OBJECTIVE To investigate the effects and potential mechanism of Qiangxin decoction on mitochondrion of rats with chronic heart failure （CHF）. METHODS The CHF model was established by ligating the left anterior descending branch of the coronary artery. Modeled rats were divided into model group， Qiangxin decoction low-dose and high-dose groups （12.25， 24.50 g/kg， calculated by crude drug）， and chemical medicine group （Sacubitril valsartan sodium tablets， 10.42 mg/kg）， with 10 rats in each group； control group was set up without treatment. Each group of rats was orally administered with the corresponding medication or normal saline twice a day for 28 consecutive days. After the last medication， the contents of N-terminal pro-brain natriuretic peptide （NT-proBNP） and adenosine triphosphate （ATP） in serum and phosphatidic acid （PA） and cardiolipin （CL） in myocardial tissue were all detected； the pathological damage and collagen fibrosis of rat myocardial tissue were observed； the apoptosis of myocardial cells was determined； the ultrastructure of myocardial tissue was observed； the protein expressions of mitofusin 1 （Mfn1）， Mfn2， optic atrophy protein 1 （OPA1） and dynamin-related protein 1 （Drp1） were all detected in myocardial tissue. RESULTS Compared with control group，the serum content of NT-proBNP， apoptotic rate of myocardial cells， and relative expressions of S-OPA1 and Drp1 proteins were all increased significantly； serum content of ATP，contents of PA and CL， and relative expressions of Mfn1， Mfn2 and L-OPA1 proteins were all significantly reduced （P＜0.05）. There were abnormal membrane tissue structure in various layers of myocardial tissue， degeneration and necrosis of myocardial cells， and severe fibrosis； the mitochondria were swollen， with reduced or absent cristae， and uneven matrix density. After intervention with Qiangxin decoction， the levels of the aforementioned quantitative indicators in serum and myocardial tissue of rats （excluding CL content in the Qiangxin decoction low- dose group） were significantly reversed （P＜0.05）； the pathological damage of myocardial tissue had significantly improved， fibrosis had significantly reduced， mitochondrial morphology tended to be normal， cristae had increased， and matrix density was uniform. CONCLUSIONS Qiangxin decoction can regulate myocardial mitochondrial function and structural integrity of CHF rats， thereby improving myocardial energy metabolism and antagonizing myocardial fibrosis， the mechanism of which may be associated with activating PA/Mfn/CL signaling pathway.

OBJECTIVE To investigate the effects and potential mechanism of Qiangxin decoction on mitochondrion of rats with chronic heart failure （CHF）. METHODS The CHF model was established by ligating the left anterior descending branch of the coronary artery. Modeled rats were divided into model group， Qiangxin decoction low-dose and high-dose groups （12.25， 24.50 g/kg， calculated by crude drug）， and chemical medicine group （Sacubitril valsartan sodium tablets， 10.42 mg/kg）， with 10 rats in each group； control group was set up without treatment. Each group of rats was orally administered with the corresponding medication or normal saline twice a day for 28 consecutive days. After the last medication， the contents of N-terminal pro-brain natriuretic peptide （NT-proBNP） and adenosine triphosphate （ATP） in serum and phosphatidic acid （PA） and cardiolipin （CL） in myocardial tissue were all detected； the pathological damage and collagen fibrosis of rat myocardial tissue were observed； the apoptosis of myocardial cells was determined； the ultrastructure of myocardial tissue was observed； the protein expressions of mitofusin 1 （Mfn1）， Mfn2， optic atrophy protein 1 （OPA1） and dynamin-related protein 1 （Drp1） were all detected in myocardial tissue. RESULTS Compared with control group，the serum content of NT-proBNP， apoptotic rate of myocardial cells， and relative expressions of S-OPA1 and Drp1 proteins were all increased significantly； serum content of ATP，contents of PA and CL， and relative expressions of Mfn1， Mfn2 and L-OPA1 proteins were all significantly reduced （P＜0.05）. There were abnormal membrane tissue structure in various layers of myocardial tissue， degeneration and necrosis of myocardial cells， and severe fibrosis； the mitochondria were swollen， with reduced or absent cristae， and uneven matrix density. After intervention with Qiangxin decoction， the levels of the aforementioned quantitative indicators in serum and myocardial tissue of rats （excluding CL content in the Qiangxin decoction low- dose group） were significantly reversed （P＜0.05）； the pathological damage of myocardial tissue had significantly improved， fibrosis had significantly reduced， mitochondrial morphology tended to be normal， cristae had increased， and matrix density was uniform. CONCLUSIONS Qiangxin decoction can regulate myocardial mitochondrial function and structural integrity of CHF rats， thereby improving myocardial energy metabolism and antagonizing myocardial fibrosis， the mechanism of which may be associated with activating PA/Mfn/CL signaling pathway.

