Tropane alkaloids (TAs) are a class of anticholinergic drugs widely used in clinical practice and mainly extracted from plant, among which Atopa belladonna is the main commercial drug source. It is of great industrial value to obtain TAs in large quantities by plant metabolic engineering. In TAs pathway, cytochrome oxidase CYP82M3 catalyze the synthesis of tropinone and then tropinone reductase I (TRI) compete with TRII for tropinone to form tropine leading to the TAs synthesis (drainage). In this study, based on the "increasing flow and drainage" metabolic engineering strategy, two genes, namely HnCYP82M3 and DsTRI from Hyoscyamus niger and Datura stramonium, respectively, were overexpressed in the hair roots of A. belladonna, with a view to promote the TAs accumulation. The HnCYP82M3 gene was cloned from the root of H. niger, and it encoded amino acid with 91.7% sequence identity with AbCYP82M3 from A. belladonna. Overexpression of HnCYP82M3 alone did not affect the content of TAs in hair roots of A. belladonna, indicating that CYP82M3 was not a key enzyme in TAs biosynthesis. Simultaneous overexpression of HnCYP82M3 and DsTRI greatly promoted the accumulation of the three TAs, and the contents of hyoscyamine, anisodamine and scopolamine were 4.97 times, 2.83 times and 2.19 times that of the control, respectively, and the increase amplitude was greater than that of single overexpression of DsTRI. This study showed that the "increasing flow and drainage" strategy of enzyme genes co-expression at branch points was a promising metabolic engineering method to effectively improve the biosynthesis of TAs in A. belladonna, and laid a theoretical and technical foundation for the large-scale industrial acquisition of TAs.

OBJECTIVE: Myocardial ischemia/reperfusion injury (MIRI) is an obstacle to the success of cardiac reperfusion therapy. This study explores whether luteolin can mitigate MIRI by regulating the p53 signaling pathway. METHODS: Model mice were subjected to a temporary surgical ligation of the left anterior descending coronary artery, and administered luteolin. The myocardial infarct size, myocardial enzyme levels, and cardiac function were measured. Latent targets and signaling pathways were screened using network pharmacology and molecular docking. Then, proteins related to the p53 signaling pathway, apoptosis and oxidative stress were measured. Hypoxia/reoxygenation (HR)-incubated HL1 cells were used to validate the effects of luteolin in vitro. In addition, a p53 agonist and an inhibitor were used to investigate the mechanism. RESULTS: Luteolin reduced the myocardial infarcted size and myocardial enzymes, and restored cardiac function in MIRI mice. Network pharmacology identified p53 as a hub target. The bioinformatic analyses showed that luteolin had anti-apoptotic and anti-oxidative properties. Additionally, luteolin halted the activation of p53, and prevented both apoptosis and oxidative stress in myocardial tissue in vivo. Furthermore, luteolin inhibited cell apoptosis, JC-1 monomer formation, and reactive oxygen species elevation in HR-incubated HL1 cells in vitro. Finally, the p53 agonist NSC319726 downregulated the protective attributes of luteolin in the MIRI mouse model, and both luteolin and the p53 inhibitor pifithrin-α demonstrated a similar therapeutic effect in the MIRI mice. CONCLUSION Luteolin effectively treats MIRI and may ameliorate myocardial damage by regulating apoptosis and oxidative stress through its targeting of the p53 signaling pathway. Please cite this article as: Zhai P, Ouyang XH, Yang ML, Lin L, Li JY, Li YM, Cheng X, Zhu R, Hu DS. Luteolin protects against myocardial ischemia/reperfusion injury by reducing oxidative stress and apoptosis through the p53 pathway. J Integr Med. 2024; 22(6): 652-664.
Luteolin/pharmacology* ; Animals ; Myocardial Reperfusion Injury/metabolism* ; Oxidative Stress/drug effects* ; Tumor Suppressor Protein p53/genetics* ; Apoptosis/drug effects* ; Mice ; Signal Transduction/drug effects* ; Male ; Disease Models, Animal ; Mice, Inbred C57BL ; Myocardial Infarction/prevention & control* ; Reactive Oxygen Species/metabolism*

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Branchial Region ; Branchioma/diagnosis* ; Humans ; Sleep ; Snoring

