1.Analysis of chloroplast genomes from Salvia miltiorrhiza and its congeneric species
Jindong YANG ; Zhenxi FANG ; Chengyang NIE ; Ruibing CHEN ; Qing LI ; Lei ZHANG
Journal of Pharmaceutical Practice and Service 2025;43(6):275-282
Salvia miltiorrhiza Bunge (Lamiaceae) is a medicinal plant widely used in Traditional Chinese Medicine for treating cardiovascular and cerebrovascular diseases. Chloroplasts are double-membrane-bound, chlorophyll-containing organelles and responsible for photosynthesis in plant cells. The structural information of chloroplast genomes serves as the foundation for precise exogenous gene insertion, site selection, and chloroplast genome modification. In this study, a comprehensive analysis and comparison of 125 chloroplast genomes from S. miltiorrhiza and 76 congeneric species were conducted, focusing on sequence characteristics, codon usage bias, simple sequence repeats (SSRs), contraction/expansion of chloroplast genome boundaries, and phylogenetic relationships, which could provide a theoretical foundation for advancing chloroplast genetic engineering, genetic diversity analysis, molecular breeding, and species identification within the Salvia genus.
2.Clinical features and prognostic analysis of adrenal metastasis tumor
Xiaofeng GUAN ; Xing LUO ; Haiqi LIANG ; Chengyang LI ; Deyun LIU
Chinese Journal of Urology 2024;45(4):314-319
Objective:To discuss the clinical features and prognostic factors of adrenal metastases.Methods:The clinical data of 37 patients with adrenal metastases admitted to the First Affiliated Hospital of Guangxi Medical University from May 2015 to August 2022 were retrospectively analyzed. There were 30 males and 7 females. The median age was 54 (43, 62) years old, including 13 cases aged ≥60 years old and 24 cases aged < 60 years old. There were 22 cases of right metastasis, 12 cases of left metastasis and 3 cases of bilateral metastasis. The maximum diameter of metastatic carcinoma was <3 cm in 10 cases, 3-6 cm in 18 cases, and >6 cm in 9 cases. There were 20 cases of synchronous metastasis (diagnosed with adrenal metastasis at the same time as the primary tumor) and 17 cases of metachronous metastasis (found after the diagnosis of primary tumor). There were 20 cases of adrenal metastasis alone and 17 cases of adrenal metastasis combined with other distant metastasis. 16 cases underwent adrenalectomy, 15 cases underwent adrenalectomy combined with other organ resection, and 6 cases underwent ultrasound-guided needle biopsy. 18 patients were treated with surgery alone, and 19 patients were treated with combined treatment (surgery, chemotherapy, radiotherapy, immunotherapy, targeted therapy, interventional therapy and seed implantation). There were 12 cases of hepatocellular carcinoma, 8 cases of renal clear cell carcinoma, 5 cases of lung adenocarcinoma, 2 cases of colon adenocarcinoma, 2 cases of neuroblastoma, 1 case of renal pelvis carcinoma, 1 case of gastric adenocarcinoma, 1 case of cholangiocarcinoma, 1 case of renal papillary cell carcinoma, 1 case of colon adenosquamous carcinoma, 1 case of thymic small cell carcinoma, 1 case of endometrial carcinoma, and 1 case of diffuse large B-cell lymphoma. The Kaplan-Meier method was used to draw the survival curve, the log-rank test was used to analyze the relationship between the clinical characteristics of patients and overall survival (OS) and progression-free survival (PFS), and the Cox proportional hazards regression model was used to analyze the factors affecting OS.Results:The median follow-up time of the 37 patients was 10 (4, 18.5) months.The overall survival rates at 6 months, 1 year and 2 years were 59.5% (22/37), 43.2% (16/37) and 32.4% (12/37), respectively. The 6-month, 1-year and 2-year overall survival rates of 18 patients who underwent surgery alone were 66.7% (12/18), 44.4% (8/18) and 27.8% (5/18), respectively. The 6 months, 1 year and 2 years overall survival rates of 19 patients with comprehensive treatment were 52.6% (10/19), 42.1% (8/19) and 36.8% (7/19), respectively. There was no significant difference in overall survival rate between the two groups ( P=0.773). Kaplan-Meier survival curve analysis showed that the side of metastatic cancer was a risk factor for OS ( P=0.012) and PFS ( P=0.013), and the time of diagnosis