Protective Mechanism of Paeoniflorin on Mice with Ulcerative Colitis Based on AMPK/mTOR Autophagy Pathway / 中国实验方剂学杂志

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg-1）， paeoniflorin （50 mg·kg-1） + inhibitor （20 mg·kg-1） group， and high dose （50 mg·kg-1）， medium dose （25 mg·kg-1）， and low dose （12.5 mg·kg-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

Effect of Linggui Zhugantang on Mitochondrial Fusion-fission and Sirt3/ AMPK Signaling Pathway in Chronic Heart Failure Rats After Myocardial Infarction / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg-1） and Captopril group （0.002 57 g·kg-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.

Danggui Shaoyaosan Regulates Autophagy via AMPK/mTOR/ULK1 Signaling Pathway in Rat Model of Metabolism-associated Fatty Liver Disease / 中国实验方剂学杂志

ObjectiveTo investigate the regulatory effect of Danggui Shaoyaosan on adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase-1 （ULK1） signaling pathway in the rat model of metabolism-associated fatty liver disease （MAFLD）. MethodSixty SD rats were randomized into control， model， western medicine （polyene phosphatidylcholine capsules，0.144 g·kg-1）， and low-， medium-， and high-dose （2.44， 4.88， 9.76 g·kg-1， respectively） Danggui Shaoyaosan groups. After being fed with a high-fat diet for 8 weeks， the rats in each group were administrated with corresponding drugs for 4 weeks. At the end of drug treatment， serum and liver tissue were collected for subsequent determination of related indicators. ResultCompared with the control group， the model group showed increased contents of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C） and activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum， increased contents of TC， TG， and free fatty acids （FFAs） in the liver （P<0.01）， and decreased content of high-density lipoprotein cholesterol （HDL-C） in the serum （P<0.01）. Furthermore， the model group showed down-regulated protein levels of p-AMPK， microtubule-associated protein 1 light chain 3B （LC3B） Ⅱ， Beclin1， and ULK1 （P<0.01） and up-regulated protein levels of p-mTOR and ubiquitin-binding protein p62 in the liver （P<0.01）. The hepatic steatosis was obvious and the NAFLD activity score （NAS） and oil red O staining area increased in the model group， （P<0.05， P<0.01）. Compared with the model group， Danggui Shaoyaosan reduced the contents of TC and TG and the activities of ALT and AST in the serum， lowered the levels of TC， TG， and FFA in the liver， down-regulated the protein levels of p-mTOR and p62 （P<0.01）， elevated the serum HDL-C level， and up-regulated the protein levels of p-AMPK， LCBⅡ， Beclin1， and ULK1 in the liver （P<0.05， P<0.01）. Moreover， it alleviated hepatic steatosis and decreased the NAS and oil red O staining area （P<0.05， P<0.01）. ConclusionDanggui Shaoyaosan has therapeutic effect on MAFLD rats by regulating AMPK/mTOR/ULK1 signaling pathway to enhance autophagy.

Effect of Modified Gegen Qinliantang on TGR5/cAMP/GLP-1 Signaling Pathway in Pancreatic Tissue of Type 2 Diabetes Mellitus db/db Mice / 中国实验方剂学杂志

ObjectiveTo discuss the effect of modified Gegen Qinliantang （MGQT） on blood glucose and lipids and Takeda G protein-coupled receptor 5 （TGR5）-related pathways in pancreatic tissue of obese type 2 diabetes mellitus （T2DM） mice. MethodA total of 10 male specific pathogen free （SPF） m/m mice （7 weeks old） and 50 male SPF （7 weeks old） were adaptively fed for one week in SPF laboratory. The m/m mice were included in the blank group. T2DM was induce d in the 50 db/db mice. The model mice were randomized into the model group， metformin group （0.2 g·kg-1）， high-dose， medium-dose， and low-dose （31.9， 19.1， 6.4 g·kg-1） MGQT groups， with 10 in each group， and the drug dose was10 mL·kg-1. The model group and the blank group received distilled water of the same volume. The administration lasted 12 weeks （once/day）. Fasting blood glucose （FBG） was detected regularly. After 12 weeks of administration， serum levels of glycated serum protein （GSP）， serum glucose （GLU）， total cholesterol （TC）， triglycerides （TG）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） were detected. Pathological changes in the pancreatic tissue were based on hematoxylin-eosin （HE） staining. Western blot was used to determine the protein expression of TGR5， protein kinase A （PKA）， phosphorylated （p）-PKA， cyclic-AMP response element binding protein （CREB）， p-CREB， proprotein convertase 1/3 （PC1/3）， and glucagon-like peptide-1 （GLP-1） in pancreatic tissues. The level of cyclic adenosine monophosphate （cAMP） in pancreatic tissue was determined by enzyme-linked immunosorbent assay （ELISA）. ResultCompared with the blank group， the model group had pathological changes in pancreatic tissue， high levels of FBG， GSP， GLU， TC， TG， and LDL-C （P<0.01）， low level of HDL-C （P<0.05）， low protein expression of TGR5， p-PKA （Thr197）/PKA， p-CREB （Ser133）/CREB， PC1/3， and GLP-1 in pancreatic tissue （P<0.01）， and low content of cAMP in the pancreas （P<0.01）. Pancreatic tissue lesion in the treatment groups were milder than that in the model group. Both the high-dose MGQT and metformin can reduce the levels of FBG， GSP， GLU， TC， TG， and LDL-C in db/db mice （P<0.05， P<0.01） and increase the level of HDL-C （P<0.01）. Except the GLP-1 protein in the medium-dose MGQT group， the protein expression of TGR5， p-PKA （Thr197）/PKA， p-CREB （Ser133）/CREB， PC1/3， and GLP-1 in the high-dose and medium-dose MGQT groups and the metformin group increased compared with that in the model group （P<0.05， P<0.01）. The content of cAMP in the pancreatic tissue of the high-dose and medium-dose MGQT groups and the metformin group was raised compared with that in model group （P<0.05， P<0.01）. ConclusionMGQT can improve the glucose homeostasis in db/db mice with T2DM by regulating TGR5/cAMP/GLP-1 signaling pathway-related protein expression.