Sishen Pills and its separated prescriptions are classic prescriptions of traditional Chinese medicine to treat intestinal diseases. In this study, a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry(HPLC-ESI-MS/MS) technology was used to identify the components of Sishen Pills, Ershen Pills, and Wuweizi Powder. The positive and negative ion sources of electrospray ionization were simultaneously collected by mass spectrometry. A total of 11 effective components were detected in Sishen Pills, with four effective components detected in Ershen Pills and eight effective components detected in Wuweizi Powder, respectively. To explore the effects of Sishen Pills and its separated prescriptions on the human intestinal flora, an in vitro anaerobic fermentation model was established, and the human intestinal flora was incubated with Sishen Pills, Ershen Pills, and Wuweizi Powder in vitro. The 16S rDNA sequencing technology was used to analyze the changes in the intestinal flora. The results showed that compared with the control group, Sishen Pills, and its separated prescriptions could decrease the intestinal flora abundance and increase the Shannon index after fermentation. The abundance of Bifidobacterium was significantly increased in the Sishen Pills and Ershen Pills groups. However, the abundance of Lactobacillus, Weissella, and Pediococcus was significantly increased in the Wuweizi Powder group. After fermentation for 12 h, the pH of the fermentation solution of three kinds of liquids with feces gradually decreased and was lower than that of the control group. The decreasing amplitude in the Wuweizi Powder group was the most obvious. The single-bacteria fermentation experiments further confirmed that Sishen Pills and Wuweizi Powder had inhibitory effects on Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, and the antibacterial activity of Wuweizi Powder was stronger than that of Sishen Pills. Both Sishen Pills and Ershen Pills could promote the growth of Lactobacillus brevis, and Ershen Pills could promote the growth of Bifidobacterium adolescentis. This study provided a more sufficient theoretical basis for the clinical application of Sishen Pills and its separated prescriptions.
Humans ; Gastrointestinal Microbiome/drug effects* ; Drugs, Chinese Herbal/chemistry* ; Fermentation/drug effects* ; Bacteria/drug effects* ; Chromatography, High Pressure Liquid ; Tandem Mass Spectrometry ; Intestines/microbiology*

Brasilicardin A, a diterpene glycoside isolated from pathogenic actinomycete Nocardia brasiliensis IFM 0406, has become a novel immunosuppressant candidate due to its significant immunosuppressive activity, low toxicity and unique mechanism of action. However, brasilicardin A and its analogues have become a research hotspot to the development of this promising immunosuppressant because of the low-yield production in the natural pathogenic producer and the synthetically challenging skeleton. According to the reported biosynthetic pathway of brasilicardin A, the function of involved diterpene synthase was analyzed by bioinformatics. Then the genes bra1-5 that synthesize the brasilicardin A skeleton were directionally amplified from the pathogenic strain N. brasiliensis IFM 0406, and heterologous expression was achieved successfully in Streptomyces albus R1. The compounds were isolated and purified by using various column chromatographies including silica gel column chromatography and semi-preparative HPLC. Six new brasilicardins were established and named brasilicardin H-M. The activity of brasilicardins was screened using lipopolysaccharide (LPS)-activated mouse primary macrophage inflammation model. Brasilicardin H-M exhibited good inhibitory activity on nitric oxide (NO) release with IC₅₀ values of 28.24 ± 3.70, 37.44 ± 2.00, 39.85 ± 4.02, 26.77 ± 4.40, 65.25 ± 1.48 and 15.24 ± 2.72 μmol·L^-1, respectively (indomethacin as the positive control with IC₅₀ value of 34.28 ± 4.10 μmol·L^-1). The results indicated that six compounds had potential anti-inflammatory activity. This study laid a foundation for the elucidation of the brasilicardin A biosynthetic pathway and evaluation of the structure-activity relationship as well as new drug developments.