of metastatic cancer was a risk factor for OS ( P=0.021). Univariate Cox regression analysis showed that, time to diagnosis of metastases ( HR=2.5, 95% CI 1.1-5.4, P=0.021), side of metastases ( HR=3.6, 95% CI 1.5-8.5, P=0.004), pulmonary metastases ( HR=3.6, 95% CI 1.1-11.0, P=0.032) and adenocarcinoma of the primary tumor ( HR=3.2, 95% CI 1.2-8.8, P=0.025) were risk factors for OS. Multivariate Cox regression analysis showed that simultaneous presence of metastases ( HR=2.6, 95% CI 1.1-6.2, P=0.033) and metastases located on the left side ( HR=3.6, 95% CI 1.5-8.6, P=0.005) were independent risk factors for OS. Conclusions:Hepatocellular carcinoma is the most common pathological type of adrenal metastases in this study. Patients may benefit from combined therapy based on surgery. The time of diagnosis, side, tumor origin and pathological type of primary tumor are the prognostic factors of adrenal metastases. Simultaneous presence of metastases and left-sided metastases are independent risk factors for OS in patients with adrenal metastases.
3.Research progress of choroidal neovascularization secondary to central serous chorioretinopathy
Chengyang SUN ; Tong LI ; Xiaodong SUN
Chinese Journal of Experimental Ophthalmology 2024;42(8):763-767
Central serous chorioretinopathy (CSC) is a self-limiting disease characterized by serous detachment of the neurosensory retina affecting the macular area.Choroidal neovascularization (CNV) is a common complication of CSC which would lead to a poor visual prognosis.Recent studies have shown that disease duration is a main important risk factor for CNV secondary to CSC.Although the mechanism is not fully understood, it may involve various pathological mechanisms including choroidal ischemia and hypoxia and impaired retinal barrier function.Multimodal imaging techniques can improve the detection rate of CNV secondary to CSC, while optical coherence tomography showing hyperreflective flat irregular pigment epithelial detachment requires high vigilance for the possibility of secondary CNV.The utilization of photodynamic therapy and anti-vascular endothelial growth factor drugs improves its visual prognosis, but it still lacks consensus on treatment regimen.This article reviews the research progress in risk factors, pathogenesis, multimodal imaging for diagnosis and treatment of CNV secondary to CSC to help better understand the mechanism of CNV formation and optimize the clinical management of such situation.
4.Levels of serum VEGF and NO in patients with early diabetic retinopathy and its application value
Chunyan LI ; Jinjing FU ; Chengyang WANG
International Journal of Biomedical Engineering 2023;46(3):221-225
Objective:To detect the levels of vascular endothelial growth factor (VEGF) and nitric oxide (NO) in the serum of patients with early diabetic retinopathy (DR) and to explore their value in the treatment of DR.Methods:Fifty-nine patients with type 2 diabetes mellitus from March 2019 to June 2020 in the Yulin Second Hospital were collected and divided into the non-diabetic retinopathy (NDR) group (32 cases) and background stage diabetic retinopathy (BDR) group (27 cases) according to the DR grading criteria, and 29 healthy subjects were enrolled in the healthy control (HC) group. Serum VEGF levels were measured by a double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and serum NO levels were measured by the nitrate reductase method (one-step method). Pearson analysis was used to correlate serum VEGF with NO levels in each group, and a logistic regression model was used to analyze the risk factors for DR.Results:Compared with the HC group, the differences in serum VEGF and NO levels of patients in the BDR group were statistically significant (all P < 0.05). The serum VEGF and NO levels were positively correlated in all groups (all P < 0.01). Duration of diabetes, smoking, glycated hemoglobin A1c (HbA1c), serum VEGF, and NO levels were high risk factors for DR. Conclusions:Serum VEGF and NO levels may be potential pathological mechanisms for the development of microvascular complications in patients with type 2 diabetes, and the interaction may induce or accelerate the progression of DR. This study is expected to provide a new idea of complementary or combined treatment in the treatment of DR.
