BACKGROUND:Studies have shown that mitochondrial oxidative stress has an important role in the development of knee osteoarthritis,and Hemin can regulate the expression of mitochondria-related proteins. OBJECTIVE:To study the regulatory effect of Hemin on oxidative stress in mouse chondrocytes and its interventional effect and mechanism in knee osteoarthritis. METHODS:(1)In vitro cell experiment:Primary chondrocytes from C57BL/6 mice were extracted and induced with 10 ng/mL interleukin-1β to construct an in vitro chondrocyte model of osteoarthritis.The optimal concentration of Hemin(0,1,10,20,40,80,and 160 μmol/L)for the intervention in mouse chondrocytes was determined by cell counting kit-8 method.Chondrocytes were randomly divided into control group,model group(interleukin-1β)and Hemin group(interleukin-1β+Hemin).Reactive oxygen species,mitochondrial membrane potential and apoptosis of chondrocytes in each group were detected.(2)In vivo experiment:Adult C57BL/6 mice were randomly divided into normal group,model group(osteoarthritis)and Hemin group(osteoarthritis+Hemin),with eight mice in each group.After 4 weeks of Hemin treatment,the behavioral test and histopathological observation of the knee joint were performed in each group.Changes in extracellular matrix-related protein expression and apoptosis in chondrocytes and the expression level of Nrf2/HO-1 protein in cartilage tissue were detected. RESULTS AND CONCLUSION:In vitro experiment:the optimal concentration of Hemin on primary chondrocytes was 40 μmol/L.Compared with the model group,the level of reactive oxygen species was significantly reduced,the mitochondrial membrane potential was significantly improved,and the apoptosis of chondrocytes was reduced in the hemin-treated interleukin-1β-induced chondrocytes.In vivo experiment:After 4 weeks of treatment,compared with the model group,the lower limb function of mice in the Hemin group was significantly improved,the histopathological score was significantly improved,and the apoptosis of knee chondrocytes was significantly reduced.All these findings indicate that Hemin can alleviate oxidative stress,restore mitochondrial function and reduce apoptosis in mouse chondrocytes induced by interleukin-1β.Hemin can improve extracellular matrix degradation,promote chondrocyte anabolism,reduce catabolism and reduce chondrocyte apoptosis in knee osteoarthritis.It may act by activating the chondrocyte Nrf2/HO-1 signaling pathway in the inflammatory environment.

AIM: To identify the primary active components and underlying mechanisms of Yijing Decoction(YJD)in treating early diabetic retinopathy(DR)based on liquid chromatography-mass spectrometry and network pharmacology.METHODS: Active components of YJD were characterized through LC-MS. Components with optimal ADME(absorption, distribution, metabolism, excretion)properties were selected as key bioactive candidates. Network pharmacology approaches were employed to predict YJD-DR therapeutic targets. Protein-protein interaction(PPI)networks, gene ontology(GO)enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were subsequently conducted to predict core targets and networks. Critical targets and pathways were experimentally validated through Western blot.RESULTS: Ten core therapeutic targets were identified, including TNF, Alb, EGFR, STAT3, PTGS2, ESR1, PPAR, MMP9, TLR4, and MAPK. YJD was related to cancer-related signaling, fluid shear stress and atherosclerosis, and neurodegenerative diseases, encompassing key biological processes such as inflammatory response regulation, programmed cell death activation, and enhanced cell migration. Furthermore, Western blot analysis confirmed that YJD significantly inhibited high glucose-induced phosphorylation of STAT3(P-STAT3/STAT3)and ERK(P-ERK/ERK)in rat retinal microvascular endothelial cells.CONCLUSION: This study revealed YJD's pharmacodynamical basis and its multi-component, multi-target, and multi-paths pharmacology. YJD exerts therapeutic effects on DR by coordinately regulating critical signaling pathways and alleviating intraocular inflammation, thus preserving retinal vascular endothelial cells, maintaining blood-retinal barrier integrity, and facilitating retinal neurovascular repair.

