Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.

Osmoregulatory Appraisal of Some Osmoprotectants on Hydrolytic Activities of Some Enzymes on Seeds of Two Water Stressed Cultivars of Sorghum bicolor (Moench)

Enzymes play significant roles in metabolic processes of seeds. Therefore, this study evaluated osmoregulatory potential of some osmoprotectants on activities of some hydrolytic enzymes in the seeds of two cultivars (SOSAT.C-88 and CV. LCIC 9702) of sorghum bicolor. Matured seeds of the two cultivars were harvested and prepared for alpha, beta, total amylase and proteinase activities assay. The osmoprotectants produced significant variations on the enzymes at 10 and 14 days (DA) of 8 weeks after treatments (WAT). Seeds of well-watered SOSAT.C-88 produced higher alpha (2.10 IU/ml), beta (1.70 IU/ml) and total amylase activities (3.30 IU/ml) at 14 days (DA). Higher alpha (2.01 IU/ml and total amylase activities (2.61 IU/ml) were recorded in the seeds of CV. LCIC 9702 well-watered at 14 days DA 8WAT. Furthermore, total amylase activities (3.87 IU/ml) were recorded in the seeds produced by CV. LCIC 9702 well-watered at 14 days DA. Significant increase was noticed in beta (1.14 IU/ml) and alpha amylase (1.58 IU/ml) in the seeds of CV. LCIC 9702 treated with mycorrhiza. CV. LCIC 9702 well watered produced highest proteinase activities (1.57 U/ml) while least of the parameters were recorded in SOSAT.C-88 and CV. LCIC 9702 droughted. In conclusion, the osmoprotectants had regulatory effects on the activities of hydrolytic enzymes therefore the use of the osmoprotectants in farming should be encouraged.

Preliminary study of the role of IL-18/IL-18BP in mediating cytotoxic ability of NK-92MI cells against endothelial cells from GTKO porcine models / 器官移植

Objective To evaluate the role and potential mechanism of interleukin (IL)-18/IL-18 binding protein (BP) in mediating the killing effect of natural killer (NK)-92MI cells upon endothelial cells from α-1, 3- galactosyltransferase gene-knockout (GTKO) porcine models. Methods NK-92MI cells were divided into the NK, NK+IL-18, NK+GTKO, IL-18+NK+GTKO and IL-18+IL-18BP+NK+GTKO groups. The messenger ribonucleic acid (mRNA) levels of inflammation-related genes in NK-92MI cells were detected by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The killing effect of NK-92MI cells on endothelial cells from GTKO porcine models was evaluated by lactate dehydrogenase (LDH) assay. The apoptosis of endothelial cells from GTKO porcine models was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The expression levels of proteins with killing effect and apoptosis-related proteins were determined by Western blot. Results Compared with the NK, NK+IL-18 and NK+GTKO groups, the expression levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-8, IL-3, IL-6 and granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA were up-regulated in NK-92MI cells in the IL-18+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the IL-18+NK+GTKO group, the expression levels of IFN-γ, TNF-α, IL-8, IL-3, IL-6 and GM-CSF mRNA were down-regulated in NK-92MI cells in the IL-18+IL-18BP+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the NK+GTKO group, the expression levels of perforin, granzyme B and IFN-γ proteins in NK-92MI cells were up-regulated, the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was enhanced, the apoptosis rate of endothelial cells from GTKO porcine models was increased, and the ratios of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax)/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were elevated in the IL-18+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Compared with the IL-18+NK+GTKO group, the expression levels of perforin, granzyme B and IFN-γ proteins were down-regulated, the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was decreased, the apoptosis rate of endothelial cells from GTKO porcine models was decreased, and the ratios of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were declined in the IL-18+IL-18BP+NK+GTKO group, and the differences were statistically significant (all P < 0.05). Conclusions IL-18BP may block the expression of inflammation-related genes in NK-92MI cells induced by IL-18 and the killing effect of NK-92MI cells on endothelial cells from GTKO porcine models.