Purpose::The incidence of heatstroke (HS) is not particularly high; however, once it occurs, the consequences are serious. It is reported that calcitonin gene-related peptide (CGRP) is protective against brain injury in HS rats, but detailed molecular mechanisms need to be further investigated. In this study, we further explored whether CGRP inhibited neuronal apoptosis in HS rats via protein kinase A (PKA)/p-cAMP response element-binding protein (p-CREB) pathway.Methods::We established a HS rat model in a pre-warmed artificial climate chamber with a temperature of (35.5 ± 0.5) °C and a relative humidity of 60% ± 5%. Heatstress was stopped once core body temperature reaches above 41 °C. A total of 25 rats were randomly divided into 5 groups with 5 animals each: control group, HS group, HS+CGRP group, HS+CGRP antagonist (CGRP8-37) group, and HS+CGRP+PKA/p-CREB pathway blocker (H89) group. A bolus injection of CGRP was administered to each rat in HS+CGRP group, CGRP8-37 (antagonist of CGRP) in HS+CGRP8-37 group, and CGRP with H89 in HS+CGRP+H89 group. Electroencephalograms were recorded and the serum concentration of S100B, neuron-specific enolase (NSE), neuron apoptosis, activated caspase-3 and CGRP expression, as well as pathological morphology of brain tissue were detected at 2 h, 6 h, and 24 h after HS in vivo. The expression of PKA, p-CREB, and Bcl-2 in rat neurons were also detected at 2 h after HS in vitro. Exogenous CGRP, CGRP8-37, or H89 were used to determine whether CGRP plays a protective role in brain injury via PKA/p-CREB pathway. The unpaired t-test was used between the 2 samples, and the mean ± SD was used for multiple samples. Double-tailed p < 0.05 was considered statistically significant. Results::Electroencephalogram showed significant alteration of θ (54.50 ± 11.51 vs. 31.30 ± 8.71, F = 6.790, p = 0.005) and α wave (16.60 ± 3.21 vs. 35.40 ± 11.28, F = 4.549, p = 0.020) in HS group compared to the control group 2 h after HS. The results of triphosphate gap terminal labeling (TUNEL) showed that the neuronal apoptosis of HS rats was increased in the cortex (9.67 ± 3.16 vs. 1.80 ± 1.10, F= 11.002, p = 0.001) and hippocampus (15.73 ± 8.92 vs. 2.00 ± 1.00, F = 4.089, p = 0.028), the expression of activated caspase-3 was increased in the cortex (61.76 ± 25.13 vs. 19.57 ± 17.88, F = 5.695, p = 0.009) and hippocampus (58.60 ± 23.30 vs. 17.80 ± 17.62, F = 4.628, p = 0.019); meanwhile the expression of serum NSE (5.77 ± 1.78 vs. 2.35 ± 0.56, F = 5.174, p = 0.013) and S100B (2.86 ± 0.69 vs. 1.35 ± 0.34, F= 10.982, p = 0.001) were increased significantly under HS. Exogenous CGRP decreased the concentrations of NSE and S100B, and activated the expression of caspase-3 (0.41 ± 0.09 vs. 0.23 ± 0.04, F = 32.387, p < 0.001) under HS; while CGRP8-37 increased NSE (3.99 ± 0.47 vs. 2.40 ± 0.50, F = 11.991, p = 0.000) and S100B (2.19 ± 0.43 vs. 1.42 ± 0.30, F = 4.078, p = 0.025), and activated the expression caspase-3 (0.79 ± 0.10 vs. 0.23 ± 0.04, F= 32.387, p < 0.001). For the cell experiment, CGRP increased Bcl-2 (2.01 ± 0.73 vs. 2.15 ± 0.74, F= 8.993, p < 0.001), PKA (0.88 ± 0.08 vs. 0.37 ± 0.14, F= 20.370, p < 0.001), and p-CREB (0.87 ± 0.13 vs. 0.29 ± 0.10, F= 16.759, p < 0.001) levels; while H89, a blocker of the PKA/p-CREB pathway reversed the expression. Conclusions::CGRP can protect against HS-induced neuron apoptosis via PKA/p-CREB pathway and reduce activation of caspase-3 by regulating Bcl-2. Thus CGRP may be a new target for the treatment of brain injury in HS.

Objective    To investigate the clinical efficacy of minimally invasive Ivor-Lewis esophagectomy (MIILE) with reverse-puncture anastomosis. Methods    Clinical data of the patients with lower esophageal carcinoma who underwent MIILE with reverse-puncture anastomosis in our department from May 2015 to December 2020 were collected. Modified MIILE consisted of several key steps: (1) pylorus fully dissociated; (2) making gastric tube under laparoscope; (3) dissection of esophagus and thoracic lymph nodes under artificial pneumothorax with single-lumen endotracheal tube intubation in semi-prone position; (4) left lung ventilation with bronchial blocker; (5) intrathoracic anastomosis with reverse-puncture anastomosis technique. Results     Finally 248 patients were collected, including 206 males and 42 females, with a mean age of 63.3±7.4 years. All 248 patients underwent MIILE with reverse-puncture anastomosis successfully. The mean operation time was 176±35 min and estimated blood loss was 110±70 mL. The mean number of lymph nodes harvested from each patient was 24±8. The rate of lymph node metastasis was 43.1% (107/248). The pulmonary complication rate was 13.7% (34/248), including 6 patients of acute respiratory distress syndrome. Among the 6 patients, 2 patients needed endotracheal intubation-assisted respiration. Postoperative hemorrhage was observed in 5 patients and 2 of them needed hemostasis under thoracoscopy. Thoracoscopic thoracic duct ligation was performed in 1 patient due to the type Ⅲ chylothorax. TypeⅡ anastomotic leakage was found in 3 patients and 1 of them died of acute respiratory distress syndrome. One patient of delayed broncho-gastric fistula was cured after secondary operation. Ten patients with type Ⅰ recurrent laryngeal nerve injury were cured after conservative treatment. All patients were followe up for at least 16 months. The median follow-up time was 44 months. The 3-year survival rate was 71.8%, and the 5-year survival rate was 57.8%. Conclusion    The optimized MIILE with reverse-puncture anastomosis for the treatment of lower esophageal cancer is safe and feasible, and the long-term survival is satisfactory.

