This study investigates the effect of glycyrrhetinic acid(GA) combined with doxorubicin(DOX) on apoptosis in HepG2 cells and its possible mechanisms. HepG2 cells were cultured in vitro, and cell viability was assessed using the cell counting kit-8(CCK-8) method. Flow cytometry was used to measure apoptosis levels in HepG2 cells. The cells were divided into the following groups: control group(0 μmol·L~(-1)), DOX group(2 μmol·L~(-1)), GA group(150 μmol·L~(-1)), and DOX + GA combination group(2 μmol·L~(-1) DOX + 150 μmol·L~(-1) GA), with treatments given for 24 hours. The colocalization level between the endoplasmic reticulum(ER) and mitochondria was assessed by colocalization fluorescence imaging. Fluorescence probes were used to measure the Ca~(2+) content in the ER and mitochondria. The qRT-PCR and Western blot were used to determine the mRNA and protein expression of sirtuin-3(SIRT3). Co-immunoprecipitation(CO-IP) was applied to investigate the interactions between voltage-dependent anion channel 1(VDAC1) and SIRT3, as well as between VDAC1, glucose-regulated protein 75(GRP75), and inositol 1,4,5-trisphosphate receptor(IP3R). The results showed that the combination of DOX and GA promoted apoptosis in HepG2 liver cancer cells. The colocalization level between the ER and mitochondria was significantly reduced, the Ca~(2+) content in the ER was significantly increased, and the Ca~(2+) content in the mitochondria was significantly decreased. The relative expression of VDAC1, GRP75, and IP3R was significantly reduced, and interactions between VDAC1, GRP75, and IP3R were observed. SIRT3 mRNA and protein expression levels were significantly increased, and an interaction between SIRT3 and VDAC1 was detected. The acetylation level of VDAC1 was significantly decreased. In conclusion, GA combined with DOX induces apoptosis in HepG2 cells by mediating the deacetylation of VDAC1 through SIRT3, weakening the interactions among VDAC1, GRP75, and IP3R. This regulates the formation of endoplasmic reticulum-mitochondrial membrane domains(ERMMDs), affects Ca~(2+) transport between the ER and mitochondria, and ultimately triggers cell apoptosis.
Humans ; Apoptosis/drug effects* ; Hep G2 Cells ; Glycyrrhetinic Acid/pharmacology* ; Doxorubicin/pharmacology* ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/physiopathology* ; Mitochondria/metabolism* ; Endoplasmic Reticulum/metabolism* ; Cell Survival/drug effects* ; Membrane Proteins/genetics*

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets

Persistent and maladaptive drug-related memories represent a key component in drug addiction. Converging evidence from both preclinical and clinical studies has demonstrated the potential efficacy of the memory reconsolidation updating procedure (MRUP), a non-pharmacological strategy intertwining two distinct memory processes: reconsolidation and extinction-alternatively termed "the memory retrieval-extinction procedure". This procedure presents a promising approach to attenuate, if not erase, entrenched drug memories and prevent relapse. The present review delineates the applications, molecular underpinnings, and operational boundaries of MRUP in the context of various forms of substance dependence. Furthermore, we critically examine the methodological limitations of MRUP, postulating potential refinement to optimize its therapeutic efficacy. In addition, we also look at the potential integration of MRUP and neurostimulation treatments in the domain of substance addiction. Overall, existing studies underscore the significant potential of MRUP, suggesting that interventions predicated on it could herald a promising avenue to enhance clinical outcomes in substance addiction therapy.
Humans ; Substance-Related Disorders/psychology* ; Memory Consolidation/physiology* ; Animals ; Extinction, Psychological/physiology*

Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P)H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.
Nicotiana/genetics* ; Photosynthesis/physiology* ; Malate Dehydrogenase/biosynthesis* ; Plant Leaves/genetics* ; Scenedesmus/enzymology* ; Carbon Cycle/genetics* ; Lipid Metabolism/genetics* ; Plants, Genetically Modified/metabolism*