5.Influence of Mode of Delivery on Children’s Attention Deficit Hyperactivity Disorder and Childhood Intelligence
Min XU ; Xuemei YU ; Benjie FAN ; Guimei LI ; Xinxin JI
Psychiatry Investigation 2023;20(8):714-720
Objective:
To investigate whether differences exist in attention deficit hyperactivity disorder (ADHD) and intelligence between children born by cesarean delivery and those born by vaginal delivery.
Methods:
This retrospective study included singleton children that were born between January 2013 and December 2014. The Chinese version of the Conners’ Parent Rating Scale–Revised (CPRS-48) was required on the probability of psychological and behavioral problems. The China–Wechsler Intelligence Scale for Children (C-WIRS) was used for evaluation of crystallized intelligence and Raven’s Standard Progressive Matrices for evaluation of fluid intelligence.
Results:
A total of 10,568 valid questionnaires were obtained. CPRS-48 ADHD index and detection rate were higher in cesarean delivery group than those in vaginal delivery group. Cesarean delivery groups had a lower performance intelligence quotient score according to C-WISC.
Conclusion
Children born by cesarean delivery were more likely to have a risk of ADHD and a lower performance intelligence quotient compared with those born by vaginal delivery.
6.Adjustable "paper clip" techniques suturing the dorsal vein complex in radical prostatectomy
Yang CHEN ; Fangxing ZHANG ; Tianyu LI ; Chengyang LI ; Deyun LIU ; Haibiao YAN ; Zhanbin YANG ; Hua MI ; Linjian MO ; Naikai LIAO ; Shubo YANG ; Jiwen CHENG
Journal of Modern Urology 2023;28(8):707-712
【Objective】 To investigate the efficacy of the adjustable "paper clip" techniques in the suture of dorsal vein complex (DVC) and retention of urethral function in robot-assisted laparoscopic radical prostatectomy (RALRP). 【Methods】 A total of 30 cases of prostate cancer treated with RALRP were enrolled, all of which used the adjustable "paper clip" techniques. During operation, the DVC was sewed with barbed suture, and then a reverse suture was made through two sides of the prostatic ligaments. A Hem-o-lock was used to fasten the suture, which would be flexible to control the degree of tightness for the ligature. Perioperative and follow-up data of urinary continence and symptoms were collected and analyzed. 【Results】 All operations were successful. The estimated blood loss was (123.3±80.7) mL, 53.6% patients recovered continence in 1 month, and the continence rate increased to 92.9% and 96.3% at month 3 and 6. 92.9 of patients had no risk of incontinence 3 months after surgery. 【Conclusion】 The adjustable "paper clip" techniques have advantages in reducing blood loss, maintaining clear surgical field, preserving urethral function, and improving urinary continence.