Determination of multi-index components and quality evaluation of Dilong formula granules / 药学实践杂志

Objective To establish the method for the simultaneous determination of hypoxanthine, inosine, guanosine and adenosine in Dilong formula granules by HPLC and compare the fingerprints of Dilong formula granules from different manufacturers by HPLC chromatogram. Methods The contents of hypoxanthine, inosine, guanosine and adenosine were determined by Thermo AcclaimTM120C18 column (4.6 mm×250 mm 5 μm). The mobile phase was acetonitrile-water. Gradient elution with flow rate of 0.6 ml/min was used. Column temperature was 25 ℃. Detection wavelength was 254 nm. 10 batches of samples were tested. The HPLC chromatogram were compared and analyzed by using the similarity Evaluation system of chromatographic fingerprint of traditional Chinese Medicine (version 2012.130723). Results The linear ranges for the detection of hypoxanthine, inosine, guanosine and adenosine showed good linear relationships within their own ranges (r≥0.999 9). The average recoveries were 99.20%~102.98% with RSD of 0.26 %~0.71%. The contents of 4 components in 10 batches of samples were 0.740 0~4.457 4 mg/g, 2.132 3~7.805 0 mg/g, 0.325 4~1.596 1 mg/g, 0.537 2~2.222 9 mg/g respectively. The similarity between HPLC chromatogram and control fingerprints of Dilong formula granules from different manufacturers was greater than 0.91. Conclusion The method could be used to determine the contents of hypoxanthine, inosine, guanosine and adenosine in Dilong formula granule. HPLC fingerprints could be used to evaluation the quality in Dilong formula granule. The similarity of HPLC fingerprints from different manufacturer production of Dilong formula granule is high, but 4 contents in composition are difference.

Tangbikang Granules Modulates AMPK/NF-κB Pathway to Alleviate Sciatic Nerve Inflammation in Diabetic Rats / 中国实验方剂学杂志

ObjectiveTo explore the effect of Tangbikang granules （TBK） on sciatic nerve inflammation in diabetic rats through modulation of adenosine monophosphate-activated protein kinase （AMPK）/nuclear factor （NF）-κB pathway. MethodSD rats were fed with high-fat and high-sugar diet for 8 weeks and then treated with streptozotocin （STZ， ip） at 35 mg·kg-1 for modeling. Then the rats were randomized into diabetes group， low-dose （0.625 g·kg-1）， medium-dose （1.25 g·kg-1）， and high-dose （2.5 g·kg-1） TBK groups， and lipoic acid group （0.026 8 g·kg-1） according to body weight and blood glucose level， and a normal group was designed. After modeling， administration began and lasted 12 weeks. The body mass， blood glucose level， and thermal withdrawal latency （TWL） of the rats were detected before treatment and at the 4th， 8th， and 12th week of administration. At the 12th week， the sciatic nerve was collected for hematoxylin-eosin （HE） and Luxol fast blue （LFB） staining， and the structural changes of sciatic nerve were observed under scanning electron microscope. The levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in sciatic nerve were measured by enzyme-linked immunosorbent assay （ELISA）， and the levels of AMPK， phosphorylated （p）-AMPK， and NF-κB proteins in the sciatic nerve were measured by Western blot. ResultThe blood glucose concentration and TWL in the model group were higher than those in the normal group at each time point （P<0.01）. The levels of IL-1β， TNF-α， and NF-κB protein in sciatic nerve in the model group were higher than those in the normal group （P<0.01）， and the p-AMPK/AMPK ratio was smaller than that in the normal group （P<0.01）. Compared with the model group， TBK of the three doses lowered the TWL （P<0.05， P<0.01） and the levels of IL-1β， TNF-α， and NF-κB protein in sciatic nerve of rats （P<0.05， P<0.01）， and high-dose and medium-dose TBK raised p-AMPK/AMPK （P<0.05， P<0.01）. The sciatic nerve fibers were orderly and compact with alleviation of demyelination in rats treated with TBK compared with those in the model group. ConclusionTBK improves the function of sciatic nerve and alleviates neuroinflammation in diabetic rats. The mechanism is the likelihood that it up-regulates the expression of AMPK in the AMPK/NF-κB pathway and inhibits the expression of downstream NF-κB， thereby alleviating the neuroinflammation caused by high levels of inflammatory factors such as IL-1β and TNF-α due to NF-κB activation.

