Mechanism of Hedysari Radix Polysaccharide on Diabetic Nephropathy in db/db Mice Based on JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Hedysari Radix polysaccharide （HRP） on the Janus kinase 2 （JAK2）/signal transducer and activator of transcription protein 3 （STAT3） signaling pathway in diabetic nephropathy db/db mice. MethodFifty db/db mice were randomly divided into model group， irbesartan group （irbesartan suspension， 22.75 mg·kg-1）， and high-， medium-， and low-dose HRP groups （HRP suspension， 200， 100， 50 mg·kg-1） according to the body weight， with 10 mice in each group. Another 10 C57BL/6 mice were assigned to the normal group. The mice were treated with corresponding drugs by gavage， while those in the normal group and the model group received distilled water at 5 mL·kg-1. The mice in the six groups were administered once a day by gavage for 12 consecutive weeks. The uric acid （UA）， triglycerides （TG）， and total cholesterol （TC） were detected. Periodic acid-Schiff （PAS） staining and Masson staining were used to observe the pathological changes in kidney tissues. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to detect the protein and mRNA expression levels of JAK2， STAT3， suppressor of cytokine signaling 3 （SOCS3）， and tumor necrosis factor-α （TNF-α） in the kidney. ResultAfter 12 weeks of treatment， compared with the normal group， the model group showed significant pathological ultrastructural changes in kidney tissues and increased UA， TG， and TC levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed improvement in pathological ultrastructure of kidney tissues and reduced UA， TG， and TC levels （P<0.05， P<0.01）. Compared with the normal group， the model group showed a decrease in SOCS3 protein and mRNA expression levels and an increase in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.01）. Compared with the model group， the high- and medium-dose HRP groups and the irbesartan group showed an increase in SOCS3 protein and mRNA expression levels and a decrease in JAK2， STAT3， and TNF-α protein and mRNA expression levels （P<0.05， P<0.01）. ConclusionHRP can alleviate renal damage in diabetic nephropathy to a certain extent， and its mechanism may be related to the inhibition of the activation of the JAK2/STAT3 signaling pathway.

Regulatory Effect of Herbal Pair Astragali Radix-Puerariae Lobatae Radix on PI3K/Akt/FoxO1 Signaling Pathway in Rats with T2DM Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of herbal pair Astragali Radix-Puerariae Lobatae Radix （AR-PLR） against type 2 diabetes mellitus （T2DM） based on network pharmacology and experimental verification. MethodThe active ingredients and targets of AR and PLR were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. The related targets of T2DM were retrieved from disease databases and the common targets of drugs and diseases were extracted. The protein-protein interaction （PPI） network was analyzed and constructed by STRING and the network topology of key targets was analyzed by Cytoscape 3.7.1. Then gene ontology（GO） and Kyoto encyclopedia of genes and genomes（KEGG） enrichment analyses of core targets were carried out by DAVID to explore its possible molecular mechanism. The T2DM model was induced in rats by the high-fat diet combined with tail intravenous injection of streptozocin. The rats were divided into a normal group，a model group，a metformin group，and high-，medium- and low-dose AR-PLR groups. After four weeks of intragastric administration，the serum levels of fasting blood sugar （FBS），fasting insulin（FINS），aspartate aminotransferase（AST），alanine aminotransferase（ALT），triglyceride（TG），total cholesterol（TC），low-density lipoprotein cholesterin（LDL-C），high-density lipoprotein cholesterin （HDL-C），interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） of rats in each group were measured. The protein expression of insulin receptor substrate-2（IRS-2），phosphatidylinositol 3-kinase（PI3K），protein kinase B（Akt）， and forkhead box transcription factor O1（FoxO1） in rat liver was detected by Western blot. ResultA total of 131 core targets of AR-PLR in the treatment of T2DM were screened out by network pharmacology， where Akt1，mitogen-activated protein kinase 1 （MAPK1），TNF-α，and IL-6 were critical. As revealed by KEGG enrichment analysis， AR-PLR exerted the hypoglycemic effect mainly through the PI3K/Akt，TNF， and FoxO signaling pathways. Compared with the model group，the high- and medium-dose AR-PLR groups showed reduced FBS and FINS levels and increased glycogen level （P<0.05，P<0.01），all the AR-PLR groups showed decreased levels of AST，ALT，TG， and LDL-C （P<0.05，P<0.01）， the high- and low-dose AR-PLR groups showed decreased TC levels （P<0.05）. Compared with the model group， the high- and medium-dose AR-PLR groups showed reduced levels of IL-6 and TNF-α（P<0.05，P<0.01）， and the high-dose AR-PLR group showed increased expression of IRS-2， Akt， p-Akt， PI3K， and p-PI3K， and decreased expression of FoxO1 protein（P<0.05）. ConclusionAR-PLR has the characteristics of multi-component，Multi-target and multi-pathway in the treatment of T2DM. This herbal pair may regulate the PI3K/Akt/FoxO1 signaling pathway through IL-6， TNF-α， and other targets to affect insulin resistance， glycogen synthesis， gluconeogenesis， glucose transport， inflammation， immune response， and other processes， thereby treating T2DM.