Estrogens/pharmacology* ; Humans ; Smell

Cell death occurs in various tissues and organs in the body. It is a physiological or pathological process that has different effects. It is of great significance in maintaining the morphological function of cells and clearing abnormal cells. Pyroptosis, apoptosis, and necrosis are all modes of cell death that have been studied extensively by many experts and scholars, including studies on their effects on the liver, kidney, the heart, other organs, and even the whole body. The heart, as the most important organ of the body, should be a particular focus. This review summarizes the mechanisms underlying the various cell death modes and the relationship between the various mechanisms and heart diseases. The current research status for heart therapy is discussed from the perspective of pathogenesis.
Apoptosis ; Autophagy ; Cardiovascular Diseases ; Humans ; Necrosis ; Pyroptosis

【Objective】 To explore the maximum tolerated dose(MTD) of pegylated liposome doxorubicin(PLD) when combined with cisplatin as a modified regimen for osteosarcoma. 【Methods】 A total of 14 patients with pathologically confirmed metastatic or unresectable osteosarcoma received one cycle of PLD combined with cisplatin therapy. The study used a traditional 3+3 design, with three PLD dose levels(40, 50, and 60 mg/ m², day 1). It was designed to recruit three patients initially at each dose level. Cisplatin was given at a dose of 100 mg/m²(administered within four days) for each patients. Patients received prophylactic granulocyte stimulating factor therapy 48 h after chemotherapy. Toxicities were documented according to the the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0(NCI CTCAE v4.0) . 【Results】 Of the 14 patients, 9 were male and 5 female, with a median age of 20 years(range 14~43). Both of the patients at dose level of 60 mg/m² experienced dose-limiting toxicity(DLT) (grade 3 oral mucositis and grade 4 neutropenic fever). Only 1 of the 9 patients at dose level of 50 mg/m² experienced DLT(grade 4 thrombocytopenia lasting for more than 3 days) and thus the MTD was 50 mg/m². Most common grade 3~4 adverse events across all cohorts included neutropenia(12 cases, 12/14), thrombocytopenia(7 cases, 7/14), anemia(4 cases, 4/14) and oral mucositis(2 cases, 2/14). All the adverse events were relieved after symptomatic and supportive treatment. No treatment-related death was observed. 【Conclusions】 For advanced osteosarcoma, when combined with cisplatin, the MTD of PLD was 50 mg/m². The main DLT was oral mucositis and neutropenic fever. The adverse events can be relieved after symptomatic treatment.

Objective To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus (E. granulosus). Methods We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1 (DNASTAR Inc., Madison, WI), and the phylogenetic analysis was performed by using MEGA5.1 (CEMI, Tempe, AZ, USA). In addition, linear and conformational epitopes were analysed, including secondary structure, NXT/S glycosylation, fibronectin type III (FnIII) domain and glycosylphosphatidylinositol anchor signal (GPI-anchor). The secondary structure was predicted by PSIPRED method. Results Our results indicated that most isolates overall shared 72.6–100% identity in EG95 gene sequence with the published standard EG95 sequence, X90928. However, EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates, respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine, X90928, and they belonged to Subgroup 1. However, in comparison to X90928, several amino acid mutations occurred in most isolates besides oncosphere, which potentially altered the immunodominant linear epitopes, glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates, and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Background:Reduced expression of tripartite motif-containing 3 (TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma (HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines (SK-Hep1,Hep3B,Huh7,HepG2,Bel-7402) and one normal liver cell line (L02) were detected with Western blotting.HepG2 and Bel-7402 cells with IowTRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3 (LV-TRIM3),whereas Huh7 and Hep3B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA (siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect of TRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed thatTRIM3 overexpression induced G0/G1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3B cells by siTRIM3 led to significantly decreased percentages of both cells in the G0/G1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed thatTRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G0/G1 phase.

Fourteen compounds were isolated from 95% ethanol extract by silica gel, MCI, and ODS column chromatography. These compounds were respectively identified as quercetin (1), kaempferol (2), (+)-catechin (3), fraxin (4), protocatechuic acid (5), gallic acid (6), methyl gallate (7), ethyl gallate (8), apocynol A (9), baccatin (10), cerevisterol (11), ellagic acid (12), 3, 3',4'-tri-0-methylellagic acid(13) and N-benzoyl-L-phenylalaninyl-N-benzoyl-L-phenylalaninate(14) by analyzing their spectral data and comparing with the previously reported literatures. Except for gallic acid (6), all other compounds were isolated from this plant for the first time. Compounds 1, 2 and 6 showed moderate anti-proliferation activities on tumor cells.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; chemistry ; toxicity ; Euphorbiaceae ; chemistry ; Humans ; Plants, Medicinal ; chemistry ; Spectrometry, Mass, Electrospray Ionization