Mechanism of Shaoyaotang in Regulating Water Metabolism and Intestinal Epithelial Permeability in Ulcerative Colitis Through cAMP/PKA/CREB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the role of cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA）/cAMP-response element binding protein （CREB） signaling pathway in water metabolism and intestinal epithelial permeability in ulcerative colitis （UC） and the intervention mechanism of Shaoyaotang based on the theory of large intestine governing fluids. MethodSixty male SD rats were divided into blank group， model group， mesalazine group （0.42 g·kg-1）， Shaoyaotang low-dose group （11.1 g·kg-1）， Shaoyaotang medium-dose group （22.2 g·kg-1） and Shaoyaotang high-dose group （44.4 g·kg-1）， with 10 in each group. The UC rat model of internal retention of dampness-heat was established by compound factors. The blank group and the model group were given normal saline （ig）. The mesalazine group was given mesalazine （ig）， and Shaoyaotang low-， medium- and high-dose groups were administrated with corresponding doses of Shaoyaotang （ig）. The treatment lasted for 14 days. The diarrhea score and fecal moisture content of rats in each group were observed. The contents of diamine oxidase （DAO） and D-lactic acid in plasma were detected by enzyme-linked immunosorbent assay （ELISA）. The protein expressions of aquaporin （AQP）8， AQP4， ZO-1 and Occludin in colon tissues were detected by immunohistochemistry， while those of cAMP， PKA and CREB in colon tissues were determined by Western blot. ResultCompared with the normal group， the model group had elevated diarrhea score and fecal moisten content （P<0.01）， increased contents of DAO and D-lactic acid in plasma （P<0.01） and decreased protein expressions of ZO-1， Occludin， AQP8， AQP4， cAMP， PKA and CREB in colon （P<0.01）. Compared with the conditions in the model group， the contents of DAO and D-lactic acid in plasma in each administration groups were lower （P<0.01）， while the protein expressions of ZO-1， Occludin， AQP8， AQP4， cAMP， PKA and CREB in colon were higher （P<0.01）. ConclusionShaoyaotang alleviates the diarrhea in UC， probably through activating cAMP/PKA/CREB signaling pathway， up-regulating expressions of AQPs， enhancing tight junctions in intestinal epithelium and thus improving the water metabolism in colon and the intestinal mucosal permeability.

Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of salvianolate on the protein expressions of adenosine monophosphate （AMP）-activated protein kinase （AMPK）， silent information regulator 1 （SIRT1） and peroxisome proliferator-activated receptor-γ coactivator-1α （PGC-1α）， autophagy and apoptosis in kidney tissue of rats with membranous nephropathy （MN）， and to explore its possible molecular mechanism against MN. MethodEighty male SD rats were randomly divided into normal group， model group， benazepril hydrochloride group （10 mg·kg-1）， and salvianolate low-， medium-， and high-dose groups （16.7， 33.3 and 66.7 mg·kg-1）. The rats were modeled by injection of cationized bovine serum albumin （C-BSA） into the tail vein. After successful modeling， rats in the administration groups were given corresponding doses of drugs for 4 consecutive weeks， and then 24-hour urine， serum and kidney tissue were collected for the detection of 24-hour urinary protein （UTP）， blood urea nitrogen （BUN）， serum creatinine （SCr）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， C reactive protein （CRP）， glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， and malondialdehyde （MDA）. The pathological changes of kidneys were observed by light microscope， electron microscope and immunofluorescence. Western blot was used to detect the protein expressions of phospho-AMPK （p-AMPK）， AMPK， phospho-SIRT1 （p-SIRT1）， SIRT1 and PGC-1α in rat kidney tissue. The protein expressions of autophagy-specific gene （Beclin-1）， microtubule-associated protein 1 light chain 3 （LC3） Ⅱ， ubiquitin-binding protein （p62）， B cell lymphoma （Bcl-2）， Bcl-2-associated X （Bax）， and cysteine aspartic protease-7 （Caspase-7） in rat kidney tissue were determined by immunohistochemistry （IHC）. ResultCompared with the conditions in the normal group， the levels of UTP， IL-6， TNF-α， CRP and MDA in the model group were increased （P<0.05） while the levels of SOD and GSH-Px were decreased （P<0.05）， and there was no difference in BUN and SCr. Compared with the model group， the administration groups had lowered UTP， IL-6， TNF-α， CRP and MDA （P<0.05） while elevated SOD and GSH-Px （P<0.05）. It could be seen from hematoxylin and eosin （HE） staining， Masson staining， immunofluorescence and electron microscopy that the pathological damage of rat kidney tissue in the model group was significant， but after treatment with benazepril hydrochloride and salvianolate， the pathological damage of kidney cells was gradually improved. The expressions of p-AMPK/AMPK， p-SIRT1/SIRT1， PGC-1α， Bcl-2， Beclin-1 and LC3Ⅱ in rat kidney in the model group were lower than those in the normal group （P<0.05） while the expressions of Bax， Caspase-7 and p62 were higher （P<0.05）. Compared with the model group， benazepril hydrochloride group and salvianolate groups had an up-regulation in the expressions of p-AMPK/AMPK， p-SIRT1/SIRT1， PGC-1α， Bcl-2， Beclin-1 and LC3Ⅱ in the kidney （P<0.05） while a down-regulation in the expressions of Bax， Caspase-7 and p62 （P<0.05）. ConclusionThe protective effect of salvianolate on the kidneys of MN rats may be related to the activation of AMPK/SIRT1/PGC-1α signaling pathway， the up-regulation of autophagy and the reduction of apoptosis.