7.Effect of L-carnitine on ferroptosis in renal tubular epithelial cells induced by calcium oxalate
Moran HUANG ; Jiawen ZHAO ; Chengyang LI
Chinese Journal of Urology 2023;44(4):292-300
Objective:To investigate the effect of L-carnitine on calcium oxalate-induced ferroptosis in renal tubular epithelial cells (HK-2).Methods:The effects of calcium oxalate(0, 2, 4 and 8 mmol/L) on the expression of ferroptosis-related protein long chain fatty acyl-CoA synthetase 4 (ACSL4), cystine/glutamate transporter(XCT) and glutathione peroxidase 4 (GPX4) in HK-2 cells were detected by Western blotting. The experiment was then divided into four groups: ①control group, cells were cultured in normal medium for 12 hours, then continued to use normal medium; ②L-carnitine group, cells were pretreated with medium containing 5mmol/L L-carnitine for 12 hours, then changed to medium containing 5mmol/L L-carnitine; ③calcium oxalate group, cells were cultured in normal medium for 12 hours, and then replaced with medium containing 4 mmol/L calcium oxalate; ④calcium oxalate+ L-carnitine group, the cells were pretreated with medium containing 5mmol/L L-carnitine for 12 h, and then replaced with 5mmol/L L-carnitine and 4mmol/L calcium oxalate medium. After changing the culture medium for 24 hours, the cells or supernatants were collected, and the expression levels of ferroptosis-related protein quinone oxidoreductase (NQO1), ACSL4, XCT and GPX4 were detected by Western blotting. The levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde were detected by corresponding kit, and the level of reactive oxygen species in cells was detected by reactive oxygen species kit.Results:The results of Western blotting showed that the expression of ACSL4 protein in 0, 2, 4, 8 mmol/L calcium oxalate was 0.37±0.16, 0.68±0.16, 0.73±0.09, 0.89±0.03 respectively. The expression of XCT protein was 1.11±0.10, 0.91±0.14, 0.83±0.09, 0.80±0.07, respectively. The expression of GPX4 protein was 1.23±0.13, 0.99±0.17, 0.81±0.05, 0.72±0.06, respectively. Compared with 0mmol/L group, the expression of ACSL4 protein increased and the expression of XCT and GPX4 decreased in 2, 4 and 8 mmol/L groups, and the difference was more significant between 4 mmol/L group and 0 mmol/L group. So 4 mmol/L was taken as the optimal concentration for follow-up experiment. The levels of NQO1 in control group, L-carnitine group, calcium oxalate group and calcium oxalate+ L-carnitine group were (0.36±0.06, 0.54±0.05, 0.76±0.07, 0.90±0.03) respectively. There was significant difference between L-carnitine group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of ACSL4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.66±0.10, 0.58±0.08, 0.99±0.03, 0.77±0.09) respectively. There was no significant difference between L-carnitine group and control group(P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of XCT in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (0.93±0.08, 0.85±0.07, 0.76±0.06, 0.99±0.05). There was no significant difference between L-carnitine group and control group (P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GPX4 in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (1.10±0.09, 1.09±0.09, 0.85±0.03, 0.99±0.02) respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of LDH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine were (100.00±5.37)%, (99.50±6.38)%, (153.77±6.06)% and (132.50±5.58)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The SOD levels in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±5.79)%, (105.80±3.26)%, (44.74±7.60)% and (85.01±5.15)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The levels of GSH in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±4.73)%, (107.10±5.48)%, (53.49±3.98)% and (85.18±5.48)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.01). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The levels of MDA in control group, L-carnitine group, calcium oxalate group and calcium oxalate + L-carnitine group were (100.00±2.36)%, (98.00±11.10)%, (129.11±2.59)% and (113.35±5.79)%, respectively. There was no significant difference between L-carnitine group and control group( P>0.05). There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). The fluorescence intensity of ferrous ion in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (39.77±0.68) AU, (68.40±3.14) AU and (48.60±4.30) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.05). The fluorescence intensity of reactive oxygen species in control group, calcium oxalate group and calcium oxalate + L-carnitine group was (63.98±9.41) AU, (145.41±8.39) AU and (85.37±4.51) AU, respectively. There was significant difference between calcium oxalate group and control group ( P<0.01). There was significant difference between calcium oxalate + L-carnitine group and calcium oxalate group ( P<0.01). Transmission electron microscopy results showed that mitochondria were wrinkled, cristae were broken or disappeared in the calcium oxalate group compared to the control group, and a double-layer membrane structure was evident. DAPI staining showed that compared with the control group, some of the nuclei in the calcium oxalate group were significantly more damaged, while compared with the calcium oxalate group, the nuclei in the calcium oxalate + L-carnitine were significantly less damaged. The results of crystal adhesion test showed that compared with the control group, calcium oxalate crystals in the calcium oxalate group adhered to the cells in black-like particles and formed clusters. Compared with the calcium oxalate group, the calcium oxalate + L-carnitine showed less black particles adhering to the cells. Conclusions:L-carnitine may reduce the effects of oxidative stress and ferroptosis induced by calcium oxalate, thus reducing cell damage and crystal adhesion.
