Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.
Isatis/genetics* ; Plant Proteins/metabolism* ; Phylogeny ; Arabidopsis/genetics* ; Flavonoids ; Cloning, Molecular

The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca²⁺, Mn²⁺ and Co²⁺) showed inhibitory effect on the activity of UGT74L2, while Mg²⁺ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the K_m value was 29.84 μmol·L^-1, the k_cat was 0.02 s^-1, and the k_cat·K_m^-1 was 572.6 mol^-1·s^-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.

【Objective】 To explore the relationship between the storage time of leukodepleted red blood cells and transfusion adverse reactions by analyzing the occurrence of transfusion adverse reactions of patients after leukodepleted red blood cells transfusion from four hospitals. 【Methods】 By using the electronic medical record management system, the collection and transfusion dates of leukodepleted red blood cells from four hospitals in Enshi Prefecture from 2018 to 2022, as well as the information on transfusion adverse reactions, were retrieved. 【Results】 From 2018 to 2022, a total of 697 61 bags of leukodepleted red blood cells were transfused in four hospitals, resulting in 166 cases of transfusion adverse reactions, among which 93 were allergic reactions, 63 were non hemolytic febrile reactions, and 10 were others, with a total incidence rate of transfusion adverse reactions at 0.24%. The average storage time of leukodepleted red blood cells with and without transfusion adverse reactions was (20.25±6.31) and (19.88±5.50) days, respectively. With a storage time of 7 days as the threshold, the incidence of transfusion adverse reactions was the lowest for a storage time of 15~21 days. The incidence of transfusion adverse reactions of leukodepleted red blood cells in two groups (with storage days ≤21 days and >21 days) was not statistically significant(P>0.05). 【Conclusion】 Allergic reactions were the main type of transfusion adverse reaction caused by leukodepleted red blood cells, and the incidence of transfusion adverse reactions decreased and then increased with the prolongation of the storage time of leukodepleted red blood cells. There was no significant difference in the incidence of transfusion adverse reactions with leukodepleted red blood cells stored for ≤ 21 days and >21 days.

Hip fractures are among the most common fractures in the elderly, presenting to be a leading cause of disability and mortality. Surgical treatment is currently the main treatment method for hip fractures. The incidence of perioperative malnutrition is increased after hip fractures in the elderly due to the comorbidities, decreased basal metabolic rate, accelerated protein breakdown, weakened anabolism and surgical stress. However, malnutrition not only increases the incidence of postoperative complications, but also leads to increased mortality, indicating an important role of perioperative nursing management of nutrition for the elderly patients with hip fractures. At present, there still lacks scientific guidance and application standards on perioperative nursing management of nutrition for the elderly patients with hip fractures. Therefore, the Orthopedic Nursing Committee of Chinese Nursing Association and the Editorial Board of Chinese Journal of Trauma organized relevant experts to formulate the Expert consensus on perioperative nursing management of nutrition for elderly patients with hip fractures ( version 2023) according to evidence-based medical evidences and their clinical experiences. Fourteen recommendations were made from aspects of nutrition screening, nutrition assessment, nutrition diagnosis, nutrition intervention and nutrition monitoring to provide guidance for perioperative nursing management of nutrition in elderly patients with hip fractures.

Objective To evaluate the efficacy and safety of the new left ventricular circulation assist device iVAC 2L in high-risk percutaneous coronaryintervention(HR-PCI)in Chinese patients.Methods We reported 6 PCIs in 5 patients supported by iVAC 2L,a new left ventricular circulation assist device,performed in Macao from September 2022 to March 2023.All patients were assessed by heart team and categorize to be high-risk for procedure.Clinical and intra-procedural data were analyzed.iVAC 2L-related complications and 30-day results were also documented.Results Insertion and removement of iVAC 2L successfully performed in all the 5 patients.Three of them underwent complete revascularization in the index procedure;one failed for the first time due to intolerance of the prolonged procedure,but succeeded for the reattempt of complete revascularization a month later,with the support of iVAC 2L.PCI was abandoned due to poor vessel condition.iVAC 2L,the new left ventricular circulation assist device,supported effectively during the 6 procedures.The patients were stable during the procedure.The success rate of hemodynamic support was 100％.No iVAC 2L-related complications and 30-day major adverse cardiac and cerebral events occurred,the 30-day survival was 100％.Conclusions Initial experience suggested that the new left ventricular circulation assist device iVAC 2L could provide effective and safe support in high-risk PCI.