Research progress on islet protection and functional maintenance / 器官移植

Islet transplantation is a promising treatment of diabetes mellitus and its complications. Nevertheless, dysfunction post-transplantation, rejection and shortage of donors are the bottleneck issues in the field of islet transplantation. Optimizing the preservation method of pancreas plays a positive role in obtaining a sufficient quantity of effective islets and maintaining their functions. During the culture stage, anti-rejection and anti-apoptosis treatment of islets, including mesenchymal stem cell (MSC), MSC-derived exosomes, anti-apoptosis drugs and gene modification, may become major approaches for islet protection and functional maintenance in clinical islet transplantation. Use of anti-instant blood-mediated inflammatory reaction (IBMIR) drugs after islet transplantation also plays a critical role in protecting islet function. In this article, the whole process from islet preparation to islet transplantation was illustrated, and relevant strategies of islet protection and functional maintenance were reviewed, aiming to provide reference for improving the quality of donors to compensate for the shortage of absolute quantity of donors and elevating the efficiency of islet transplantation.

Effect of Modified Huangqi Guizhi Wuwutang on Apoptosis-related Bax and Caspase-12 of Sciatic Nerve Cells in Diabetes Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of modified Huangqi Guizhi Wuwutang （MHGW） on the protein and mRNA expression of B-cell lymphoma-2-associated X protein （Bax） and cysteinyl aspartate specific proteinase-12 （Caspase-12） related to the apoptosis of sciatic nerve cells in diabetes rats to explore the mechanism of MHGW in the treatment of peripheral neuropathy in diabetes. MethodAnimal experiments were conducted. A diabetes model was induced in sixty male sprague-dawley （SD） rats by feeding on a high-sugar and high-fat diet combined with streptozotocin （STZ） intraperitoneal injection. Rats with random blood glucose levels ≥ 16.7 mmol·L-1 for three consecutive days were considered to have successfully developed diabetes. Forty-eight rats that successfully developed diabetes were randomly divided into a model group， an α-lipoic acid group （0.026 8 g·kg-1·d-1）， a high-dose MHGW group （2.5 g·kg-1·d-1）， and a low-dose MHGW group （1.25 g·kg-1·d-1）， with 12 rats in each group. Another 10 rats were assigned to the normal group. Body weight and random blood glucose levels of the rats were monitored. At the end of a 16-week intervention period， the sciatic nerve conduction velocity of the rats was measured using the Key point electromyography collection system. The protein and mRNA expression of Bax and Caspase-12 in the sciatic nerve cells was detected by Western blot analysis and real-time quantitative polymerase chain reaction （Real-time PCR）， respectively. ResultCompared with the normal group， the model group showed a significant decrease in body weight （P<0.01） and a significant increase in random blood glucose levels （P<0.01）. After a 16-week intervention， compared with the model group， the high-dose MHGW group exhibited a significant increase in body weight （P<0.05）， while there were no statistically significant differences in body weight changes among the other treatment groups. Random blood glucose levels significantly decreased in all treatment groups （P<0.01）. After 16 weeks of intervention， compared with the normal group， the model group had significantly reduced motor and sensory nerve conduction velocities （P<0.01）. Compared with the model group， all treatment groups showed significant increases in motor and sensory nerve conduction velocities （P<0.05， P<0.01）. The expression of Bax and Caspase-12 proteins in the sciatic nerve cells was significantly elevated in the model group compared with that in the normal group （P<0.01）. In contrast， all treatment groups showed significant reductions in the expression of Bax and Caspase-12 proteins in the sciatic nerve cells as compared with that in the model group （P<0.01）. The expression of Bax and Caspase-12 mRNA in the sciatic nerve cells significantly increased in the model group compared with that in the normal group （P<0.01）. Compared with the model group， the α-lipoic acid group and the high-dose MHGW group showed significant reductions in the expression of Bax mRNA in the sciatic nerve cells （P<0.05， P<0.01）， while the low-dose MHGW group showed a decreasing trend in the expression of Bax mRNA. The expression of Caspase-12 mRNA in the sciatic nerve cells significantly decreased in all treatment groups （P<0.01）. ConclusionMHGW may improve and repair sciatic nerve damage in diabetes rats by inhibiting sciatic nerve cell apoptosis.

