ObjectiveTo investigate the mechanism of ferroptosis mediated by long-chain acyl-CoA synthetase 4 （ACSL4） signalling pathway in rats with coronary heart disease with blood stasis syndrome and the intervention effect of Xuefu Zhuyutang. MethodsSPF male SD rats were randomly divided into normal group， sham-operation group， model group， trimetazidine group （5.4 mg·kg^-1）， low-， medium-， and high-dose group （3.51， 7.02，14.04 g·kg^-1） of Xuefu Zhuyutang. The coronary artery left anterior descending ligation method was used to prepare a model of coronary heart disease with blood stasis syndrome， and continuous treatment for 7 d was conducted， while the sham-operation group was only threaded and not ligated. The general macroscopic symptoms of the rats were observed， and indicators such as electrocardiogram， echocardiography， and blood rheology were detected. The pathological morphology of myocardial tissue was observed by hematoxylin-eosin （HE） staining， and the changes in mitochondria in myocardial tissue were observed by transmission electron microscopy. The level of iron deposition in myocardial tissue was observed by Prussian blue staining. The levels of 12-hydroxyeicosatetraenoic acid （12-HETE） and 15-HETE were detected in serum by enzyme-linked immunosorbent assay. A biochemical colourimetric assay was used to detect the levels of Fe²⁺， lipid peroxidation （LPO）， glutathione （GSH）， and T-GSH/glutathione disulfide （GSSG） in myocardial tissue. DCFH-DA fluorescence quantitative assay was employed to detect the levels of reactive oxygen species （ROS）. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was adopted to detect the protein and mRNA expressions of glutathione peroxidase 4 （GPX4）， ferritin heavy chain 1 （FTH1）， ACSL4， and ly-sophosphatidylcholine acyltransferase3 （LPCAT3） in myocardial tissue. ResultsCompared with those in the normal group， the rats in the model group were poor in general macroscopic symptoms. The electrocardiogram showed widened QRS wave amplitude and increased voltage， bow-back elevation of the ST segments， elevated T waves， J-point elevation， and accelerated heart rate. Echocardiography showed a significant reduction in left ventricular ejection fraction （LVEF） and left ventricular fraction shortening （LVFS） （P<0.01）. Blood rheology showed that the viscosity of the whole blood （low， medium， and high rate of shear） was significantly increased （P<0.01）. HE staining showed an abnormal structure of myocardial tissue. There was a large area of myocardial necrosis and inflammatory cell infiltration and a large number of connective tissue between myocardial fibers. Transmission electron microscopy showed that the mitochondria were severely atrophy or swelling. The cristae were reduced or even broken， and the matrix was flocculent or even vacuolated. Prussian blue staining showed that there were a large number of iron-containing particles， and the iron deposition was obvious. The content of 12-HETE and 15-HETE in the serum was significantly increased （P<0.01）. The content of Fe²⁺， LPO， and ROS in myocardial tissue was significantly increased （P<0.01）. The content of GSH was significantly decreased （P<0.01）， and T-GSH/GSSG was decreased （P<0.01）. The protein and mRNA expressions of GPX4 and FTH1 in myocardial tissue were both significantly decreased （P<0.05， P<0.01）， while those of ACSL4 and LPCAT3 increased significantly （P<0.01）. Compared with the model group， the general macroscopic symptoms and electrocardiogram results of rats in low-， medium- and high-dose groups of Xuefu Zhuyutang were alleviated， and the differences in LVEF/LVFS ratios were all significantly increased （P<0.05， P<0.01）. The differences in whole-blood viscosity （low， medium， and high rate of shear） were all significantly decreased （P<0.01）. The results of HE staining and transmission electron microscopy showed that the morphology， structure， and mitochondria of cardiomyocytes were improved. The content of 12-HETE and 15-HETE in serum was reduced to different degrees in low-， medium-， and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）. The content of Fe²⁺， LPO， and ROS was significantly reduced in the medium- and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）， and the content of GSH and T-GSH/GSSG was significantly increased （P<0.05， P<0.01）. The protein and mRNA expressions of GPX4 and FTH1 were significantly increased to varying degrees in the medium- and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）， and ACSL4 and LPCAT3 were decreased to different degrees in the low-， medium-， and high-dose groups of Xuefu Zhuyutang （P<0.05， P<0.01）. ConclusionXuefu Zhuyutang can regulate iron metabolism and anti-lipid oxidation reaction to mediate ferroptosis through the ACSL4 signalling pathway， thus exerting a protective effect on rats with coronary heart disease with blood stasis syndrome.