Objective: To examine the long term outcome of splenic hilar lymphadenectomy (SHL) for locally advanced Siewert type Ⅱ and Ⅲ adenocarcinoma of esophagogastric junction (AEG) with a tumor diameter ≥4 cm. Methods: A total of 489 locally advanced Siewert type Ⅱ and Ⅲ AEG patients with a tumor diameter ≥4 cm who underwent radical resection from January 2010 to April 2016 were included. There were 383 males and 106 females. There were 225 patients aged≥65 years and 264 patients aged <65 years. SHL was conducted in 270 patients(SHL group). Wilcoxon rank-sum test or χ² test were conducted for inter-group comparison. Cox proportional hazard regression was used to analyze the long term outcome of SHL and the prognosis factors of overall survival. Kaplan-Meier curve was drawn finally. The results of survival analysis were verified by Log-rank test. Results: Followed-up to April 2021,the median follow-up time was 78.0 months (range: 74.0 to 85.0 months), the follow-up rate was 95.5%(467/489). The splenic hilar lymphnode metastasis rate of the SHL group was 12.6% (34/270). Younger patients (<65 years old), less complications, higher proportion of patients received adjuvant chemotherapy were demonstrated in the SHL group (χ²: 5.644 to 6.744, all P<0.05). Multivariate analysis showed that SHL was the independent prognosis factor of overall survival for patients with Siewert type Ⅱ and Ⅲ AEG and a tumor diameter≥4 cm (HR=0.68, 95%CI: 0.52 to 0.88, P=0.004) along with preoperative CA19-9, pathological T stage, pathological N stage, adjuvant chemotherapy and postoperative complication. Further subgroup analysis demonstrated that the SHL group had better 5-year overall survival than non-SHL group (62.4% vs. 39.2%, χ²=17.983, P=0.006) in Siewert type Ⅲ AEG rather than in Siewert type Ⅱ AEG(57.3% vs. 53.7%, χ²=3.031, P=0.805). Conclusion: In experienced center, splenic hilar lymphadenectomy can improve the prognosis of Siewert type Ⅲ AEG with a tumor diameter ≥4 cm.
Adenocarcinoma/surgery* ; Aged ; Esophageal Neoplasms ; Esophagogastric Junction/surgery* ; Female ; Humans ; Lymph Node Excision/methods* ; Lymphatic Metastasis/pathology* ; Male ; Retrospective Studies ; Stomach Neoplasms/surgery* ; Survival Analysis

Precious Tibetan medicine formula is a characteristic type of medicine commonly used in the clinical treatment of central nervous system diseases. Through the summary of modern research on the precious Tibetan medicine formulas such as Ratnasampil, Ershiwuwei Zhenzhu Pills, Ershiwewei Shanhu Pills, and Ruyi Zhenbao Pills, it is found that they have obvious advantages in the treatment of stroke, Alzheimer's disease, epilepsy, angioneurotic headache, and vascular dementia. Modern pharmacological studies have shown that the mechanisms of precious Tibetan medicine formulas in improving central nervous system diseases are that they promote microcirculation of brain tissue, regulate the permeability of the blood-brain barrier, alleviate inflammation, relieve oxidative stress damage, and inhibit nerve cell apoptosis. This review summarizes the clinical and pharmacological studies on precious Tibetan medicine formulas in prevention and treatment of central nervous system diseases, aiming to provide a reference for future in-depth research and innovative discovery of Tibetan medicine against central nervous diseases.
Blood-Brain Barrier ; Brain ; Central Nervous System Diseases ; Humans ; Medicine, Tibetan Traditional ; Stroke/drug therapy*

This study aimed to investigate the effect of Tibetan medicine Ershiwuwei Songshi Pills(ESP) on the intestinal flora of non-alcoholic steatohepatitis(NASH) mice. Forty-eight male C57 BL/6 mice were randomly divided into the control group, model(methionine-choline-deficient, MCD) group, high-(0.8 g·kg~(-1)), medium-(0.4 g·kg~(-1)), and low-dose(0.2 g·kg~(-1)) ESP groups, and pioglitazone(PGZ, 10 mg·kg~(-1)) group, with eight mice in each group. Mice in the control group were fed with normal diet, while those in the remaining five groups with MCD diet for five weeks for inducing NASH. During modeling, they were gavaged with the corresponding drugs. The changes in body mass, daily water intake, and daily food intake were recorded. At the end of the experiment, the liver tissues were collected and stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by oil red O staining for observing fat accumulation in the liver. The levels of serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) and triglyceride(TG) in liver tissue were measured. The changes in intestinal flora of mice were determined using 16 S rRNA high-throughput sequencing technology. The results showed that compared with the model group, the high-, medium-and low-dose ESP groups and the PGZ group exhibited significantly lowered AST and ALT in serum and TG in liver tissues and alleviated hepatocellular steatosis and fat accumulation in the liver. As demonstrated by 16 S rRNA sequencing, the abundance index and diversity of intestinal flora decreased in the model group, while those increased in the ESP groups. Besides, the Firmicutes to Bacteroidetes ratio decreased at the phylum level. In the alteration of the composition of intestinal flora, ESP reduced the abundance of Erysipelotrichia and Faecalibaculum but increased the abundance of Desulfovibrionaceae, Rikenellaceae, Lachnospiraceae, and Ruminococcaceae. This study has revealed that ESP has a protective effect against NASH induced by MCD diet, which may be related to its regulation of the changes in intestinal flora, alteration of the composition of intestinal flora, and inhibition of the intestinal dysbiosis.
Animals ; Disease Models, Animal ; Gastrointestinal Microbiome ; Liver ; Male ; Medicine, Tibetan Traditional ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease/drug therapy*

