell assay and flow cytometry showed significantly decreased number of migratory HemEC but significantly increased apoptosis rate respectively in the 150 μmol/L resveratrol group (61 ± 5, 13.01% ± 0.45%, respectively) compared with the control group (150 ± 6, 3.93% ± 0.68%, t = 15.11, 19.34, respectively, both P < 0.001) . Conclusion:The key glycolytic enzyme Glut-1 was highly expressed in proliferating IH tissues and HemEC, and resveratrol could inhibit the proliferation and migration of HemEC, but promote their apoptosis.

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the last step key enzyme of the methylerythritol phosphate (MEP) pathway, synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which is important for regulation of isoprenoid biosynthesis. Here the full-length cDNA of, designated(GenBank Accession No. KJ933412.1), was isolated fromfor the first time. TwHDR has an open reading frame (ORF) of 1386 bp encoding 461 amino acids. TwHDR exhibits high homology with HDRs of other plants, with an N-terminal conserved domain and three conserved cysteine residues.cDNA was cloned into an expression vector and transformed into anmutant. Since loss-of-functionmutant is lethal, the result showed that transformation ofcDNA rescued themutant. This complementation assay suggests that thecDNA encodes a functional HDR enzyme. The expression ofwas induced by methyl-jasmonate (MJ) insuspension cells. The expression ofreached the highest level after 1 h of MJ treatment. These results indicate that we have identified a functional TwHDR enzyme, which may play a pivotal role in the biosynthesis of diterpenoid triptolide in.

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from().(GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by thecDNA was expressed and purified as a recombinant protein from() and showed SMT activity. The expression ofwas highly up-regulated incell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis ofand will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.