Objective To explore the application efficacy and safety of oblique ultrasound-guided techniques in PICC puncture,in order to provide guidance and references for clinical application.Methods Through convenient sampling,654 patients from a tertiary A hospital in Zhejiang Province from March to December 2022 were selected as the study subjects.The random numbers were generated through Excel table functions and they were randomly grouped into 3 groups:A,B,and C.The ultrasound short axis method,long axis method,and oblique axis method were employed to guide PICC puncture catheterization,respectively.The success rate of PICC puncture,the number of subcutaneous adjustments of the puncture needle,puncture time,and the occurrence of puncture complications(such as hematoma,puncture of the posterior wall of blood vessels,accidental injury to arteries,and accidental injury to nerves)were recorded during the catheterization process in 3 groups.Results A total of 654 patients completed the study,including 215 in group A,219 in group B,and 220 in group C.The success rate of first-time puncture in the group C(86.36％)was higher than that in group A(73.95％)and group B(63.93％),and there was a statistically significant difference among 3 groups(P＜0.001).The subcutaneous adjustment frequency of the puncture needle was 1(1,1)in group C,1(1,2)in group A,and 1(1,2)in group B.The difference between 3 groups was statistically significant(P＜0.001);the puncture time of group C was shorter than that of group A and group B,and the difference was statistically significant(P＜0.001).There was a statistically significant difference in the puncture time between 3 groups(P＜0.017);the pairwise comparison of the number of subcutaneous needle adjustments and the success rate of a puncture between 3 groups showed that there was a statistical difference between group C and group A,and between group C and group B(P＜0.017),while there was no statistical difference between group A and group B(P＞0.017).There was statistical significance(P＜0.05)among 3 groups in terms of complications such as accidental nerve injury and puncture of the contralateral vascular wall by puncture needle,but there was no statistical significance in terms of accidental arterial injury and hematoma occurrence among 3 groups.Conclusion Compared with the short axis approach and the long axis approach,the ultrasound oblique axis approach guided PICC puncture has statistical differences in the success rate of a puncture and the incidence of puncture complications,etc.It is recommended to use the ultrasound oblique axis approach during PICC puncture.

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Electroacupuncture ; Phosphatidylinositol 3-Kinase/metabolism* ; Facial Nerve Injuries/therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Beclin-1 ; Glial Cell Line-Derived Neurotrophic Factor ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Mammals/metabolism*

Objective To establish a method for testing isolated mitral valve in vitro and quantitatively evaluate the effect of transcatheter edge-to-edge repair technology(TEER)on functional mitral regurgitation(FMR)(non-A2-P2 regurgitation).Methods In this study,an FMR(non-A2-P2 regurgitation)model was developed by dilating the annulus orifice and displacing the papillary muscle in isolated porcine mitral valve.The hydrodynamics characteristics of 6 valves were tested by a pulsatile flow testing system under different physiological and pathological conditions before and after TEER.Results The results show that the valve regurgitation improved from moderate-severe[regurgitant fraction(60.2±17.5)％]to mild-moderate[regurgitant fraction(34.7±12.0)％]by repair(P＜0.001).The EOA[(3.8±1.6)cm2 vs.(2.2±0.5)cm2,P＜0.001]and the forward cross valve pressure difference[(1.8±1.3)mmHg vs.(3.8± 1.8)mmHg,P＜0.001],which characterize the forward flow performance of the valve,were compared before and after repair,and the differences were statistically significant.At the same time,the repair caused valve stenosis(the effective orifice area decreased by 40％and the positive differential pressure increased by 110％),but the valves was still within the normal physiological range,and no iatrogenic stenosis was caused.Conclusions It can be seen that TEER has an effect on FMR.This study provides validation and evaluation methods in vitro for expanding indications and improving TEER,and reference for developing standards of transcatheter valve repair testing in vitro.