Mechanism of Shenqi Yiliu Prescription Combined with Cisplatin on H22 Liver Cancer-bearing Mice Based on NLRP3/Caspase-1/GSDMD Pyroptosis Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-tumor effect and mechanism of Shenqi Yiliu prescription in the intervention of pyroptosis. MethodTen male BALB/c mice were randomly selected and assigned to the blank group. The remaining 40 mice underwent the induction of the liver cancer xenograft model. After 5 days of modeling， 40 surviving mice were randomly divided into model group， cisplatin group ［2.5×10-3 g·kg-1·（3 d）-1］， Shenqi Yiliu prescription group （27 g·kg-1·d-1）， and a combination group （Shenqi Yiliu prescription group + cisplatin）. The mice in the blank group and the model group were treated with an equal volume of normal saline for 10 days. The general conditions of mice in each group were observed. After the intervention， the tumor weight of the mice was weighed and the tumor inhibition rate was calculated. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in tumor tissues. The levels of mouse liver function indicators， including alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were detected. The TdT-mediated dUTP-biotin nick end labeling （TUNEL） assay was used to detect DNA damage in mouse tumor tissue cells. Immunohistochemistry （IHC）， immunofluorescence （IF）， and Western blot were used to detect the protein expression levels of NOD-like receptor protein 3 （NLRP3）， cysteinyl aspartate-specific protease-1 （Caspase-1）， and gasdermin D （GSDMD） in tumor tissues. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in tumor tissues were detected by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with the mice in the blank group， those in the model group were in a poor mental state， sleepy， and lazy， and their fur color was dull， with increased levels of serum ALT and AST in liver function tests （P<0.01）. Compared with the model group， the groups with drug intervention showed improved mental state， inhibited tumor growth to varying degrees， and decreased tumor weight， and the tumor inhibition rate in the combination group was the highest （P<0.01）. HE staining showed that the pathological and morphological lesions of the tumor tissues in the model group were significant， while those in all groups with drug intervention were improved to a certain extent. The karyolysis and nuclear rupture in the Shenqi Yiliu prescription group and the combination group were more significant. In the liver function test， the serum ALT and AST levels of mice in the Shenqi Yiliu prescription group and the combination group decreased （P<0.01）， and the inflammatory factors IL-1β and IL-18 in each group with drug intervention decreased （P<0.05， P<0.01）. Among them， the declining trend of IL-1β and IL-18 in the Shenqi Yiliu prescription group was the most significant （P<0.01）. TUNEL staining showed that the positive TUNEL staining in each group with drug intervention decreased after intervention （P<0.05， P<0.01）， especially the cisplatin group and Shenqi Yiliu prescription group （P<0.01）. Western blot， IHC， and IF found that the protein expression levels of NLRP3， Caspase-1， and GSDMD in each group with drug intervention decreased （P<0.05， P<0.01）. Compared with the mice in the cisplatin group， those in the Shenqi Yiliu prescription group and the combination group had better mental state and regular tumor morphology， and the tumor weight of the mice in the combination group decreased （P<0.05）. The levels of ALT and AST in the Shenqi Yiliu prescription group decreased （P<0.05）， and the levels of IL-1β and IL-18 in the Shenqi Yiliu prescription group and the combination group decreased （P<0.05， P<0.01）， especially in the combination group （P<0.01）. The results of IHC showed that the expression of GSDMD protein in the tumor tissues of mice in the combination group was reduced （P<0.01）. IF detection showed that the expression of NLRP3 in the tumor tissues of the Shenqi Yiliu prescription group was reduced （P<0.01）. The results of Western blot showed that the expression level of NLRP3 protein in the Shenqi Yiliu prescription group and the combination group decreased （P<0.01）， and the expression level of Caspase-1 protein in the combination group decreased （P<0.01）. The decrease in GSDMD protein expression was not significant， and the difference was not statistically significant. ConclusionShenqi Yiliu prescription combined with cisplatin has an obvious anti-tumor effect， which may be achieved by down-regulating the NLRP3/Caspase-1/GSDMD inflammatory pyroptosis pathway to inhibit cell pyroptosis， and relieve the inflammatory response in mice with liver cancer.

Relationship between interleukin-1β and silicosis development / 中国基层医药

Silicosis is a diffuse pulmonary fibrosis disease caused by occupational exposure to silica, which is one of the occupational diseases with high incidence in developing countries. Up to now, there is no definite drug to relieve or reverse the lung injury caused by silicosis, so it is very important to prevent, diagnose and treat pulmonary fibrosis as soon as possible. Studies have shown that a chronic inflammatory environment contributes to pulmonary fibrosis to a certain extent. Interleukin-1β is a cytokine that increases the number of inflammatory factors in the microenvironment in the immune response and plays a key role in inflammatory reaction. Therefore, the release of interleukin-1β is of great significance in the pathogenesis of silicosis. This paper aims to systematically expound the development course of silicosis, the signal pathway of interleukin-1β production, and the relationship between them.