Objective To investigate the biological basis of disease and syndrome by studying the spectrum of myocardial tissue metabolites in the rat model of coronary heart disease with heart blood stasis syndrome.Methods SD rats were randomly divided into sham-operation group and model group.The left anterior descending coronary artery was ligated to prepare the rat model of coronary heart disease with heart blood stasis syndrome.The general condition was observed,and the tongue chromaticity,electrocardiogram,cardiac function were detected.HE staining and transmission electron microscopy were used to observe myocardial tissue morphology and ultrastructure.UPLC-MS technology was used to investigate the differential metabolites in rat myocardial tissue,and enrichment analysis was conducted on metabolic pathways.Results Compared with the sham-operation group,the tongue chromaticity R,G,B values of model group rats were significantly reduced(P<0.05),ECG heart rate and ST segment elevation amplitude significantly increased(P<0.05),LVEF and LVFS significantly decreased,and LVIDs and LVIDd significantly increased(P<0.05).Myocardial tissue pathology revealed that the structure was blurred,inflammatory cells infiltrated,mitochondria swelled,ruptured,and dissolved,and crista structure fracture decreased.A total of 29 potential biomarkers with significant differences between the sham-operation group and the model group were identified in metabolomics(7 upregulated and 22 downregulated),with the majority of 10 pathways enriched in thiamine metabolism,arginine biosynthesis,purine metabolism,aminoacyl-tRNA biosynthesis,alanine,aspartate and glutamate metabolism,pentose and glucuronate interconversions,glycolysis/gluconeogenesis,valine,leucine and isoleucine degradation,TCA cycle,pyruvate metabolism.Conclusion Ligation of the left anterior descending coronary artery can mimic the pathological process of coronary heart disease with blood stasis syndrome in a good way,and its pathological mechanism involves the disruption of multi-level metabolic networks such as glucose metabolism,mitochondrial energy metabolism,amino acid metabolism,protein biosynthesis,and purine metabolism.

In the case of cardiac dysfunction， energy metabolism changes and the metabolism of myocardial substrates is reconstructed， as manifested by variation in the selection and utilization of energy substrates such as fatty acids and glucose. Persistent metabolic disorders of substrates will decrease energy supply， thus resulting in the occurrence and development of heart failure. Metabolic remodeling of substrate is resulted from the decline of visceral function and the accumulation of pathological products. Deficient Qi stagnation is the core pathogenesis. Deficient Qi （heart Qi deficiency， insufficient energy） is the root cause， which exists in the whole disease course. Stagnation （phlegm， blood stasis， fluid， lipid toxic products， lactic acid， etc.） is the symptom， which evidences the aggravation of the disease. Deficient Qi and stagnation are intertwined and causal， which form a spiral vicious circle. The typical syndrome is excess resulted from deficiency and deficiency-excess in complexity. The treatment principle is reinforcing healthy Qi and tonifying deficiency， dredging and removing pathogen. At the early stage， the method of reinforcing healthy Qi and tonifying deficiency （benefiting Qi） should be used， and the method of dredging and removing pathogen （activating blood） can be applied according to the conditions of patients. At the middle and late stages， both reinforcing healthy Qi and tonifying deficiency （benefiting Qi and warming Yang） and dredging and removing pathogen （activating blood， resolving stasis， and excreting water） should be emphasized. Chinese medicine can be applied according to the pathogenesis， thereby promoting the utilization of fatty acids， glucose， and other substrates and reducing the accumulation of toxic products derived from metabolic remodeling of substrate. Thus， both the root cause and symptoms can be alleviated， further improving cardiac energy metabolism and heart function.