Objective:Study on the correlation between the active components of Salviea Miltiorrhizae Radix et Rhizoma screened by high-throughput sequencing and the regulation of lncRNA-mRNA in human lung adenocarcinoma A549 cells.Methods:A549 cells were cultured, and the IC 50 dose of cryptotanshinone and tanshinone ⅡA was confirmed according to the cell proliferation experiment. A549 cells were randomly divided into blank control group, cryptotanshinone group, and tanshinone IIA group using a random number table method. After 24 hours of intervention, the cell cycle was detected by flow cytometry. High-throughput sequencing technique was used to detect the expressions of lncRNA and mRNA in A549 cells in intervention group and non-intervention group. By analyzing the expression profiles of differential genes related to cryptotanshinone and tanshinone ⅡA, the obtained differential genes were analyzed by GO and KEGG. Results:The cell cycle results showed that the proportion of G0/G1 phase cells in cryptotanshinone and Tanshinone ⅡA was increased ( P<0.01), the proportion of S phase cells was decreased ( P<0.01), and the proportion of G2/M phase cells in cryptotanshinone was decreased ( P<0.01). The results of high-throughput screening showed that cryptotanshinone could up-regulate 4 698 lncRNA, down-regulate 1 557 lncRNA, up-regulate 4 810 mRNA and down-regulate 5 644 mRNA. Tanshinone ⅡA could up-regulate 1 348 lncRNA, down-regulate 1 299 lncRNA, up-regulate 4646 mRNA and down-regulate 4 741 mRNA. The function and pathway enrichment analysis of differential lncRNA and mRNA showed that the differentially expressed genes of cryptotanshinone and tanshinone ⅡA were mainly related to cell cycle, autophagy, AMPK signaling pathway, FoxO signaling pathway and EGFR signaling pathway. GAS5 may be one of the targets for the inhibitory effects of cryptotanshinone and tanshinone ⅡA. Conclusion:Cryptotanshinone and tanshinone ⅡA have certain inhibitory effects on A549 cells, and there are differentially expressed genes of lncRNA-mRNA, which are closely related to cell cycle and signal pathway.

AIM: To investigate the mechanism by which Yijing Decoction antagonist high glucose-induced damage to the basement membrane(BM)in an in vitro inner blood-retinal barrier(iBRB)model.METHODS:Rat retinal microvascular pericytes(RMPs)and endothelial cells(ECs)were isolated and cultured to establish an in vitro iBRB model. The cells were randomly divided into four groups: low glucose group(LG), high glucose group(HG), minocycline group(MG)and Yijing Decoction group(YG). The LG group received 25 mmol/L glucose, the HG group received 60 mmol/L glucose, the MG group received 60 mmol/L glucose + 10 μg/mL minocycline, and the YG group received 60 mmol/L glucose + 10% Yijing Decoction-containing serum. Incubation for each group were terminated after intervention for 12 h. Next, the Western blot analysis was performed to assess the protein expression of BM-related proteins, including collagen Ⅳ(CⅣ)and laminin(LN), as well as matrix metalloproteinase(MMPs)/tissue inhibitor of matrix metalloproteinases(TIMPs)such as MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2.RESULTS:Compared to the LG group, the protein expressions of CⅣ increased in the HG, MG, and YG groups, as did LN in the HG and MG groups(all P<0.05). Both Yijing Decoction and minocycline effectively inhibited the elevated expression of CⅣ and LN induced by high glucose, and the difference between the YG, MG, and HG groups was statistically significant(all P<0.05). Futhermore, compared to the LG group, the protein expressions of MMP-2, MMP-3, and MMP-9 increased in the HG, MG, and YG groups(all P<0.05). Yijing Decoction specifically attenuated the high glucose-induced increase in MMP-2, MMP-3 and MMP-9 protein expression, and there were statistically significant differences between the YG and HG group(all P<0.05). No significant difference were observed in the expressions of TIMP-1 and TIMP-2 among the LG, HG, MG, and YG groups(all P>0.05).CONCLUSION:Yijing Decoction can potentially intervene in DR by modulating the protein expression of MMP-2, MMP-3, MMP-9, CⅣ, and LN, suppressing high glucose-induced BM remodeling, and mitigating damage to iBRB.