8.Introduction to Health Research Priority Setting Methods and Application Recommendations of Methods in Field of Traditional Chinese Medicine
Chengyang JING ; Xue WU ; Le ZHANG ; Miaomiao LI ; Zeqi DAI ; Xing LIAO
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(6):167-175
Health research priority setting, based on the existing disease burden or healthcare needs, screens out specific areas or topics with relatively high research priority by scientific and systematic methods, and optimizes the allocation of health resources by influencing healthcare decision-making, so as to alleviate the imbalance between regional or global health and development. Many developed countries have carried out related research and practical work on different scales, and the World Health Organization (WHO) attaches great importance to the transformation and application of relevant achievements in developing countries. As the largest developing country in the world, China's research in this field started relatively late, and only a small number of scholars have carried out part of the localization methodology research and practice according to the specific national conditions. However, health research priority setting has not yet attracted the attention of large-scale research institutions or government organizations in China. Although the priority setting is rarely mentioned in the research on traditional Chinese medicine (TCM), the research and decision-making on the diseases responding specifically to TCM can also be regarded as the practical work of exploring the priority of TCM. Policymakers have a sense of priority support in the "priority of TCM research", but the decisions from the top design are mainly based on the consensus reached by high-level think tanks. There is a lack of extensive research, and moreover, the data of multiple stakeholders are not included. Therefore, it is urgent to introduce appropriate priority setting methods to solve the problem of transparency and scientificity in the decision-making process. Given the perspective of the specific implementation, the present study introduced three international priority setting methods, i.e., the James Lind Alliance and Priority Setting Partnerships(JLAPSP,)the Child Health and Nutrition Research Initiative(CHNRI), and the Council on Health Research and Development (COHRED), and presented relevant recommendations on how to apply them in the research of TCM, which is expected to provide references for the local research.
9.Mechanism of calcium oxalate crystal-induced ferroptosis in human renal tubular epithelial cell
Kai ZHOU ; Jiawen ZHAO ; Chengyang LI
Chinese Journal of Urology 2023;44(8):622-629
Objective:To investigate the role of ferroptosis in calcium oxalate (Calcium Oxalate, CaOx) crystal-induced injury of human renal tubular epithelial cells (HK-2 cells).Methods:From March 2021 to September 2021, I used calcium oxalate crystal suspension to intervene HK-2 cells to build a HK-2-CaOx reaction model. Set the concentration gradient group and time gradient of calcium oxalate crystal intervention in HK-2 cells: 7 groups of calcium oxalate crystals with different concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L) were used to intervene HK-2 cells24 hours, the HK-2 cell protein was extracted after the intervention; HK-2 cells were intervened with calcium oxalate crystals at optimum concentration, and extract proteins at different time points (0, 3, 6, 9, 12, 24, 48 h) after intervention, the expression of intracellular ferroptosis marker protein glutathione peroxidase 4 (GPX4) was detected by Western blot. Intervention of HK-2 cells with ferroptosis inducer Erastin and ferroptosis inhibitor ethyl 3-amino-4-cyclohexylaminobenzoate (Ferrostatin-1, Fer-1) to regulate intracellular ferroptosis Level. HK-2 cells were divided into 4 groups: normal control group (NC; no intervention treatment, cultured in complete medium only); calcium oxalate crystal stimulation group (CaOx; cultured in complete medium containing 4.0 mmol/L CaOx crystals); calcium oxalate crystals + erastine treatment group (CaOx+ Erastin; cultured in complete medium