Guiqi Baizhu Prescription Combined with Oxaliplatin Protects Intestinal Barrier of Tumor-bearing Mice with Gastric Cancer by Regulation of VIP/cAMP/PKA/AQP Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Guiqi Baizhu prescription combined with oxaliplatin on the intestinal barrier of tumor-bearing mice with gastric cancer by regulating downstream aquaporin 3 （AQP3） and aquaporin 4 （AQP4） through the vasoactive intestinal peptide （VIP）/cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA） signaling pathway. MethodThe gastric cancer cell lines MFC with a density of 1×107/mL were prepared into cell suspension. The tumor-bearing mouse model of gastric cancer was established by inoculating 0.2 mL cell suspension under the right axilla of mice. After successful modeling， mice were randomly divided into 5 groups， namely， model group， oxaliplatin group （10 mg·kg-1）， and high， medium， and low-dose oxaliplatin + Guiqi Baizhu prescription groups （17.68， 8.84， 4.42 g·kg-1）， with 10 mice in each group， and the remaining 10 mice were set as a blank group. Mice in each group were treated with Chinese medicine， oxaliplatin， or normal saline by gavage or intraperitoneal injection for 14 d. The next day after the last dose， blood was taken from the eyeball to separate serum and take colonic samples. Hematoxylin-eosin （HE） staining was used to observe the changes in tissue morphology. The content of D-lactate acid （D-LA） and diamine oxidase （DAO） in the serum was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of VIP， cAMP， PKA， AQP3， and AQP4 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the blank group， the model group showed edema in the colonic submucosa， disordered arrangement of intestinal glands in the mucosal layer， loss of goblet cells， infiltration of inflammatory cells， and villus shedding. However， there were different degrees of improvement in each administration group. As compared with the blank group， the serum levels of DAO and D-LA in the model group were significantly increased （P<0.01）. As compared with the model group， the levels of DAO and D-LA in the high-dose oxaliplatin + Guiqi Baizhu prescription group and the level of D-LA in the medium-dose oxaliplatin + Guiqi Baizhu prescription group were decreased （P<0.05， P<0.01）. As compared with the oxaliplatin group， the levels of D-LA in the high and medium-dose oxaliplatin + Guiqi Baizhu prescription groups were decreased （P<0.05）， and the levels of DAO and D-LA in other administration groups were decreased as well， but the difference had no statistical significance. As compared with the blank group， the mRNA and protein expression levels of VIP， cAMP， PKA， AQP3， and AQP4 in the model group were significantly decreased （P<0.05， P<0.01）. As compared with the model group， the mRNA and protein expression levels of VIP， cAMP， PKA， AQP3， and AQP4 in each administration group were increased， and those in the high-dose oxaliplatin + Guiqi Baizhu prescription group were significantly increased （P<0.05， P<0.01）， while the protein expression level of cAMP in the medium-dose oxaliplatin + Guiqi Baizhu prescription group were increased （P<0.05）. As compared with the oxaliplatin group， the protein expression levels of cAMP in the high-dose oxaliplatin + Guiqi Baizhu prescription group were increased （P<0.05）， and the mRNA and protein expressions of these indexes in the other groups were also increased but the differences were not statistically significant. ConclusionGuiqi Baizhu prescription combined with oxaliplatin can regulate AQP3 and AQP4 through the VIP/cAMP/PKA signaling pathway to protect the intestinal barrier of tumor-bearing mice with gastric cancer.

Significance of high expression of C5orf46 in gastric cancer and potential intervention of tarditional Chinese medicine based on bioinformatics, molecular docking, and cell experiments / 中国中药杂志