Effect of Maxing Shigantang on Airway Injury and EGFR Expression in Asthma Model Rats / 中国实验方剂学杂志

ObjectiveTo explore the optimal formula of Maxing Shigantang in regulating epidermal growth factor receptor（EGFR）expression and alleviating airway injury in asthmatic rats and to reveal the underlying mechanism. MethodSD male rats were randomly divided into normal group, model group, dexamethasone group (5×10-4 g·kg-1) and Maxing Shigantang 1∶0.5, 1∶1, 1∶2 groups (group A, B, C, 10 g·kg-1), with 8 rats in each group. The other groups except the normal group received nebulization of 2% acetylcholine chloride and 0.4% histamine phosphate for the modeling of asthma. One hour before modeling， the normal group and the model group were given the same amount of normal saline， and the other groups were given the same amount of corresponding drugs， once a day for 7 days. On the 7th day， the model was established and the incubation period of asthma was recorded. The rats were then immediately anesthetized， and arterial blood and tracheal tissue were collected. Enzyme-linked immunosorbent assay （ELISA） was employed to detect the levels of interleukin-2 （IL-2）， interleukin-4 （IL-4）， and tumor necrosis factor-α （TNF-α） in serum. Pathological sections were prepared for the observation of the pathological changes of tracheal tissues and the ultrastructure of epithelial cells in each group. Terminal-deoxynucleotidyl transferase-mediated nick-end labeling （TUNEL） was adopted to detect epithelial cell apoptosis， and in situ hybridization and Western blot were employed to determine the mRNA and protein levels of epidermal growth factor receptor （EGFR）， respectively. ResultCompared with the model group， groups A， B and C prolonged the incubation period of asthma （P<0.05，P<0.01）. Compared with the control group， the model group showed declined IL-2 level （P<0.01）， risen IL-4 and TNF-α levels （P<0.05，P<0.01）， increased airway pathology score， collagen volume fraction， and airway epithelial cell apoptosis index （P<0.01）， and up-regulated mRNA and protein levels of EGFR in trachea tissue （P<0.01）. Compared with the model group， group A showed increased IL-2 level （P<0.05） and declined IL-4 （P<0.05，P<0.01） level， and group B showed declined IL-4 level （P<0.05）. The level of TNF-α in groups A， B， and C declined compared with that in the model group （P<0.01）. Maxing Shigantang repaired the tracheal tissue to different degrees （P<0.05）. Among the three groups， group A inhibited tracheal fibrosis （P<0.05） and had the most significant effect of repairing the ultrastructural changes of airway epithelial cells. Groups A， B and C all inhibited the apoptosis of airway epithelial cells （P<0.05）. All the three groups inhibited the up-regulation of EGFR mRNA level （P<0.05，P<0.01）， and groups B and C inhibited the up-regulation of EGFR protein level （P<0.05，P<0.01）. ConclusionMaxing Shigantang can inhibit the abnormal changes of airway epithelial structure， alleviate airway injury， and can down-regulate the expression of EGFR in the tracheal tissue of asthma model rats. In this study， the optimal compatibility of Maxing Shigantang to repair airway epithelial injury in asthmatic rats was group A， with the Ephedrae Herba-Armeniacae Semen Amarum-Glycyrrhizae Radix et Rhizoma-Gypsum Fibrosum ratio of 1∶0.5∶4∶1.