containing 10.0 μmol/L erastine and 4.0 mmol/L calcium oxalate crystals); calcium oxalate crystals + Fer-1 Treatment group (CaOx+ Fer-1; cultured in complete medium containing 1.0 μmol/L Fer-1 and 4.0 mmol/L calcium oxalate crystals). After 24 hours, the expression of ferroptosis-related protein GPX4, long-chain fatty acyl-CoA synthase 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) in HK-2 cells was analyzed by western blot and immunofluorescence techniques; the content of glutathione in HK-2 cells was detected; DCFH-DA fluorescence staining was used to observe the expression of reactive oxygen species (ROS) in HK-2 cells. The adhesion of calcium oxalate in HK-2 cells in each group was observed by light microscope, and the nuclear damage of HK-2 cells was detected by DAPI staining.Results:The expression levels of GPX4 in cells in the concentration gradient of 0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 mmol/L were5.67±1.05, 5.60±0.02, 4.99±0.94, 4.82±0.93, 4.50±0.70, 4.14± 0.53, 0.97±0.53. The expression difference of GPX4 between the 4.0 mmol/L group and the 0 mmol/L group was statistically significant ( P=0.026). 4.0 mmol/L was selected as the optimal concentration to intervene the cells. The expression levels of GPX4 in the time gradient (0, 3, 6, 9, 12, 24, 48 h) cells were 11.73±1.29, 11.68±1.32, 11.72±1.30, 10.97±1.28, 10.63±1.21, 8.79±1.10, 8.03±1.06. The expression difference of GPX4 between the 24h intervention group and the 0h intervention group was statistically significant( P=0.090), so 24h was chosen as the optimal intervention time for calcium oxalate crystals. Compared with the NC group, the CaOx+ Erastin group had higher expression of ACSL4 (9.71±0.68 vs. 3.96±0.17, P<0.01); SLC7A11 (5.76±1.31 vs. 9.18±1.54, P=0.001) and GPX4 (3.61±0.25 vs. 9.26±0.13, P<0.01) the expression level decreased. Compared with the CaOx group, the CaOx+ Fer-1 group had higher protein expression levels of GPX4 (7.52±0.23 vs. 3.61±0.25, P<0.01), SLC7A11 (7.85±1.34 vs. 5.76±1.31, P=0.012), ACSL4 (5.84 ±0.62 vs. 9.71±0.68, P=0.002) protein expression was significantly decreased. Compared with CaOx group, CaOx+ Erastin group had significantly lower protein expression of GPX4 (2.71±0.18 vs. 3.61±0.25, P=0.001), SLC7A11 (3.82±1.60 vs. 5.75±1.31, P=0.017), ACSL4(11.15±0.44 vs.9.71±0.68, P<0.01) protein expression increased. The results of glutathione determination showed that compared with the NC group, the glutathione content in the CaOx group was significantly reduced [(53.38±3.53) mmol/L vs. (81.88±4.02) mmol/L, P<0.01]. Compared with the CaOx group, the CaOx+ Fer-1 group had significantly higher glutathione content [(68.26±4.55)mmol/L vs. (53.38±3.53)mmol/L, P=0.001]. Compared with the CaOx group, the glutathione content was decreased [(38.22±2.95)mmol/L vs.(53.38±3.53)mmol/L, P=0.01]. The results of DCFH-DA fluorescence staining showed that compared with the NC group (63.36±5.17 vs. 22.72±3.73, P<0.01), the CaOx group had a significantly higher fluorescence intensity, Compared with the CaOx group (45.32±4.33 vs. 63.36±5.17, P=0.002), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly weakened, Compared with the CaOx group (82.38±6.25 vs.63.36±5.17, P=0.002), the fluorescence intensity of the cells in the CaOx+ Erastin group was significantly increased. The results of immunofluorescence showed that the CaOx group was significantly weakened compared with the NC group (31.63±2.86 vs. 50.36±4.23, P<0.01), and the CaOx+ Fer-1 group was significantly weakened compared with the CaOx group (39.89±3.35 vs. 31.63±2.86), P=0.038), the fluorescence intensity of cells in the CaOx+ Fer-1 group was significantly enhanced, the CaOx+ Erastin group was compared with the CaOx group (23.36±3.74 vs. 31.63±2.86, P=0.022), the cell fluorescence in the CaOx+ Erastin group was The intensity is significantly reduced. DAPI staining to calculate the damage ratio of each group of nuclei: NC group (2.85%), CaOx group (11.96%), CaOx+ Fer-1 group (8.76%), CaOx+ Erastin group (16.27%). Conclusion:CaOx crystals can induce ferroptosis in HK-2 cells by increasing the level of oxidative stress in HK-2 cells.