Objective:To evaluate the prognostic value of pretreatment systemic immune-inflammation index (SII) and lactate dehydrogenase (LDH) in non-metastatic nasopharyngeal carcinoma (NPC).Methods:We retrospectively collected the data of 839 patients with non-metastatic NPC from National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Sun Yat-sen University Cancer Center between January 2007 and October 2015. All patients received intensity modulated radiation based treatment. Optimal cutoff value of SII and LDH were determined by X-title software. The association between SII, LDH and clinical prognosis of non-metastatic NPC patients were analyzed. Kaplan-Meier method was used for survival analysis, and Log rank test was used for comparison of survival rates between groups. Propensity score matching (PSM) analysis was carried out to minimize the effects of confounding factors. The risk stratification model of prognosis by combining N stage, SII and LDH was constructed to compare the prognosis of patients in high risk group, middle risk group and low risk group, and the receiver operating characteristic (ROC) curve analysis was used to evaluate its prognostic value.Results:The optimal cutoff value of SII is 447.2×10 9/L for predicting the 5-year overall survival (OS) of NPC patients, and the best cutoff value of LDH is 198.9 U/L. The proportion of patients with stage T3-4 and stage III-IVB in high SII group was higher than that in low SII group ( P<0.001). Multivariate Cox regression analysis showed that N stage, SII and LDH were independent factors of OS, progression-free survival (PFS) and distant metastasis-free survival (DMFS) of NPC patients (N stage, HR=1.705, 95% CI: 1.247-2.332; HR=1.755, 95% CI: 1.342-2.295; HR=2.161, 95% CI: 1.515-3.082. SII, HR=1.525, 95% CI: 1.097-2.119; HR=1.518, 95% CI: 1.150-2.004; HR=1.837, 95% CI: 1.272-2.653. LDH, HR=2.041, 95% CI: 1.403-2.968; HR=1.725, 95% CI: 1.233-2.414; HR=2.492, 95% CI: 1.690-3.672, respectively). After PSM, SII was still an independent prognostic factor of OS, PFS and DMFS in NPC patients ( HR=1.52, 95% CI: 1.09-2.12; HR=1.52, 95% CI: 1.15-2.00; HR=1.82, 95% CI: 1.26-2.63, respectively). Combined with N 2-3 stage, SII (>447.2×10 9/L), and LDH (>198.9 U/L), patients were divided into high-(3 risk factors), intermediate- (2 risk factors) and low-risk (0-1 risk factors) groups. The 5-year OS rates of patients in low-, intermediate- and high-risk groups were 86.1%, 79.8% and 41.2% respectively, the 5-year PFS rates were 80.7%, 70.2% and 33.9% respectively, and the 5-year DMFS rates were 88.9%, 79.2% and 47.5% respectively. There were significant differences in OS, PFS and DMFS among these three groups ( P<0.001). Distant metastasis was the main failure pattern in low-, intermediate- and high-risk groups, and the highest rate of distant metastasis was 83.3% (15/31) in high-risk group. ROC curve of the risk stratification model for predicting 5-year OS of NPC patients is 0.610, which is higher than TNM stage (0.609), SII (0.574) and LDH (0.558). Conclusions:Pretreatment SII and LDH are significantly correlated with the prognosis of patients with non-metastatic NPC. The combination of SII, LDH and N stage can stratify the prognostic risk of NPC patients. The risk stratification model can enhance the accuracy of prognosis.

Objective:To evaluate the prognostic value of pretreatment systemic immune-inflammation index (SII) and lactate dehydrogenase (LDH) in non-metastatic nasopharyngeal carcinoma (NPC).Methods:We retrospectively collected the data of 839 patients with non-metastatic NPC from National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital and Sun Yat-sen University Cancer Center between January 2007 and October 2015. All patients received intensity modulated radiation based treatment. Optimal cutoff value of SII and LDH were determined by X-title software. The association between SII, LDH and clinical prognosis of non-metastatic NPC patients were analyzed. Kaplan-Meier method was used for survival analysis, and Log rank test was used for comparison of survival rates between groups. Propensity score matching (PSM) analysis was carried out to minimize the effects of confounding factors. The risk stratification model of prognosis by combining N stage, SII and LDH was constructed to compare the prognosis of patients in high risk group, middle risk group and low risk group, and the receiver operating characteristic (ROC) curve analysis was used to evaluate its prognostic value.Results:The optimal cutoff value of SII is 447.2×10 9/L for predicting the 5-year overall survival (OS) of NPC patients, and the best cutoff value of LDH is 198.9 U/L. The proportion of patients with stage T3-4 and stage III-IVB in high SII group was higher than that in low SII group ( P<0.001). Multivariate Cox regression analysis showed that N stage, SII and LDH were independent factors of OS, progression-free survival (PFS) and distant metastasis-free survival (DMFS) of NPC patients (N stage, HR=1.705, 95% CI: 1.247-2.332; HR=1.755, 95% CI: 1.342-2.295; HR=2.161, 95% CI: 1.515-3.082. SII, HR=1.525, 95% CI: 1.097-2.119; HR=1.518, 95% CI: 1.150-2.004; HR=1.837, 95% CI: 1.272-2.653. LDH, HR=2.041, 95% CI: 1.403-2.968; HR=1.725, 95% CI: 1.233-2.414; HR=2.492, 95% CI: 1.690-3.672, respectively). After PSM, SII was still an independent prognostic factor of OS, PFS and DMFS in NPC patients ( HR=1.52, 95% CI: 1.09-2.12; HR=1.52, 95% CI: 1.15-2.00; HR=1.82, 95% CI: 1.26-2.63, respectively). Combined with N 2-3 stage, SII (>447.2×10 9/L), and LDH (>198.9 U/L), patients were divided into high-(3 risk factors), intermediate- (2 risk factors) and low-risk (0-1 risk factors) groups. The 5-year OS rates of patients in low-, intermediate- and high-risk groups were 86.1%, 79.8% and 41.2% respectively, the 5-year PFS rates were 80.7%, 70.2% and 33.9% respectively, and the 5-year DMFS rates were 88.9%, 79.2% and 47.5% respectively. There were significant differences in OS, PFS and DMFS among these three groups ( P<0.001). Distant metastasis was the main failure pattern in low-, intermediate- and high-risk groups, and the highest rate of distant metastasis was 83.3% (15/31) in high-risk group. ROC curve of the risk stratification model for predicting 5-year OS of NPC patients is 0.610, which is higher than TNM stage (0.609), SII (0.574) and LDH (0.558). Conclusions:Pretreatment SII and LDH are significantly correlated with the prognosis of patients with non-metastatic NPC. The combination of SII, LDH and N stage can stratify the prognostic risk of NPC patients. The risk stratification model can enhance the accuracy of prognosis.