Kidney ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) with a poor prognosis and high mortality rate. Recent studies have reported that chrysophanol may have a renal protective effect, but its specific impact and mechanism on IRI remain unclear. This study aimed to explore the effects and mechanisms of chrysophanol on AKI induced by IRI. By utilizing a unilateral kidney IRI mouse model, histopathological changes in the kidney, serum levels of creatinine and urea nitrogen, and protein expressions of apoptosis and mitophagy in kidney tissue were examined. Additionally, a hypoxia/reoxygenation (H/R) model of human kidney-2 (HK-2) cells was established to measure mitochondrial membrane potential levels and reactive oxygen species (ROS). Functional enrichment analysis was performed to screen relevant targets of chrysophanol and AKI, and to verify key targets and pathways. The animal experiments conducted in this study were ethically approved by the Experimental Animal Ethics Committee of Peking University (No. LA2021503). The findings indicate that the IRI group exhibited elevated levels of creatinine and urea nitrogen in serum, significant renal tissue damage, and increased expression of renal injury markers (KIM1), apoptosis-related proteins (cleaved-caspase 3, caspase 3, cytochrome C), and mitochondrial autophagy protein (PINK1) compared to the sham surgery group. Chrysophanol treatment ameliorated the aforementioned pathological changes in a dose-dependent manner in an IRI model. Additionally, it exhibited significant improvements in mitochondrial membrane potential and inhibition of ROS production in HK-2 cells subjected to H/R conditions. Through network pharmacological analysis, HSP90AA1 and PIK3R1 were identified as key targets primarily enriched in the phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) pathway. Real-time quantitative PCR (qPCR) validation confirmed that chrysophanol significantly decreased HSP90AA1 and PIK3R1 mRNA levels in HK-2 cells under H/R conditions, while also enhancing the protein expressions of p-PI3K, PI3K, p-Akt, and Akt. In conclusion, chrysophanol has the potential to enhance AKI by selectively modulating HSP90AA1 and PIK3R1, activating the PI3K/Akt pathway, decreasing apoptosis, regulating mitochondrial autophagy, enhancing mitochondrial membrane potential, and suppressing ROS production. These findings suggest that chrysophanol could serve as a promising therapeutic option for the treatment of AKI.

Purpose::To explore the effect of green channel for stroke patients on the treatment of severe aneurysmal subarachnoid hemorrhage.Methods::This is a retrospective case-control study. The clinical data of patients with severe aneurysmal subarachnoid hemorrhage admitted to the emergency department of our hospital from January 2015 to June 2022 were retrospectively analyzed. Patients diagnosed with subarachnoid hemorrhage, confirmed intracranial aneurysm by preoperative CT angiography or digital subtraction, graded Hunt-Hess grade III, IV, and V, < 72 h from the onset to the time of consultation received surgical treatment in our hospital were included in this study. Patients with serious underlying diseases, such as heart, liver, kidney diseases, or malignant tumors, traumatic subarachnoid hemorrhage, previous history of cerebral hemorrhage, and incomplete data were excluded. The control group included patients with severe aneurysmal subarachnoid hemorrhage admitted from January 2015 to December 2018 before the establishment of the green channel for stroke patients, and the observation group included patients with severe aneurysmal subarachnoid hemorrhage admitted from January 2019 to June 2022 after the establishment of the green channel. The control group received routine treatment in the emergency department; the observation group received improved treatment of green channel for stroke patients. Gender, age, Hunt-Hess grade on admission, modified Rankin scale (mRS) on admission, aneurysm location, aneurysm size and whether accompanied by intracerebral hemorrhage, the time from onset to emergency department, the time from emergency department to vascular diagnostic examination, the time from onset to surgery, the time from emergency department to surgery, the time from hospital admission to surgery, length of hospital stay, complications, treatment effect were analyzed and compared between the 2 groups. SPSS 23.0 software was utilized to conduct comparisons between the 2 groups. The t-test, Chi-square test, or Mann-Whitney U test was chosen based on the data type. Statistical significance was established when p < 0.05. Results::A total of 71 patients were included in this study, of whom 37 were in the control group and 34 were in the observation group. There were no statistical differences in age, gender, Hunt-Hess grade, mRS scores, aneurysm location, aneurysm size, intracerebral hemorrhage, the time from onset to emergency department, length of hospital stay, complications between the observation group and the control group (all p > 0.05). The time (min) from visit to vascular diagnostic test (60.50 vs. 120.00, p =0.027), the time (min) from onset to surgery (1792.00 vs. 2868.00, p =0.023), the time (min) from emergency department to surgery (1568.50 vs. 2778.00, p =0.016), the time (min) from hospital admission to surgery (1188.50 vs. 2708.00, p =0.043), all of them were shorter in the observation group than those in the control group. The relative values of admission and 7-day postoperative mRS scores and the relative values of admission and discharge mRS scores ≥ 2 were used as the criteria for determining better efficacy, and the treatment effect was better than that in the control group, and the differences were statistically significant (admission to 7 days postoperative mRS score ≥ 2, 17 (50.0 %) vs. 8 (21.6 %), p =0.012; admission to discharge mRS score ≥ 2, 19 (55.9 %) vs. 11 (29.7 %), p =0.026). Conclusion::The green channel for stroke patients with severe aneurysmal subarachnoid hemorrhage can effectively shorten the time from arrival at the emergency department to vascular diagnostic examination and the time from the emergency department to surgery, and achieve a better therapeutic effect, which is worth popularizing and applying.