Enzymatic Digestion Method Coupled with Artificial Intelligence Techniques in Forensic Drowning Diatom Detection / 中山大学学报(医学科学版)

ObjectiveArtificial intelligence （AI） full smear automated diatom detection technology can perform forensic pathology drowning diatom detection more quickly and efficiently than human experts.However， this technique was only used in conjunction with the strong acid digestion method， which has a low extraction rate of diatoms. In this study， we propose to use the more efficient proteinase K tissue digestion method （hereinafter referred to as enzyme digestion method） as a diatom extraction method to investigate the generalization ability and feasibility of this technique in other diatom extraction methods. MethodsLung tissues from 6 drowned cadavers were collected for proteinase K ablation and made into smears， and the smears were digitized using the digital image matrix cutting method and a diatom and background database was established accordingly.The data set was divided into training set， validation set and test set in the ratio of 3：1：1， and the convolutional neural network （CNN） models were trained， internally validated， and externally tested on the basis of ImageNet pre-training. ResultsThe results showed that the accuracy rate of the external test of the best model was 97.65 %， and the area where the model features were extracted was the area where the diatoms were located. The best CNN model in practice had a precision of more than 80 % for diatom detection of drowned corpses. ConclusionIt is shown that the AI automated diatom detection technique based on CNN model and enzymatic digestion method in combination can efficiently identify diatoms and can be used as an auxiliary method for diatom detection in drowning identification.

The role of enzymes in the angiogenic activity of Hancornia speciosa latex

The Hancornia speciosa latex has shown angiogenic activity. Angiogenesis plays a major role in wound healing, and materials that stimulate this process could be used to develop drugs. This study aimed to explain the role of proteins in the H. speciosa serum fraction latex in angiogenesis. Hence, this material was treated with proteinase K and the proteins were inactivated. After protein inactivation, angiogenic activity was assessed with the chick chorioallantoic membrane assay. The result showed that the proteins in the serum fraction are responsible for angiogenic activity. Then, the total protein content in the serum fraction and its enzymatic activity were investigated. The low protein content observed in the H. speciosa serum fraction latex suggests that this biomaterial could be used to develop new drugs with a hypoallergenic response. Despite the low protein content, there was a significant enzymatic activity of at least three enzymes in the serum fraction latex: ß-1,3 glucanase, ß-glucosidase, and proteases. These enzymes seem to influence the healing process, assisting debridement, extracellular matrix remodeling, and collagen deposition, and decreasing the chances of contamination by microorganisms. In conclusion, the enzymes in the H. speciosa serum latex are associated with the angiogenic activity of this biomaterial and may be used to assist the wound healing process.

Correlation between serum cystatin C level and clinicopathological features of IgA nephropathy / 中华检验医学杂志

Objective:To investigate the correlation between serum cystatin C (CysC) and clinical and pathological features of IgA nephropathy.Methods:Four hundred and twenty-one cases of primary IgA nephropathy diagnosed by renal biopsy in Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from January 2010 to January 2021 were retrospectively analyzed. According to the serum CysC level at the time of renal biopsy, the patients were divided into high serum CysC group and normal serum CysC group, and the clinical data and pathological indices of the patients were compared. Spearman correlation analysis was used to analyze the correlation between estimated glomerular filtration rate (eGFR) and serum CysC. The clinicopathological factors related to the serum CysC level were analyzed by multiple linear regression. The area under the receiver operator characteristic curve (AUC) was used to evaluate the ability of serum CysC level to predict related pathological injury.Results:The age, prevalence of hypertension, serum creatinine, urea and uric acid levels of high serum CysC group were significantly higher than those of normal serum CysC group, while the eGFR level was significantly lower than that of normal serum CysC group ( P<0.05). Spearman correlation analysis showed that serum CysC was negatively correlated with eGFR ( r=-0.744, P<0.001). In terms of pathological injury, the degree of renal tubular atrophy and renal interstitial fibrosis (T) and renal arteriole wall thickening (A) in high serum CysC group were more serious than those in normal serum CysC group ( P<0.05). Multiple linear regression analysis showed that the prevalence of hypertension, serum creatinine, urea, uric acid, T and A were correlated with serum CysC levels (standard regression coefficient β=0.048, 0.299, 0.260, 0.134, 0.195, 0.068, respectively, P<0.05). After adding serum CysC on the basis of clinical features, the prediction efficiency of renal tubular atrophy and renal interstitial fibrosis was higher (AUC were 0.829 [95% CI 0.787-0.870], 0.847 [95% CI 0.808-0.886], P<0.05). Conclusions:Patients with older age, hypertension, poor renal function and severe pathological damage are more likely to have elevated serum CysC levels. Serum CysC was related to the prevalence of hypertension, creatinine, urea, uric acid, T and A. Combined with serum CysC level can effectively improve the ability prediction of T.