Objective:To investigate the effectiveness and safety of the coaxial needle technique in percutaneous liver biopsy for patients with coagulation function abnormalities.Methods:Clinical data of 210 patients who underwent percutaneous liver biopsy using the coaxial needle technique under ultrasound guidance from December 2018 to May 2021 in 3 centers were collected. A retrospective analysis was conducted to compare the puncture success rate, number of samples obtained, pathology qualification rate, intraoperative and postoperative bleeding rates between the group with coagulation function abnormalities and the group with normal coagulation function.Results:After propensity score matching, there were 105 patients in each group, with a puncture success rate of 100% in both groups. The pathology qualification rate was 100% for all samples.Intraoperative bleeding occurred in 78 cases (74.3%, 78/105) in the coagulation function abnormalities group and in 64 cases (61.0%, 64/105) in the normal coagulation function group, with a statistically significant difference between the two groups ( P=0.006). Postoperative bleeding occurred in 3 cases (2.9%, 3/105) in the coagulation function abnormalities group and in 0 case in the normal coagulation function group, with no statistically significant difference between the two groups ( P=0.081). Conclusions:The use of the coaxial needle technique for percutaneous liver biopsy in patients with coagulation function abnormalities not only allows for obtaining an adequate tissue sample but also demonstrates good safety.

Objective Intracranial hemorrhage(ICH),the second most common subtype of stroke,exacerbates the disruption of the blood-brain barrier(BBB),leading to vasogenic edema,plasma protein extravasation,and infiltration of neurotoxic substances.The clearance capacity of the brain plays a crucial role in maintaining BBB homeostasis and facilitating patient recovery after hemorrhage.This study aimed to investigate the effect of circadian rhythms on BBB function,neuronal damage,and clearance capabilities. Methods The transwell model and hemoglobin were co-cultured to simulate the BBB environment after ICH.After intervention with different light groups,neuronal apoptosis was determined,glial phagocytosis was analyzed,the expression of endogenous clearing-related proteins aquaporin 4(AQP4)and low-density lipoprotein receptor-related protein 1(LRP1)was detected by western blotting and immunofluorescence dual standard method,and the expression of the tight junction protein occludin and melatonin receptor 1A(MTNR1A)was quantitatively analyzed. Results Circadian rhythms play a key role in maintaining the integrity of the BBB,reducing oxidative stress-induced neuronal damage,and improving microglial phagocytosis.Meanwhile,the expression of occludin and MTNR1A in neurovascular unit(NVU)co-cultured with hemoglobin improved the expression of AQP4 and LRP1,the key proteins in the NVU's endogenous brain clearance system. Conclusion Circadian rhythm(alternating black and white light)protects the NVU BBB function after ICH,promotes the expression of proteins related to the clearance of the hematoma,provides new evidence for the clinical treatment of patients recovering from ICH,and improves the circadian rhythm to promote brain metabolism and hematoma clearance.