10.Analysis of the changes in intestinal microecology in the early stage of sepsis rat based on 16S rDNA sequencing
Hongyi LI ; Ruiqing ZHAI ; Huoyan LIANG ; Yanhui ZHU ; Yan YAN ; Chengyang WANG ; Xianfei DING ; Gaofei SONG ; Tongwen SUN
Chinese Critical Care Medicine 2022;34(1):28-34
Objective:To investigate the changes of intestinal microecology in the early stage of sepsis rat model by 16S rDNA sequencing.Methods:Sixty male Sprague-Dawley (SD) rats were randomly divided into cecal ligation and puncture (CLP) group and sham operation group (Sham group), with 30 rats in each group. In the CLP group, sepsis rat model was reproduced by CLP method; the rats in the Sham group only underwent laparotomy without CLP. At 24 hours after the operation, the intestinal feces and serum samples of 8 rats in each group were collected. The survival rate of the rest rats was observed until the 7th day. The level of serum tumor necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA). Intestinal feces were sequenced by 16S rDNA sequencing technology. The operational taxonomic unit (OTU) data obtained after sequence comparison and clustering was used for α diversity and β diversity analysis, principal coordinate analysis and linear discriminant analysis effect size analysis (LEfSe) to observe the changes of intestinal microecology in early sepsis rats and excavate the marker flora.Results:At 24 hours after the reproduction of the model, the rats in the CLP group showed shortness of breath, scattered hair and other manifestations, and the level of serum TNF-α increased significantly as compared with that in the Sham group (ng/L: 43.95±9.05 vs. 11.08±3.27, P < 0.01). On the 7th day after modeling, the cumulative survival rate of the Sham group was 100%, while that of the CLP group was 31.82%. Diversity analysis showed that there was no significant difference in α diversity parameter between the Sham group and the CLP group (number of species: 520.00±52.15 vs. 492.25±86.61, Chao1 richness estimator: 707.25±65.69 vs. 668.93±96.50, Shannon index: 5.74±0.42 vs. 5.79±0.91, Simpson index: 0.93±0.03 vs. 0.94±0.05, all P > 0.05). However, the β diversity analysis showed that the difference between groups was greater than that within groups whether weighted according to OTU or not (abundance weighted matrix: R = 0.23, P = 0.04; abundance unweighted matrix: R = 0.32, P = 0.01). At the phylum level, the abundance of Proteobacteria and Candidatus_sacchari in the CLP group increased significantly as compared with the Sham group [18.100% (15.271%, 26.665%) vs. 6.974% (2.854%, 9.764%), 0.125% (0.027%, 0.159%)% vs. 0.018% (0.008%, 0.021%), both P < 0.05]. At the genus level, the abundance of opportunistic pathogen including Helicobacter, Ruthenium, Streptococcus, Clostridium ⅩⅧ in the CLP group was significantly higher than that in the Sham group [5.090% (1.812%, 6.598%) vs. 0.083% (0.034%, 0.198%), 0.244% (0.116%, 0.330%) vs. 0.016% (0.008%, 0.029%), 0.006% (0.003%, 0.010%) vs. 0.001% (0%, 0.003%), 0.094% (0.035%, 0.430%) vs. 0.007% (0.003%, 0.030%), all P < 0.05], and the abundance of probiotics such as Alloprevotella and Romboustia was significantly lower than that in the Sham group [7.345% (3.662%, 11.546%) vs. 22.504% (14.403%, 26.928%), 0.113% (0.047%, 0.196%) vs. 1.229% (0.809%, 2.29%), both P < 0.01]. LEfSe analysis showed that the probiotics belonging to Firmicutes were significantly enriched in the Sham group, and Romboustia was the most significantly enriched species. Opportunistic pathogens such as Helicobacter, Streptococcus and Clostridium ⅩⅧ were significantly enriched in the CLP group, Helicobacter_NGSU_ 2015 was the most significantly enriched species. Conclusion:In the early stage of sepsis, the intestinal microbiota structure of rats is significantly changed, which mainly shows that the abundance of Alloprevotella and other probiotics is significantly reduced, while that of Helicobacter and other opportunistic pathogens is significantly increased.

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