Research process on pathogenesis of gastroesophageal reflux disease / 中国医师杂志

Gastroesophageal reflux disease (GERD) is a gastrointestinal motility disorder that results from the reflux of stomach contents into the esophagus or oral cavity, causing symptoms or complications. The typical symptoms of GERD are heartburn and regurgitation of gastric contents into the oropharynx. Heartburn is the sensation of burning or discomfort behind the sternum. Heartburn may radiate into the neck, is typically worse after meals or when in a reclining position, and may be eased by antacids. Regurgitation is the backflow of gastric contents into the mouth or hypopharynx. Epigastric pain can also be a symptom of GERD. Extraesophageal symptoms of GERD include dental erosions, laryngitis, cough, and asthma. In recent years, great progress has been made in understanding the molecular basis of GERD, suggesting that its pathogenesis is more complex and multifactorial. In this paper, the molecular pathogenesis was taken as the starting point, including the mechanism of genes in the pathogenesis and development of GERD, the mechanism of NF-κB pathway in the pathogenesis and development of GERD, the role of proteinase-activated receptor-2 in the pathogenesis of GERD, the association between abnormal serotonin pathway and GERD, and the relationship between reactive oxygen species and GERD, to summarize the pathogenesis of gastroesophageal reflux disease.

Effect of HMGB1/Caspase-1/GSDMD signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury / 器官移植

Objective To evaluate the effect of high mobility group box 1 (HMGB1)/ cysteinyl aspartate specific proteinase (Caspase)-1/Gasdermin D (GSDMD) signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury (IRI). Methods C57BL/6 mice were randomly divided into the sham operation group (Sham group), IRI 2 h group, IRI 6 h group, IRI 12 h group, glycyrrhizic acid (GA)+Sham group and GA+IRI 12 h group (n=8 in each group). AML12 cells were evenly divided into the Sham group, IRI 12 h group, GA+Sham group and GA+IRI 12 h group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β and IL-6 in each group were detected by enzyme-linked immune absorbent assay(ELISA). The messenger ribonucleic acid (mRNA) levels of IL-1β and IL-6 were detected by reverse transcription polymerase chain reaction(RT-PCR). The pathological score of liver ischemia and cell apoptosis were compared among all groups. The expression level of HMGB1 in the liver tissues of each group was determined by immunohistochemistry. The expression levels of HMGB1, Caspase-1 and GSDMD proteins in the mouse liver tissues and AML12 cells were measured by Western blot. Results Compared with the Sham group, the serum levels of ALT, AST, IL-1β and IL-6 and the relative expression levels of IL-1β and IL-6 mRNA in the liver tissues were all significantly up-regulated after IRI in each group (all P < 0.05), and showed significant time-dependent pattern along with the prolongation of reperfusion time. Compared with the Sham group, the pathological score of hepatic ischemia and the apoptosis rate of hepatocytes were significantly increased after IRI in each group (all P < 0.05). Immunohistochemical results showed that the expression level of HMGB1 in the liver tissues was significantly up-regulated after IRI, which showed an increasing trend along with the prolongation within the period of 2-12 h. Western blot showed that compared with the Sham group, the relative expression levels of HMGB1, Caspase-1 and GSDMD proteins in vivo and in vitro were up-regulated in the IRI 12 h group. The relative expression level of HMGB1 protein was significantly up-regulated, whereas those of Caspase-1 and GSDMD proteins were significantly down-regulated in the GA+IRI 12 h group compared with those in the IRI 12 h group (all P < 0.05). Conclusions Hepatocytes probably activate the Caspase-1/GSDMD signaling pathway by releasing HMGB1, thereby triggering hepatocyte pyroptosis and leading to liver IRI. Inhibition of extracellular release of HMGB1 by GA may mitigate liver IRI.