Objective:To explore the involvement of miR-126 and the role of mammalian target of rapamycin (mTOR)/hypoxia-induced factor 1 α (HIF-1 α) pathway in regulating human umbilical cord mesenchymal stem cells (hUCMSCs) exosomes (Exo) on vascular endothelial growth factor (VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells (HRECs).Methods:The hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1% oxygen concentration. The cell model of high glucose and low oxygen was established. After modeling, divided HRECs into Exo group, phosphate buffer saline (PBS) group, PBS+anti-miR126 group, Exo+anti-miR126 group, PBS+anti-mTOR group, and PBS+anti-HIF-1 α group. High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo. PBS+anti-mTOR group, PBS+anti-HIF-1 α group: 500 nmol/L mTOR inhibitor ADZ2014, 25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h, and then co-culture with PBS after High-glucose and hypoxia-induced. PBS+anti-miR126 group, Exo+anti-miR126 group: miR-126 LNA power inhibitor probe was transfected with high glucose, and co-cultured with PBS and hUCMSC Exo 6 h after transfection. Real-time polymerase chain reaction (qPCR) measured miRNA-126 expression levels in PBS, and Exo groups for 0, 8, 16 and 24 h. After 24 h of co-culture, the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence, Western blot and qPCR, respectively. Western blot, qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups. The expression of VE GF-A, mTOR, and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR. Comparison between two groups was performed by t-test; one-way ANOVA was used for comparison between multiple groups. Results:At 0, 8, 16 and 24 h, the relative mRNA expression of miR-126 gradually increased in the Exo group ( F=95.900, P<0.05). Compared with the PBS group, The mTOR, HIF-1 α protein ( t=3.466, 6.804), mRNA in HRECs in the Exo group, VEGF-A mRNA expression ( t=8.642, 7.897, 6.099) were all downregulated, the difference was statistically significant ( P<0.05). The relative expression level of VEGF-A protein ( t=3.337, 7.380) and mRNA ( t=8.515, 10.400) was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group, differences were statistically significant ( P<0.05). The relative expression of VEGF-A, mTOR, and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group, the differences were all statistically significant ( t=4.664, 6.136, 6.247; P<0.05). Conclusions:miR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.

Objective:To investigate the value of multi-stage dynamic contrast enhanced MRI (DCE-MRI) histogram in predicting survival of patients undergoing surgical treatment of colorectal cancer (CRC).Methods:The retrospective cohort study was conducted. The clinico-pathological data of 81 patients with CRC who were admitted to the Jiuquan City People′s Hospital from January 2018 to February 2019 were collected. There were 47 males and 34 females, aged (62±6)years. All patients underwent routine MRI and DCE-MRI examination to extract relevant imaging parameters. Observation indicators: (1) treatment, imaging examination and follow-up; (2) imaging factors influencing postoperative disease-free survival of patients with CRC. Measurement data with normal distribution were represented as Mean± SD, and measurement data with skewed distribution were represented as M(range). Count data were described as absolute numbers. Univariate and multivariate analyses were conducted using the COX proportional risk model. Pearson correlation test was used to analyze and exclude factors with correlation in univariate analysis, and the multi-variate analysis was conducted on the rest of factors. The Kaplan-Meier method was used to calculate survival rates, and Log-Rank test was used for survival analysis. Results:(1) Treatment, imaging examination and follow-up. All 81 patients underwent preoperative MRI plain scan, enhanced imaging, and diffusion weighted imaging. After complete examination, all patients underwent radical resection of CRC and received postoperative chemotherapy using the FOLFOX regimen. All 81 patients were followed up for 42(range, 11-61)months after surgery. The 1-, 3-, 5-year overall survival rate of 81 patients after surgery was 98.8%, 96.3%, 93.8%, respectively. During the follow-up period, 56 of the 81 patients survived from disease-free and 25 patients had disease progressed. (2) Imaging factors influencing postoperative disease-free survival of patients with CRC. Results of multivariate analysis showed that the kurtosis value and skewness value of positive enhancement integral (PEI) were independent imaging factors influencing postoperative disease-free survival of patients of CRC ( odds ratio=1.840, 1.243, 95% confidence interval as 1.403-2.412, 1.020-1.516, P<0.05). Taking the median values of kurtosis value and skewness value of PEI as 4.864 and 5.042 for further analysis. The postoperative 5-year disease-free survival rate of patients with kurtosis value of PEI <4.864 and ≥4.864 was 89.7% and 10.3%, showing a significant difference between them ( χ2=31.265, P<0.05). The postoperative 5-year disease-free survival rate of patients with skewness value of PEI<5.042 and ≥5.042 was 63.4% and 36.6%, showing a significant difference between them ( χ2=8.164, P<0.05). Conclusions:The kurtosis value and skewness value of PEI in DCE-MRI are independent imaging factors influencing postoperative disease-free survival of patients of CRC. The DCE-MRI histogram can effectively evaluate the postoperative prognosis of patients of CRC.