This study aims to investigate the expression, prognosis, and clinical significance of C5orf46 in gastric cancer and to study the interaction between the active components of C5orf46 and tarditional Chinese medicine. The ggplot2 package was utilized for differential expression analysis of C5orf46 in gastric cancer tissues and normal tissues. The survival package was used for survival analysis, univariate regression analysis, and multivariate regression analysis. Nomogram analysis was used to assess the connection between C5orf46 expression in gastric cancer and overall survival. The abundance of tumor-infiltrating lymphocytes was calculated by GSVA package. Coremine database, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and PubChem database were used to search the potential components corresponding to C5orf46 gene and tarditional Chinese medicine. Molecular docking was performed to explore the binding affinity of potential components to C5orf46. Cell experiments were performed to explore the expression of C5orf46 gene in cells of the blank group, model group, and drug administration groups. As compared with normal tissues, C5orf46 expression was higher in gastric cancer tissues, which had more significant predictive effects in the early stages(T2, N0, and M0). The more advanced the tumor node metastasis(TNM) stage, the higher the C5orf46 expression and the lower the probability of survival of patients with gastric cancer. The expression of C5orf46 positively correlated with the helper T cells1 in gastric cancer and the macrophage infiltration level in gastric cancer, and negatively correlated with B cells, central memory T cells, helper T cells 17, and follicular helper T cells. Seven potential components of C5orf46 were obtained, and three active components were obtained after the screening, which matched five tarditional Chinese medicines, namely, Sojae Semen Nigrum, Jujubae Fructus, Trichosanthis Fructus, Silybi Fructus, and Bambusae Concretio Silicea. Molecular docking revealed that sialic acid and adeno-sine monophosphate(AMP) had a good binding ability to C5orf46. The results of real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot showed that, as compared with the model group, the mRNA and protein expression levels of C5orf46 were significantly lower in the drug administration groups. The lowest expression level was found at the concentration of 40 μmol·L~(-1). The results of this study provide ideas for the clinical development of traditional Chinese medicine compounds for the treatment of gastric cancer as well as other cancers.

Bone-derived MSCs encapsulated in alginate hydrogel prevent collagen-induced arthritis in mice through the activation of adenosine A2A/2B receptors in tolerogenic dendritic cells

Tolerogenic dendritic cells (tolDCs) facilitate the suppression of autoimmune responses by differentiating regulatory T cells (Treg). The dysfunction of immunotolerance results in the development of autoimmune diseases, such as rheumatoid arthritis (RA). As multipotent progenitor cells, mesenchymal stem cells (MSCs), can regulate dendritic cells (DCs) to restore their immunosuppressive function and prevent disease development. However, the underlying mechanisms of MSCs in regulating DCs still need to be better defined. Simultaneously, the delivery system for MSCs also influences their function. Herein, MSCs are encapsulated in alginate hydrogel to improve cell survival and retention in situ, maximizing efficacy in vivo. The three-dimensional co-culture of encapsulated MSCs with DCs demonstrates that MSCs can inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines. In the collagen-induced arthritis (CIA) mice model, alginate hydrogel encapsulated MSCs induce a significantly higher expression of CD39+CD73+ on MSCs. These enzymes hydrolyze ATP to adenosine and activate A2A/2B receptors on immature DCs, further promoting the phenotypic transformation of DCs to tolDCs and regulating naïve T cells to Tregs. Therefore, encapsulated MSCs obviously alleviate the inflammatory response and prevent CIA progression. This finding clarifies the mechanism of MSCs-DCs crosstalk in eliciting the immunosuppression effect and provides insights into hydrogel-promoted stem cell therapy for autoimmune diseases.

Clinical application value of Huanglian Jiedu Pills in improving syndrome of excess heat and fire toxin based on phase Ⅱ clinical trial study on plasma ATP, 4-HNE, and ACTH levels / 中国中药杂志

A randomized, double-blind, placebo-controlled, multi-center phase Ⅱ clinical trial design was used in this study to recruit subjects who were in line with the syndrome of excess heat and fire toxin, and were diagnosed as recurrent oral ulcers, gingivitis, and acute pharyngitis. A total of 240 cases were included and randomly divided into a placebo group and a Huanglian Jiedu Pills group. The clinical efficacy of Huanglian Jiedu Pills in treating the syndrome of excess heat and fire toxin was evaluated by using the traditional Chinese medicine(TCM) syndrome scale. Enzyme-linked immunosorbent assay(ELISA) was used to determine and evaluate the levels of adenosine triphosphate(ATP), 4-hydroxynonenal(4-HNE), and adrenocorticotropic hormone(ACTH) in plasma of the two groups before and after administration and to predict their application value as clinical biomarkers. The results showed that the disappearance rate of main symptoms in the Huanglian Jiedu Pills group was 69.17%, and that in the placebo group was 50.83%. The comparison between the Huanglian Jiedu Pills group and the placebo group showed that 4-HNE before and after administration was statistically significant(P<0.05). The content of 4-HNE in the Huanglian Jiedu Pills group decreased significantly after administration(P<0.05), but that in the placebo group had no statistical significance and showed an upward trend. After administration, the content of ATP in both Huanglian Jiedu Pills group and placebo group decreased significantly(P<0.05), indicating that the energy metabolism disorder was significantly improved after administration of Huanglian Jiedu Pills and the body's self-healing ability also alleviated the increase in ATP level caused by the syndrome of excess heat and fire toxin to a certain extent. ACTH in both Huanglian Jiedu Pills group and placebo group decreased significantly after administration(P<0.05). It is concluded that Huanglian Jiedu Pills has a significant clinical effect, and can significantly improve the abnormal levels of ATP and 4-HNE in plasma caused by the syndrome of excess heat and fire toxin, which are speculated to be the effective clinical biomarkers for Huanglian Jiedu Pills to treat the syndrome of excess heat and fire toxin.