Mechanism of Tingli Dazao Xiefeitang on Cell Pyrolysis and Apoptosis Induced by Caspase-1 in A549 Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of coking death and apoptosis of A549 cells induced by Tingli Dazao Xiefeitang. MethodA549 cells were randomized into blank group， traditional Chinese medicine（TCM） low， medium， and high concentration groups， which were treated with 20， 40， 60 mg·L-1 Tingli Dazao Xiefeitang， and TCM low， medium， and high concentration groups， respectively， and blank group was treated with equal volume culture medium. After 48 h of treatment， cell migration was detected by scratch assay and cell apoptosis was detected by flow cytometry. The relative expression levels of cysteine aspartate protease-1（Caspase-1）， NOD-like receptor protein 3 （NLRP3）， dermoderin D （GSDMD）， Survivin protein and nuclear transcription factor -κB （NF-κB） pathway proteins were detected by Western blot. The levels of intracellular reactive oxygen species （ROS） were determined by DCFH-DA fluorescence probe， and the contents of tumor necrosis factor -β （TNF-β） and interleukin-1β （IL-1β） in supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with blank group， the scratch healing rate， apoptosis rate， relative expression of Survivin protein， Caspase-1， GSDMD， NLRP3， ROS and NF-κB phosphorylation levels were significantly increased in low， medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. Compared with the low concentration group， the scratch healing rate， apoptosis rate， Survivin protein relative expression， Caspase-1， GSDMD， NLRP3 relative expression， ROS and NF-κB phosphorylation levels were significantly increased in the medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. Compared with the TCM group， the scratch healing rate， apoptosis rate， Survivin protein relative expression， Caspase-1， GSDMD， NLRP3 relative expression， ROS and NF-κB phosphorylation levels were significantly increased in the high concentration group. The contents of TNF-β and IL-1β in supernatant were significantly increased （P<0.05）. ConclusionTingli Dazao Xiefeitang can improve NLRP3 protein expression， inhibit Survivin protein expression and promote apoptosis of A549 cells. At the same time， it can activate NF-κB pathway and ROS system， up-regulate the expression of Caspase-1 and GSDMD， mediate scortosis of A549 cells.

Research progress in the relationship between deubiquitinase OTUB1 and urological cancer / 国际生物医学工程杂志

Ovarian tumor-associated protease B1(OTUB1) is a member of the deubiquitinase family. Its highly specific recognition and cleavage function of polyubiquitinated chains has attracted widespread attention, and it can regulate a variety of important signaling pathways, such as the epithelial-mesenchymal transition (EMT) pathway, the MAPK signaling pathway, and the p53-related signaling pathway. In recent years, it has gradually become a new direction of oncology research. More and more studies have proved that OTUB1 is closely related to various tumors such as hepatocellular carcinoma, colorectal cancer, and breast cancer. It regulates the occurrence, development, and prognosis of tumors. OTUB1 could be a potential treatment for tumors. Urinary tract tumors mainly include prostate cancer, bladder cancer, and renal cell carcinoma. In this review, the research progress on the correlation between OTUB1 and urological tumors was reviewed, including its important role in the occurrence and development of urological tumors and the possibility of treating urological tumors with OTUB1.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Modified Wenjingtang Affects Caspase-8， MMP-9， N-cadherin and E-cadherin in Local Microenvironment of EM Rats with Kidney Deficiency and Blood Stasis / 中国实验方剂学杂志