OBJECTIVE： To establish a method for the determination of related substances in rifabutin crude drug and capsules by HPLC. METHODS： HPLC method was adopted. The determination was performed on Agilent XDB-C8 column with mobile phase consisted of acetonitrile-0.1 mol/L potassium dihydrogen phosphate solution （pH 6.5±0.1） （50 ∶ 50， V/V） at the flow rate of 1.0 mL/min. The detection wavelength was set at 254 nm， the column temperature was 30 ℃， and sample size was 20 μL. The mobile phase was used as solvent to prepare the sample solution with a mass concentration of 1.0 mg/mL. The system suitability test was performed by using newly established method and current method （mass concentration of sample solution 0.5 mg/mL， C18 column） stated in quality standard of rifabutin crude drug and capsules. Related substance test was conducted for 6 batches of rifabutin crude drug and capsule （peak area normalization method）. RESULTS： The linear range of rifabutin was 0.8-16 μg/mL（r＝1.000 0）， RSDs of precision， reproducibility and stability tests （12 h） were all lower than 2.0％ （n＝6）； the limits of detection and quantification were 0.025 4， 0.085 2 μg/mL. In system suitability test， by using new method and current method， separation degree of rifabutin peak and pre-degradation product peak were 7.50 and 3.47. When 6 batches of samples were determined， the number of impurities detected by this method was 1-5 more than that by the current method， and the total amount of impurities was 0.19％-0.55％ higher. CONCLUSIONS： Established new method is well-separated and sensitive， and can be used for the determination of related substance in rifabutin crude drug and capsules， which helps the quality control of drugs.

OBJECTIVETo investigate the effects of angiotensin II type 1 receptor (AT1R) knockdown on the first-phase insulin secretion in isolated islets of db/db mice and explore the possible mechanisms.

METHODSIslets were isolated from db/db and db/m mice and the expression level of AT1R in the islets was assayed. A recombinant adenovirus containing siRNA targeting AT1R (Ad-siAT1R) and a recombinant adenovirus with nonspecific siRNA (Ad-siControl) were constructed to infect the isolated islets for 72 h. AT1R, GLUT-2, and GCK expressions in the islets were investigated and islet perifusion was performed to evaluate the kinetics of insulin release.

RESULTSThe expression level of AT1R in the isolated islets from db/db mice was twice that of islets from db/m mice. The islets treated with Ad-siAT1R showed significantly decreased AT1R mRNA and protein levels and significantly increased expression of GLUT-2 (by 190%) and GCK (by 121%) compared to those treated with Ad-siControl (P<0.05). In response to stimulation with 16.7 mmol/L glucose, the first-phase insulin secretion was impaired in both Ad-siControl group and mock infected group with the peak insulin levels only 1.8 times of the basal level; the first-phase insulin secretion was markedly improved in islets treated with Ad-siAT1R, with a peak insulin level reaching 2.8 times of the basal level.