Hypoglycemic effect of electroacupuncture at "Tianshu" (ST 25) combined with metformin on rats with type 2 diabetes mellitus based on AMPK / 中国针灸

OBJECTIVE@#To observe the hypoglycemic effect of electroacupuncture (EA) at "Tianshu" (ST 25) combined with metformin on rats with type 2 diabetes mellitus (T2DM) as well as its effect on expression of adenosine monophosphate activated protein kinase (AMPK) in liver and pancreas.@*METHODS@#Thirty-six male SD rats were randomly divided into a blank group (6 rats) and a model establishing group (30 rats). The rats in the model establishing group were fed with high-fat diet and treated with intraperitoneal injection of low-dose streptozotocin (STZ) to establish T2DM model. The rats with successful model establishment were randomly divided into a model group, a control group, a metformin group, an EA group and a combination group, 6 rats in each group. The rats in the EA group were treated with EA at "Tianshu" (ST 25), dense-disperse wave, 2 Hz/15 Hz in frequency and 2 mA in current intensity, 20 min each time. The rats in the metformin group were treated with intragastric administration of metformin (190 mg/kg) dissolved in 0.9% sodium chloride solution (2 mL/kg). The rats in the combination group were treated with EA at "Tianshu" (ST 25) and intragastric administration of metformin. The rats in the control group were treated with intragastric administration of 0.9% sodium chloride solution with the same dose. All the treatments were given once a day for 5 weeks. After the intervention, the body mass and random blood glucose were detected; the serum insulin level was detected by ELISA; the expression of AMPK and phosphorylated adenosine monophosphate activated protein kinase (p-AMPK) in liver and pancreas was detected by Western blot method; the expression of protein gene product 9.5 (PGP9.5) was detected by immunofluorescence.@*RESULTS@#①Compared with the blank group, the body mass in the model group was decreased (P<0.05); compared with the model group, the body mass in the EA group and the combination group was decreased (P<0.05); the body mass in the EA group and the combination group was lower than the metformin group (P<0.05). Compared with the blank group, the random blood glucose in the model group was increased (P<0.01); compared with the model group, the random blood glucose in the metformin group, the EA group and the combination group was decreased (P<0.01). The random blood glucose in the combination group was lower than the metformin group and the EA group (P<0.05). ②Compared with the blank group, the insulin level in the model group was decreased (P<0.05); compared with the model group, the insulin level in the metformin group, the EA group and the combination group was all increased (P<0.05). The insulin level in the combination group was higher than the metformin group and the EA group (P<0.05). ③Compared with the blank group, the protein expression of AMPK and p-AMPK in liver tissue was decreased (P<0.05), and the protein expression of AMPK and p-AMPK in pancreatic tissue was increased (P<0.05) in the model group. Compared with the model group, the protein expression of AMPK and p-AMPK in liver tissue in the metformin group, the EA group and the combination group was increased (P<0.05, P<0.01); the protein expression of AMPK in pancreatic tissue in the metformin group was increased (P<0.05); the protein expression of AMPK in pancreatic tissue in the EA group and the combination group was decreased (P<0.05); the protein expression of p-AMPK in pancreatic tissue in the metformin group, the EA group and the combination group was decreased (P<0.05). The protein expression of AMPK and p-AMPK in liver tissue in the combination group was higher than that in the metformin group and the EA group (P<0.05); the protein expression of AMPK in pancreatic tissue in the EA group and the combination group was less than that in the metformin group (P<0.05), and the expression of p-AMPK protein in pancreatic tissue in the combination group was less than that in the metformin group and the EA group (P<0.05). ④Compared with the blank group, the expression of PGP9.5 in pancreatic tissue in the model group was increased (P<0.01); compared with the model group, the expression of PGP9.5 in pancreatic tissue in the metformin group, the EA group and the combination group was decreased (P<0.05, P<0.01). The expression of PGP9.5 in pancreatic tissue in the EA group was lower than the metformin group and the combination group (P<0.05).@*CONCLUSION@#Electroacupuncture at "Tianshu" (ST 25) could promote the effect of metformin on activating AMPK in liver tissue of T2DM rats, improve the negative effect of metformin on AMPK in pancreatic tissue, and enhance the hypoglycemic effect of metformin. The mechanism may be related to the inhibition of pancreatic intrinsic nervous system.