Objective:To investigate the therapeutic effect and mechanism of modified Wenjingtang on endometriosis （EM） rats with kidney deficiency and blood stasis. Method:Healthy non-pregnant female Sprague-Dawley （SD） rats of SPF grade were randomly divided into the blank group and experimental group. After being modeled via soaking in ice water and subcutaneous injection of epinephrine hydrochloride， the ones in the experimental group were further divided into the sham operation group and EM model group， with the former only undergoing laparotomy and the latter further receiving autologous endometrial transplant for triggering EM. The successfully modeled rats with EM due to kidney deficiency and blood stasis were randomized into the positive drug （danazol， 63 mg·kg^-1） group and low- （5 g·kg^-1）， medium- （10 g·kg^-1）， and high-dose （20 g·kg^-1） modified Wenjingtang groups. The corresponding drugs were administered by gavage， once per day， for four weeks. Then the ectopic and eutopic endometrial tissues were stained with hematoxylin-eosin （HE） to observe the histopathological changes. The protein and mRNA expression levels of cysteinyl aspartate-specific proteinase-8 （Caspase-8）， matrix metalloproteinase-9 （MMP-9）， N-cadherin， and E-cadherin were detected by immunohistochemistry （IHC）， Western blotting， and real-time polymerase chain reaction （Real-time PCR）， respectively. Result:The IHC and Western blot revealed that the protein expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly increased as compared with those in the sham operation group （<italic>P</italic><0.01）， while the levels of Caspase-8 and E-cadherin was significantly decreased （<italic>P</italic><0.01）. Compared with the model group， the danazol and low-， medium-， and high-dose modified Wenjingtang groups exhibited obviously down-regulated MMP-9 and N-cadherin protein expression in eutopic and ectopic endometrial tissues （<italic>P</italic><0.05，<italic>P</italic><0.01）， but up-regulated Caspase-8 and E-cadherin （<italic>P</italic><0.05， <italic>P</italic><0.01）. Real-time PCR uncovered that the mRNA expression levels of MMP-9 and N-cadherin in eutopic and ectopic endometrial tissues of the model group were significantly elevated as compared with those in the sham operation group （<italic>P</italic><0.01）， whereas the levels of Caspase-8 and E-cadherin significantly declined （<italic>P</italic><0.01）. The comparison with the eutopic endometrial tissue in the model group showed that the mRNA expression levels of MMP-9 and N-cadherin in the danazol group and high- and medium-dose modified Wenjingtang groups were significantly down-regulated， while those of Caspase-8 and E-cadherin were significantly up-regulated （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Modified Wenjingtang alleviates the immunosuppression and blocks the angiogenesis in EM rats with kidney deficiency and blood stasis syndrome by regulating the expression of such cytokines as Caspase-8， MMP-9， N-cadherin， and E-cadherin， thus exerting the therapeutic effect against EM. The above-mentioned micro-indicators are potential markers reflecting the disease （EM）， syndrome （kidney deficiency and blood stasis）， and pathological mechanisms （immunosuppression and angiogenesis）.

Mechanism of Didangtang in Inhibiting Aortic NLRP3 Inflammasome Cascade of Mice with Diabetic Macrovascular Disease / 中国实验方剂学杂志

Objective:To explore the progression of diabetic macrovascular disease and the effects of Didangtang at different doses on it. Method:Four-week-old male apolipoprotein-E knockout （ApoE^-/-） mice with diabetic macrovascular disease induced by exposure to high-fat diet combined with streptozotocin （STZ） were randomly divided into the model， simvastatin， as well as high-， medium-， and low-dose Didangtang groups. The age-matched ApoE^-/- mice of the same batch only fed with a high-fat diet were classified into the ApoE^-/- （model control） group， and C57BL/6 mice with the same genetic background receiving a regular diet into the normal group. The sampling was conducted at the 8th and 20th weeks of the experiment for observing the pathological characteristics of the aorta and the proportion of plaque area in mice of each group at different time points， followed by the comparison of blood glucose， blood lipid， and oxidized low-density lipoprotein （ox-LDL） levels. The aortic NOD-like receptor protein 3 （NLRP3） and cysteinyl aspartate-specific proteinase-1 （Caspase-1） protein expression was detected by Western blot assay， and the serum interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， interleukin-18 （IL-18）， interleukin-1<italic>α</italic> （IL-1<italic>α</italic>）， and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） levels by enzyme-linked immunosorbent assay （ELISA）. Result:The comparison with the normal group revealed that the proportions of plaque area in the ApoE^-/- group and the model group were increased （<italic>P</italic><0.01）， while the proportion of plaque area in each administration group was significantly reduced in contrast to that of the model group （<italic>P</italic><0.05）. The aortic NLRP3 and Caspase-1 protein expression levels as well as the serum IL-1<italic>β</italic>， IL-18， IL-1<italic>α</italic>， and TNF-<italic>α </italic>levels in the ApoE^-/- group and the model group were significantly higher than those in the normal group （<italic>P</italic><0.01）. Compared with the model group， each administration group exhibited a significant reduction in aortic NLRP3 and Caspase-1 protein expression and serum IL-1<italic>β</italic>， IL-18， IL-1<italic>α</italic>， and TNF-<italic>α</italic> levels （<italic>P</italic><0.05）， with the strongest inhibitory effect detected in the medium-dose Didangtang group （<italic>P</italic><0.05）. Conclusion:Didangtang directly alleviates diabetic macrovascular disease possibly by down-regulating NLRP3 and Caspase-1 protein expression and easing the inflammatory cascade.

