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.

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.

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 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.

Therapeutic Effect of Modified Yinchenhao Tang on Cholestatic Liver Disease in Rats / 中国实验方剂学杂志

Objective::To investigate the protective effect of modified Yinchenhao Tang on α-isothiocyanate(ANIT)-induced cholestatic liver disease (CSLD). Method::Wistar rats were randomly divided into 7 groups: blank control group, model control group, compound Glycyrrhizin capsules group(22.5, 45 mg·kg-1), modified Yinchenhao Tang low, middle and high dose groups(4.1, 8.1, 16.2 g·kg-1). A model of cholestatic liver injury was prepared by intragastric administration of ANIT (100 mg·kg-1). Glycyrrhizin capsules and modified Yinchenhao Tang were administered intragastrically on the second day of modeling for 4 consecutive days. And bile duct intubation was performed on the fifth day to measure the bile flow rate of the rats, and serum was taken to test the total bilirubin(TBIL), direct bilirubin(DBIL), indirect bilirubin(IBIL), alanine aminotransferase(ALT) and total bile acid(TBA) serological indicators of each group. Pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining. The expression levels of G protein-coupled bile acid receptor(TGR5), nucleotide binding oligomerization domain-like receptor 3(NLRP3) and cysteinyl aspartate specific proteinase-1(Caspase-1) proteins in the iver tissues were detected by Western blot. Result::Compared with the blank control group, bile flow rate in the model group decreased significantly(P<0.01). TBIL, DBIL, IBIL, ALT and TBA level in serum were significantly increased(P<0.01), liver tissue lesions were severe, and significantly increased the expression of liver tissue TGR5 and Caspase-1.Compare with model group, the compound Glycyrrhizin capsules group had no significant effect on bile flow rate and TBIL, DBIL, IBIL, ALT and TBA level in serum. Bile flow rate increased and TBIL, DBIL, IBIL, ALT and TBA level in serum decreased significantly in modified Yinchenhao Tang high dose group. The compound Glycyrrhizin capsules group and modified Yinchenhao Tang group have different extents of improvement the pathological changes of the lung tissues, and the protein expression of TGR5 and Caspase-1 were significantly decreased in the liver tissue(P<0.01). Conclusion::Modified Yinchenhao Tang can effectively treat CSLD in rats, and its mechanism may be related to bile acid and bile acid receptor TGR5-mediated inflammatory factors.

Effect of ethanolic extract of Polygonum cuspidatum on acute gouty arthritis in mice through NLRP3/ASC/caspase-1 axis / 中国中药杂志

The aim of this paper was to study the effect and mechanism of alcohol extract from Polygonum cuspidatum(PCE) on acute gouty arthritis in C57 BL/6 mice through NLRP3/ASC/caspase-1 axis. The model mice which injected with ankle joint injection of sodium urate crystals(MSU) were orally administrated with three different concentration of PCE, with colchicine as positive control. HE staining was used for observing the morphological changes of synovial tissue; concentration of IL-1β, IL-6 and TNF-α secreted by synovial tissue of the ankle joint were detected by ELISA; mRNA and protein expression of NLRP3, ASC and caspase-1 in synovial tissue were detected by RT-PCR and Western blot respectively. The results showed that the swelling degree of ankle joint in model mice were significantly elevated; expression of IL-1β, IL-6 and TNF-α were significantly increased; mRNA and protein expression of NLRP3, ASC and caspase-1 also significant increase, compared with normal control group. The swelling degree of ankle joint significantly relief; expression of IL-1β, IL-6 and TNF-α in joint synovium significantly decrease; mRNA and protein expression of NLRP3, ASC and caspase-1 were significantly decrease in PCE treatment group compared with model group. Our research implied that alcohol extract from P. cuspidatum had positive effect on acute gouty arthritis in mice, and the regulation of NLRP3/ASC/caspase-1 axis may be its mechanism.

Research progress on absent in melanoma 2 in defending pathogenic microorganism invasion / 中华临床感染病杂志

Innate immunity is on the frontline of fight against pathogenic microorganism invasion .As a DNA sensor, absent in melanoma 2 (AIM2) is an important member of innate immune system.It can recognize dsDNA of pathogenic microbes to form AIM 2 inflammasomes , which facilitates defending and clearing the invasion of pathogens by activating caspase-1 dependent pyroptosis and the mature of IL-18 and IL-1β.AIM2 inflammasomes play an important part in responding to Listeria monocytogenes, Francisella tularensis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Aspergillus fumigatus, vaccinia virus , murine cytomegalovirus , and hepatitis B virus infections .This paper introduces the components of AIM 2 inflammasomes and summarizes its function in defending the invasion of pathogenic microorganisms .