Pre-treatment of the beta3-adrenergic receptor agonist BRL37344 reduces in vivo myocardial ischemia/reperfusion injury by improving AMPK and SIRT1 activity and by suppressing mTOR and p70S6K signaling pathways

Abstract This study aimed to investigate the role and signaling pathways of β3-AR in myocardial ischemia/reperfusion (I/R) injury, which is one of the leading causes of death worldwide. 47 male rats were randomly divided into two main groups to evaluate infarct size and molecular parameters. Rats in both groups were randomly divided into 4 groups. Control (sham), I/R (30 min ischemia/120 min reperfusion), BRL37344 (BRL) (A) (5 µg/kg single-dose pre-treatment (preT) before I/R) and BRL (B) (5 µg/kg/day preT for 10 days before I/R). Infarct size was determined with triphenyltetrazolium chloride staining and analyzed with ImageJ program. The levels of AMPK, SIRT1, mTOR, and p70SK6 responsible for cellular energy and autophagy were evaluated by western blot. Infarct size increased in the I/R group (44.84 ± 1.47%) and reduced in the single-dose and 10-day BRL-treated groups (32.22 ± 1.57%, 29.65 ± 0.55%; respectively). AMPK and SIRT1 levels were decreased by I/R but improved in the treatment groups. While mTOR and p70S6K levels increased in the I/R group, they decreased with BRL preT. BRL preT protects the heart against I/R injury. These beneficial effects are mediated in part by activation of AMPK and SIRT1, inhibition of mTOR and p70S6K, and consequently protected autophagy.

Role of cardiac β1-adrenergic and A1-adenosine receptors in severe arrhythmias related to Parkinson's disease

Abstract Aims Although reduced life expectancy in Parkinson's Disease (PD) patients has been related to severe cardiac arrhythmias due to autonomic dysfunctions, its molecular mechanisms remain unclear. To investigate the role of cardiac β1-Adrenergic (β1AR) and A1-Adenosine (A1R) receptors in these dysfunctions, the pharmacological effects of stimulation of cardiac β1AR (isoproterenol, ISO), in the absence and presence of cardiac β1AR (atenolol, AT) or A1R (1,3-dipropyl-8-cyclopentyl xanthine, DPCPX) blockade, on the arrhythmias induced by Ischemia/Reperfusion (CIR) in an animal PD model were studied. Methods PD was produced by dopaminergic lesions (confirmed by immunohistochemistry analysis) caused by the injection of 6-hydroxydopamine (6-OHDA, 6 μg) in rat striatum. CIR was produced by a surgical interruption for 10 min followed by reestablishment of blood circulation in the descendent left coronary artery. On the incidence of CIR-Induced Ventricular Arrhythmias (VA), Atrioventricular Block (AVB), and Lethality (LET), evaluated by Electrocardiogram (ECG) analysis, the effects of intravenous treatment with ISO, AT and DPCPX (before CIR) were studied. Results VA, AVB and LET incidences were significantly higher in 6-OHDA (83%, 92%, 100%, respectively) than in control rats (58%, 67% and 67%, respectively). ISO treatment significantly reduced these incidences in 6-OHDA (33%, 33% and 42%, respectively) and control rats (25%, 25%, 33%, respectively), indicating that stimulation of cardiac β1AR induced cardioprotection. This response was prevented by pretreatment with AT and DPCPX, confirming the involvement of cardiac β1AR and A1R. Conclusion Pharmacological modulation of cardiac β1AR and A1R could be a potential therapeutic strategy to reduce severe arrhythmias and increase life expectancy in PD patients.

A brazilian nationwide multicenter study on deficiency of deaminase-2 (DADA2)

Abstract Introduction The deficiency of ADA2 (DADA2) is a rare autoinflammatory disease provoked by mutations in the ADA2 gene inherited in a recessive fashion. Up to this moment there is no consensus for the treatment of DADA2 and anti-TNF is the therapy of choice for chronic management whereas bone marrow transplantation is considered for refractory or severe phenotypes. Data from Brazil is scarce and this multicentric study reports 18 patients with DADA2 from Brazil. Patients and methods This is a multicentric study proposed by the Center for Rare and Immunological Disorders of the Hospital 9 de Julho - DASA, São Paulo - Brazil. Patients of any age with a confirmed diagnosis of DADA2 were eligible for this project and data on clinical, laboratory, genetics and treatment were collected. Results Eighteen patients from 10 different centers are reported here. All patients had disease onset at the pediatric age (median of 5 years) and most of them from the state of São Paulo. Vasculopathy with recurrent stroke was the most common phenotype but atypical phenotypes compatible with ALPS-like and Common Variable Immunodeficiency (CVID) was also found. All patients carried pathogenic mutations in the ADA2 gene. Acute management of vasculitis was not satisfactory with steroids in many patients and all those who used anti-TNF had favorable responses. Conclusion The low number of patients diagnosed with DADA2 in Brazil reinforces the need for disease awareness for this condition. Moreover, the absence of guidelines for diagnosis and management is also necessary (t).