Objective To investigate the role of nitric oxide synthase(NOS)in the regulation of myocardial ischemia-reperfusion(IR)injury in ovariectomized(OVX)rats.Methods A total of 132 female SD rats were subjected,and 48 of them were randomly divided into sham operation group,IR group,OVX group and combined group,with 12 in each group.In order to explore the role of endothelous NOS(eNOS)and inducible NOS(iNOS)in ovariectomization increasing myo-cardial IR injury,another 84 mice were divided into negative sham group,negative IR group,nega-tive combined group,eNOS+IR group,eNOS combined group,iNOS small interfering RNA(si-iNOS)+IR group and si-iNOS combined group,with 12 in each group.The mice of the corre-sponding groups were injected with adeno-associated virus(AAV)overexpressing eNOS or knoc-king down iNOS via tail vein before OVX modeling.Myocardial infarct size,serum levels of lac-tate dehydrogenase(LDH)and creatine phosphokinase isoenzyme(CK-MB),LVEF,LVFS,and expression levels of eNOS and iNOS in the myocardial tissues were measured.Results The com-bined group had significantly increased level of iNOS in myocardium,larger myocardial infarct size and elevated serum LDH and CK-MB levels,but decreased myocardial expression of eNOS and LVEF and LVFS values than the IR group(P＜0.05).When compared with the negative combined group,the myocardial infarct size and serum LDH and CK-MB levels were decreased[(23.51±3.22)％and(26.21±2.93)％vs(58.78±5.42)％,(176.31±15.48 and 169.52±17.12 vs 328.85±37.12 U/L,35.41±6.41 and 34.77±5.94 vs 88.73±9.14 U/L,P＜0.05],and the LVEF and LVFS values were increased[(41.31±3.12)％and(42.09±3.41)％vs(30.77± 2.15)％,(21.47±1.57)％and(21.32±1.42)％vs(15.92±1.33)％,P＜0.05]in the eNOS com-bined group and si-iNOS combined group.Conclusion The decrease of eNOS expression and in-crease of iNOS expression are related to the aggravation of myocardial IR injury in OVX rats.

Objective:To learn about the clustered regularly interspaced short palindromic repeats (CRISPR) genotyping of Yersinia pestis in Yushu Tibetan Autonomous Prefecture (Yushu for short), Qinghai Province, and to explore its genetic characteristics. Methods:In this study, 44 representative strains isolated from local natural plague focus in Yushu from 1963 to 2007 were selected as experimental objects to extract DNA. Primers targeting the three CRISPR loci (YPa, YPb, and YPc) were designed for PCR amplification. The amplified products were sequenced and analyzed to identify the CRISPR spacer, and to determine the CRISPR genotypes and clusters.Results:Twenty-three spacers including 14 of YPa, 6 of YPb and 3 of YPc were observed among 44 strains, of which 2 spacers (a106 and a107) were firstly identified. According to the spacer arrays, the strains were divided into 15 CRISPR genotypes and classified into 6 CRISPR clusters which were Cb4, Cc3', Ca7, Ca7', CaΔ5' and Ca35', respectively. Among them, Ca7 was the most epidemic dominant cluster (34 strains) in Yushu.Conclusion:The CRISPR loci of Yersinia pestis in Yushu have multiple genotypes, high genetic polymorphism, and complex population structure.