CONCLUSIONSIn isolated islets of db/db mice, selective AT1R inhibition can restore the first phase insulin secretion by up-regulating GLUT-2 and GCK, which may be one of the potential mechanisms by which AT1R blockers improve insulin secretion function.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Diabetes Mellitus, Experimental ; Gene Knockdown Techniques ; Glucose ; Glucose Transporter Type 2 ; metabolism ; Insulin ; secretion ; Islets of Langerhans ; metabolism ; Mice ; Protein-Serine-Threonine Kinases ; metabolism ; RNA, Small Interfering ; pharmacology

Objective To investigate the effects of angiotensin II type 1 receptor (AT1R) knockdown on the first-phase insulin secretion in isolated islets of db/db mice and explore the possible mechanisms. Methods Islets were isolated from db/db and db/m mice and the expression level of AT1R in the islets was assayed. A recombinant adenovirus containing siRNA targeting AT1R (Ad-siAT1R) and a recombinant adenovirus with nonspecific siRNA (Ad-siControl) were constructed to infect the isolated islets for 72 h. AT1R, GLUT-2, and GCK expressions in the islets were investigated and islet perifusion was performed to evaluate the kinetics of insulin release. Results The expression level of AT1R in the isolated islets from db/db mice was twice that of islets from db/m mice. The islets treated with Ad-siAT1R showed significantly decreased AT1R mRNA and protein levels and significantly increased expression of GLUT-2 (by 190%) and GCK (by 121%) compared to those treated with Ad-siControl (P<0.05). In response to stimulation with 16.7 mmol/L glucose, the first-phase insulin secretion was impaired in both Ad-siControl group and mock infected group with the peak insulin levels only 1.8 times of the basal level; the first-phase insulin secretion was markedly improved in islets treated with Ad-siAT1R, with a peak insulin level reaching 2.8 times of the basal level. Conclusions In isolated islets of db/db mice, selective AT1R inhibition can restore the first phase insulin secretion by up-regulating GLUT-2 and GCK, which may be one of the potential mechanisms by which AT1R blockers improve insulin secretion function.

Objective To investigate the effects of angiotensin II type 1 receptor (AT1R) knockdown on the first-phase insulin secretion in isolated islets of db/db mice and explore the possible mechanisms. Methods Islets were isolated from db/db and db/m mice and the expression level of AT1R in the islets was assayed. A recombinant adenovirus containing siRNA targeting AT1R (Ad-siAT1R) and a recombinant adenovirus with nonspecific siRNA (Ad-siControl) were constructed to infect the isolated islets for 72 h. AT1R, GLUT-2, and GCK expressions in the islets were investigated and islet perifusion was performed to evaluate the kinetics of insulin release. Results The expression level of AT1R in the isolated islets from db/db mice was twice that of islets from db/m mice. The islets treated with Ad-siAT1R showed significantly decreased AT1R mRNA and protein levels and significantly increased expression of GLUT-2 (by 190%) and GCK (by 121%) compared to those treated with Ad-siControl (P<0.05). In response to stimulation with 16.7 mmol/L glucose, the first-phase insulin secretion was impaired in both Ad-siControl group and mock infected group with the peak insulin levels only 1.8 times of the basal level; the first-phase insulin secretion was markedly improved in islets treated with Ad-siAT1R, with a peak insulin level reaching 2.8 times of the basal level. Conclusions In isolated islets of db/db mice, selective AT1R inhibition can restore the first phase insulin secretion by up-regulating GLUT-2 and GCK, which may be one of the potential mechanisms by which AT1R blockers improve insulin secretion function.

Objective:To identify proteins associated with nasopharyngeal carcinoma (NPC) metastasis,and provide scientific basis for the prevention and cure of NPC.Methods:A two-dimensional gel electrophoresis and mass spectrometry were performed to screen for differential proteins between highly metastatic 5-8F and non-metastatic 6-10B NPC cell lines.Western blot was used to confirm the differential proteins.We used siRNA to inhibit the expression of differential protein nm23-H1 to determine the association of nm23-H1 with NPC in vitro invasive ability.Immunohistochemistry and statistics were used to evaluate the correlation of nm23-H1 expression with clinicopathological features and clinical outcomes in paraffin-embedded archival tissues including 93 cases of primary NPC and 20 cases of cervical lymphonode metastatic NPC (LMNPC).Results:A total of 15 differential proteins in the 2 cell lines were identified by a proteomic approach,and 3 differential proteins were selectively confirmed.Downregulation of nm23-H1 by siRNA significantly increased the in vitro invasive ability of 6-10B.Significant nm23-H1 downregulation was observed in LMNPC compared with primary NPC.nm23-H1 downregulation in primary NPC was positively correlated with lymphonode and distant metastasis,advanced clinical stage and recurrence.Survival curves showed that patients with nm23-H1 downregulation in primary NPC had a poor prognosis.Multivariate analysis confirmed that nm23-H1 expression level in primary NPC was an independent prognostic indicator.Conclusion:nm23-H1 behaves as a metastasis suppressor in NPC,and nm23-H1 downregulation is a biomarker for poor NPC prognosis.