Effect of phosphoglycerate mutase 5 mediated pyroptosis on liver ischemia-reperfusion injury / 器官移植

Objective To investigate the effect and its molecular mechanism of phosphoglycerate mutase 5 (PGAM5) mediated pyroptosis on liver ischemia-reperfusion injury (IRI). Methods C57 mouse models of liver IRI were established and randomly divided into the 6 h reperfusion (6 h group) and 12 h reperfusion (12 h group), and sham operation group (sham group) was established too, 10 rats in each group. The effect of IRI on the parameters in the liver tissues and serum samples was evaluated. The expression levels of PGAM5 and cysteinyl aspartate specific proteinase (Caspase)-1 in the liver tissues during IRI were quantitatively detected. The IRI models of liver cells were established (IRI group). The IRI models of liver cells were established after pretreatment with Caspase-1 inhibitor Z-YVAD-FMK (inhibitor group). The untreated AML12 cells were allocated into the control group. The effect of inhibiting Caspase-1 activity on pyroptosis was analyzed. AML12 cells were transfected with PGAM5 small interfering ribonucleic acid (siRNA) (siRNA group) and siRNA-negative control (siRNA-NC) (siRNA-NC group) by liposome 3000, and then IRI models of liver cells were established. The untreated AML12 cells were assigned into the control group. The effect of PGAM5 mediated pyroptosis on IRI of liver cells was assessed. Results In the 6 h and 12 h groups, partial liver cell edema, hepatic sinusoid narrowing, central vein congestion and occasional spot necrosis were observed in the mouse liver tissues, and these changes in the 12 h group were more aggravated than those in the 6 h group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the 6 h and 12 h groups were higher than those in the sham group, and the values in the 12 h group were higher than those in the 6 h group. The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were increased in the 6 h and 12 h groups, and the values in the 12 h group were lower than those in the 6 h group. The relative expression levels of IL-1β messenger ribonucleic acid (mRNA) in the mouse liver tissues in the 6 h and 12 h groups were up-regulated, and the value in the 12 h group was lower than that in the 6 h group. The cell apoptosis rates in the liver tissues were significantly increased in the 6 h and 12 h groups, and the value in the 12 h group was remarkably lower than that in the 6 h group (P < 0.01-0.05). Compared with the sham group, the relative expression levels of PGAM5 mRNA and protein in the mouse liver tissues in the 6 h and 12 h groups were significantly up-regulated, and the values in the 12 h group were significantly higher than those in the 6 h group (P < 0.01-0.05). The protein expression levels of PGAM5 and Caspase-1 in the liver tissues were up-regulated in the 6 h and 12 h groups. Compared with the control group, the relative expression levels of NOD-like receptor protein 3 (NLRP3), cleaved Caspase-1 and Gasdermin D (GSDMD) proteins were up-regulated and the fluorescence intensity of GSDMD was increased in the IRI group. Compared with the IRI group, the relative expression levels of NLRP3, cleaved Caspase-1 and GSDMD proteins were significantly down-regulated and the fluorescence intensity of GSDMD was considerably decreased in the inhibitor group (P < 0.01-0.05). Compared with the control group, the cell survival rate was significantly decreased, and the relative expression levels of PGAM5, NLRP3, cleaved Caspase-1 and GSDMD proteins were significantly up-regulated in the siRNA-NC group (P < 0.01-0.05). Compared with the siRNA-NC group, the cell survival rate was remarkably increased, whereas the relative expression levels of PGAM5, NLRP3, cleaved Caspase-1 and GSDMD proteins were significantly down-regulated in the siRNA group (P < 0.01-0.05). Conclusions PGAM5 may aggravate the liver IRI in mouse models probably by mediating pyroptosis via PGAM5/Caspase-1/GSDMD signaling pathway and aggravating liver cell injury.

Crystal structure of SARS-CoV-2 papain-like protease

The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC

Role of NLRP3 inflammasome and related inflammatory signaling pathways in renal ischemia-reperfusion injury / 器官移植

Renal ischemia-reperfusion injury (IRI) commonly occurs in renal transplantation, which is an important pathophysiological process that causes acute renal failure and severely affects clinical prognosis of the recipients. Inflammatory response plays a critical role in the pathogenesis and pathological process of IRI. Activated NOD-like receptor protein 3(NLRP3) inflammasome can mediate the maturation and release of various pro-inflammatory cytokines, thereby regulating the inflammatory response and relevant cell functions. In this article, the mechanism underlying NLRP3 inflammasome and its related inflammatory signaling pathway in renal IRI were reviewed, aiming to provide novel ideas for clinical prevention and treatment of renal IRI.