Objective To explore the influence of intravenous injection of cis-atracurium during anesthesia induction on QT interval(QTc),hemodynamics and oxidative stress level in patients undergoing hysteroscopic surgery.Methods Patients undergoing hysteroscopic surgery were classified into control group and treatment group.The treatment group was given intravenous injection of 2 mg·kg-1 of propofol emulsion injection+3 μg·kg-1 of fentanyl+0.15 mg·kg-1 of cis-atracurium besylate for injection,while the control group was given intravenous injection of 2 mg·kg-1 of propofol emulsion injection+3 μg·kg-1 of fentanyl+the same amount of 0.9％NaCl as treatment group.Laryngeal mask was placed in the two groups after 3 min,and laryngeal mask ventilation was given for general anesthesia.QTc value and hemodynamics[mean arterial pressure(MAP)]were detected before entering the room(T0),after 1 min of propofol+fentanyl intravenous injection(T1),after 1 min of cis-atracurium besylate for injection/0.9％NaCl(T2),immediately after laryngeal mask insertion(T3)and after 3 min of laryngeal mask insertion(T4).The anesthetic effect,postoperative dynamic pain visual analogue scale(VAS)score and sore throat were counted.The levels of serum oxidative stress indicators[superoxide dismutase(SOD),malondialdehyde(MDA)]at T0 and at the end of surgery(T5)were detected,and the safety was assessed.Results Fifty-two cases in treatment group and forty-four cases in control group were included.There was no statistical difference in QTc value between treatment group and control group at T0-T4(all P＞0.05).The MAP values in treatment group and control group at T3 were(84.22±5.96)and(86.78±6.11)mmHg,respectively,with a statistical difference(P＜0.05).The laryngeal mask insertion times in treatment group and control group were(17.02±2.47)and(19.52±2.34)s;the spontaneous breathing recovery times were(8.51±1.84)and(6.18±1.15)min;the eye opening time were(10.49±2.42)and(9.53±2.17)min;the laryngeal mask removal time were(11.16±2.16)and(10.21±2.34)min;the incidence rates of postoperative sore throat were 13.46％and 31.82％,and the differences were statistically significant(all P＜0.05).The levels of SOD in treatment group and control group at T5 were(36.63±4.17)and(33.26±4.86)nU·mL-1;the levels of MDA were(7.42±2.14)and(8.59±2.83)mmol·L-1(all P＜0.05).The adverse drug reactions in treatment group were mainly nausea and vomiting,respiratory depression and hypotension,and the adverse drug reactions in control group were mainly nausea and vomiting and respiratory depression.The incidence rates of adverse reactions in treatment group and control group were 13.46％and 11.36％,respectively(P＞0.05).Conclusion Cis-atracurium intravenous injection during anesthesia induction has no influence on QTc in patients undergoing hysteroscopic surgery,and has advantages in stabilizing intraoperative hemodynamics,reducing postoperative sore throat and improving serum oxidative stress indicators.

In industrial production,the expression level of drug proteins in Chinese hamster ovary cells(CHO)is influenced by many factors:the regulatory elements on transcription and translation,the ge-nomic integration sites,and the expression system.Transcription,as the first step of gene expression,largely affects protein expression,and the promoter plays a crucial role in the initiation of transcription.Most of the promoters were screened through transient transfection or random integration,but the presence of unclear copy number or random integration sites makes it difficult to accurately evaluate the promoter activity.To some extent,site-specific integration can reduce the impact of positional effects on exogenous genes and may potentially increase the expression level of exogenous genes.In the early stage of our re-search,multiple sites that can stably express exogenous proteins were identified and verified in the CHO cell genome.In this study,one of these sites(2c6)was selected for the evaluation of promoter activity.The CRISPR/Cas9 gene editing technique was used to site-specifically integrate the reporter gene(EG-FP)regulated by the simian virus early promoter(SV40),mouse elongation factor-1α(mEF-1α),chicken β-actin(cACTB)promoter,and human phosphoglycerate kinase promoter(hPGK)into the 2c6 site,respectively.The mean fluorescence intensity of the cells was analyzed by flow cytometry,and the mRNA level of EGFP was detected by qPCR to comprehensively evaluate the activity of the promoter.The results showed that the activities of the mEF-1α and mACTB promoters were better than those of SV40 and hPGK.The results of the secondary flow cytometry sorting showed that site-specific integration can more accurately evaluate the activity of the promoter in the CHO cell expression system.