A chronic cholestasis model was induced in mice by feeding a diet containing 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine(DDC). The effects of Ershiwuwei Songshi Pills(ESP) on endogenous metabolites in mice with chronic cholestasis were investigated by metabolomics analysis based on liquid chromatography-mass spectrometry(LC-MS). The results showed that ESP was effective in improving pathological injury and reducing serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), and total bile acid in the model mice. Meanwhile, 13 common differential metabolites were revealed in metabolomic screening between the model/control group and the model/ESP group, including uric acid, glycolaldehyde, kynurenine, flavin adenine dinucleotide, L-3-phenyllactic acid, I-urobilin, leukotriene D4(LTD4), taurocholic acid, trioxilin A3, D-inositol-1,4-diphosphate, PC [16:0/20:2(11Z,14Z)], PC[14:0/22:2(13Z,16Z)], and PC[20:4(5Z,8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)]. After ESP intervention, the levels of all 13 differential metabolites were significantly retraced, and pathway analysis showed that ESP achieved its therapeutic effect mainly by affecting arachidonic acid metabolism, glycerophospholipid metabolism, tryptophan metabolism, and primary bile acid biosynthesis. This study elucidated the mechanism of action of ESP against chronic cholestasis based on metabolites.
Animals ; Bile Acids and Salts ; Cholestasis/drug therapy* ; Chromatography, Liquid ; Medicine, Tibetan Traditional ; Metabolomics ; Mice

To study the protective effect of Ershiwuwei Zhenzhu Pills on ischemic stroke rats. Ninety 4-weeks-old SPF male SD rats were randomly divided into 6 groups(n=15):sham operation group, model group, nimodipine group(12 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills high-dose group(400 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills medium-dose group(200 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills low-dose group(100 mg·kg~(-1)).The permanent middle cerebral artery occlusion model(PMCAO) was established in the model group, nimodipine group, and Ershiwuwei Zhenzhu Pills groups by the improved thread plug method, while the sham operation group did not insert the thread plug.Nimodipine group and Ershiwuwei Zhenzhu Pills groups were given intragastric administration once a day for 24 days before the modeling operation, and once 1 hour before the modeling operation, while sham operation group and model group were given equal volumes of distilled water.The neuroethology of the surviving rats was measured; The volume of cerebral infarction in rats was measured by TTC method; The histopathology of rat brain was observed by HE method; The expression levels of tumor necrosis factor α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),malondialdehyde(MDA),superoxide dismutase(SOD) and catalase(CAT) in serum were detected by ELISA;The mRNA expressions of Notch 1,Jagged 1,Hes 1 and Bcl-2 in rat brain were detected by RT-PCR;Western blot was used to detect the expression levels of caspase-3 protein in rat brain; the expression levels of vascular endothelial growth factor(VEGF) and CD34 positive cells in rat brain were detected by immunofluorescence.The low, medium and high dose groups of Ershiwuwei Zhenzhu Pills and nimodipine group could significantly reduce the neurobehavioral score and cerebral infarction volume of rats with permanent middle cerebral artery occlusion, reduce the morphological changes of nerve cells, decrease the expression of TNF-α,IL-1β and IL-6 in rat serum, increase the activity of SOD and CAT,and reduce the level of MDA.Furthermore, the expression levels of Notch l, Jagged l, Hes l and Bcl-2 mRNA were significantly increased, and the expression level of caspase-3 protein was decreased.Meanwhile, the number of VEGF and CD34 positive cells increased in the treatment group.The differences were statistically significant. Ershiwuwei Zhenzhu Pills has a protective effect on ischemic stroke rats, and its mechanism may be related to anti-inflammation, anti-oxidation, promotion of nerve cell proliferation, inhibition nerve cell apoptosis and promotion of angiogenesis.
Animals ; Caspase 3/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Infarction, Middle Cerebral Artery/drug therapy* ; Interleukin-6/metabolism* ; Ischemic Stroke/drug therapy* ; Male ; Nimodipine/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Vascular Endothelial Growth Factor A/metabolism*