Mechanism of Gegen Qinliantang in Improving Ectopic Lipid Accumulation in Liver of db/db Mice with Type 2 Diabetes Mellitus by Regulating AMPK-FoxO3a Autophagy Axis / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Gegen Qinliantang （GQT） in improving ectopic lipid accumulation in the liver of db/db mice with type 2 diabetes mellitus （T2DM） by regulating the adenosine monophosphate-activated protein kinase （AMPK）-forkhead box O3a （FoxO3a） autophagy axis， to provide a scientific basis for clarifying the hypoglycemic mechanism of GQT and its clinical application. MethodSeventy-five spontaneous T2DM db/db mice and 15 normal db/m mice were selected and maintained on a regular diet for one week， followed by the measurement of blood glucose. They were then randomly divided into six groups， with 15 mice in each group， including normal group （0.2 g·kg-1 saline）， metformin group （0.2 g·kg-1）， high-， medium， and low-dose GQT group （31.9， 19.1， 6.9 g·kg-1）， and model group （0.2 g·kg-1 saline）. The mice were orally administered the corresponding drugs once daily for 12 weeks. Fasting blood glucose （FBG） and glycated hemoglobin （HbA1c） were detected. Fasting insulin （FINS） and free fatty acid （FFA） levels were measured by enzyme-linked immunosorbent assay （ELISA）. Pathological changes in liver tissues were observed by hematoxylin-eosin （HE） staining. The protein expression levels of phosphorylated （p）-AMPK， p-FoxO3a， and autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ） and p62 were detected using Western blot. Immunofluorescence was used to detect the expression of hypoxia-inducible factor-1α （HIF-1α） in liver tissues. Real-time polymerase chain reaction （Real-time PCR） was performed to detect the mRNA expression of AMPK， FoxO3a， and LC3 in liver tissues. ResultCompared with the normal group， the model group showed pathological changes in liver tissues， increased FBG， HbA1c， FINS， and FFA levels （P<0.01）， increased protein expression levels of p-AMPK， p62， and HIF-1α， decreased protein expression levels of p-FoxO3a and LC3Ⅱ in liver tissues （P<0.01）， decreased mRNA expression of AMPK， and increased expression of FoxO3a （P<0.01）. Compared with the model group， the treatment groups showed relieved liver tissue lesions and decreased FBG， HbA1c， FINS， and FFA levels （P<0.01）. The expression of p-AMPK， p62， and HIF-1α increased， while the expression of p-FoxO3a showed a dose-dependent decrease in the high-dose GQT group. The expression of LC3Ⅱ increased in the metformin group and the high-dose GQT group （P<0.01）. The mRNA expression of AMPK showed a dose-dependent increase， and the expression of FoxO3a showed a dose-dependent decrease in the treatment groups （P<0.01）. ConclusionGQT can improve ectopic lipid accumulation in the liver of T2DM db/db mice， which may be related to the regulation of the AMPK-FoxO3a autophagy axis.

The value of peripheral blood and pleural effusion T-SPOT.TB combined with pleural effusion adenosine deaminase in diagnosis of tuberculous pleurisy / 中国热带医学

@#Abstract: Objective To explore the relationship between peripheral blood and pleural effusion tuberculosis (TB) infection effector T cells, and to further evaluate the value of combined pleural effusion adenosine deaminase (ADA) for rapid diagnosis of tuberculous pleurisy. Methods The test data of 80 cases of tuberculous pleurisy and 70 cases of nontuberculous pleurisy treated in the Sixth People's Hospital of Nantong City from January 2017 to December 2020 were analyzed. The TBinfected effector T cells were also detected simultaneously in the peripheral blood and the pleural effusion by the T-SPOT technique, and the pleural effusion ADA was detected by the rate method. The subject operating characteristic curve (ROC) was applied to take the optimal pleural effusion ADA threshold to compare the sensitivity and specificity of different critical values. Person phase analysis was applied to analyze the correlation between peripheral blood and pleural effusion T-SPOT.TB. Data of peripheral blood, pleural effusion T-SPOT.TB and ADA were integrated. Results When pleural effusion ADA>45 U/L, the sensitivity and specificity for the diagnosis of tuberculous pleurisy were 50.0% and 94.3%, respectively; when ADA > 25.15 U/ L, the sensitivity and specificity were 80.0% and 72.9%. When ADA > 45 U / L, pleural/ blood T-SPOT.TB spot ratio (spot forming cells, SFCs) > 2 times, the specificity for the diagnosis of tuberculous pleurisy was 100% (highest); when 25.15 U/L< pleural effusion ADA ≤ 45 U/L, pleural/blood T-SPOT.TB spot ratio > 2 times, the specificity for the diagnosis of tuberculous pleurisy was 92.3% (second). When pleural effusion ADA ≤ 25.15 U/L, and the pleural effusion/blood T-SPOT.TB spot number ratio > 2 times, with 83.3% specificity (the lowest of the three groups). Conclusions The level of pleural effusion ADA is one of the most used methods for diagnosing tuberculous pleurisy. Further combination of pleural effusion and blood T-SPOT.TB, if the ratio of pleural effusion / blood T-SPOT. TB spots is greater than 2 times, it can further improve the diagnosis rate of tuberculous pleurisy.

Role of purine signaling in liver immune regulation / 临床肝胆病杂志

Purines are mainly composed of ATP, NAD + , and nucleic acid. In addition to their key intracellular functions, NAD + , ATP, and their hydrolyzed products (including ADP, AMP, and adenosine) are important extracellular signals involved in physiological processes and pathological conditions. Purine signaling plays an important role in immune regulation of liver microenvironment. This article mainly summarizes the regulatory effect of purine signaling on immune cells in the liver and the effect of purine signaling on the progression of liver diseases by regulating the inflammatory and anti-inflammatory responses of immune cells in the liver.