Effect of Modifide Wenjingtang on JAK2/STAT3 Signal Pathway and Downstream Factors Protein Expression in Endometriosis Rats / 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:The 10 from 105 SPF female healthy SD rats were randomly selected as the blank group. The rest constructed the rat model of kidney deficiency and blood stasis by compound factorial method. After the model was successfully established， 10 rats were randomly selected as the sham operation group， with only laparotomy and no intima suture， and the remaining rats were established with EM kidney deficiency and blood stasis type by autologous intimal transplantation. Fifty rats which were randomly selected from 56 successful rats were treated with the modified Wenjingtang （5，10，20 g·kg^-1） and danazol group（63 mg·kg^-1）， 1 time daily ， for 4 weeks. The endometrial tissues of each group were stained with hematoxylin eosin （HE） to observe the histopathology. The levels of inflammatory factors interleukin-10 （IL-10） and interleukin-17 （IL-17） in serum supernatant were detected by enzyme linked immunosorbent assay （ELISA）. Measuring the length（D₁），width （D₂） and height （D₃） of the heterotopic foci in each group before and after treatment. Then calculating the volume of them. The expression of tyrosine kinase 2（JAK2），transcription factor 3 （STAT3），phosphorylation transcription factor 3 （p-STAT3）， vascular endothelial growth factor （VEGF）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and thrombospondin-1 （TSP-1） were detected by immunohistochemistry （IHC）. The expression of VEGF，TNF-<italic>α</italic> and TSP-1 was detected by Western blot. Result:Microscopic pathological observation showed that the endometrial glandular cells of the blank group were arranged in order， and the glandular and stromal cells grew well， compared with the blank group， the endometrial structure of the model group was complete， showing a cavity like or annular closed structure， with cyst formation， and the epithelium was cubic or columnar epithelium， most of the epithelial cells had secretion， the stroma was dense， and the matrix showed a little fibrosis There were a few glands and inflammatory cell infiltration. Compared with the blank group， the content of IL-10 in serum of model group was significantly decreased （<italic>P</italic><0.01）， and the content of IL-17 was significantly increased （<italic>P</italic><0.01）， the protein expression of JAK2， STAT3，p-STAT3， VEGF， TNF-<italic>α</italic> in endometrial tissue of model group was significantly increased （<italic>P</italic><0.05）， and the expression of TSP-1 protein was significantly decreased （<italic>P</italic><0.05）. Compared with the model group， the serum IL-10 content of rats in modified Wenjingtang treatment group increased significantly （<italic>P</italic><0.01）， the IL-17 content decreased significantly <italic>（P</italic><0.01）， and the volume of ectopic foci decreased significantly （<italic>P</italic><0.01）. While the level of JAK2，STAT3，p-STAT3，TNF-<italic>α</italic>，VEGF protein in intimal tissue of modified Wenjingtang high and middle dose group decreased significantly （<italic>P</italic><0.05） and the level of TSP-1 protein increased significantly （<italic>P</italic><0.05）. Conclusion:Modified Wenjingtang can inhibit the invasion of ectopic foci in EM rats with kidney deficiency and blood stasis， the mechanism may be related to the intervention of immune barrier and block angiogenesis function mediated by JAK2/STAT3 signaling pathway activation.

Effect of Different Extracts and Monomers of Lepidium meyenii on Proliferation of Mouse Splenic Lymphocyte and Induction of IL-2 and TNF-α / 中国实验方剂学杂志

Objective:To explore effects of different extracts and monomers of <italic>Lepidium meyenii </italic>（Maca） on the proliferation of mouse splenic lymphocytes and induction of interleukin-2 （IL-2） and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） by observing their immunomodulatory effects. Method:An octadecylsilyl （ODS） column was used to enrich the methanol extract of <italic>L. meyenii</italic> in stages to obtain six fractions and three monomers. Different groups of extracts and monomers of <italic>L. meyenii </italic>at different doses were set up. Cell counting Kit-8 （CCK-8） was used to detect the effect on the proliferation of mitogen-free， concanavalin A （Con A）-induced， and lipopolysaccharides （LPS）-induced mouse splenic lymphocytes. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of IL-2 and TNF-<italic>α</italic>. Result:<italic>L. meyenii </italic>extracts Fr₃ and Fr₆， and monomers <italic>N</italic>-benzyl hexadecanamide and 1，2-dihydro-4-carboxaldehyde-3-benzyl-<italic>N</italic>-hydroxypyridine slightly promoted the proliferation of Con A-induced T lymphocytes and LPS-induced B lymphocytes （<italic>P</italic><0.01） as compared with the conditions in the model group. <italic>L. meyenii</italic> extracts and monomers significantly induced the secretion of IL-2 and TNF-<italic>α</italic> by splenic lymphocytes （<italic>P</italic><0.01）. Conclusion:<italic>L. meyenii</italic> extracts and monomers can achieve immunological enhancement by promoting the secretion of IL-2 and TNF-<italic>α</italic>， and facilitate the proliferation of splenic lymphocytes. The active components are presumedly macamides and pyridine alkaloids， and the specific mechanism still needs to be further explored.

Effect of acupuncture on patients with cancer-related fatigue and serum levels of CRP, IL-6, TNF-α and sTNF-R1 / 中国针灸

OBJECTIVE@#To observe the therapeutic effect of acupuncture on cancer-related fatigue (CRF) and to explore its possible mechanism.@*METHODS@#A total of 80 patients with CRF were randomized into an observation group and a control group, and finally 67 patients completed the trial (36 patients in the observation group, 31 patients in the control group). Patients in the control group were treated with conventional chemoradiotherapy and symptomatic treatment, while no particular anti-fatigue intervention was adopted. On the basis of treatment in the control group, acupuncture was applied at Baihui (GV 20), Guanyuan (CV 4), Qihai (CV 6), Fengchi (GB 20), Zusanli (ST 36), Sanyinjiao (SP 6) in the observation group, once a day, 5 times as one course, with 2 days interval between each course, totally 4 courses were required. Before and after treatment, scores of functional assessment of cancer therapy-fatigue (FACT-F) in Chinese and McGill quality of life questionnaire (MQOL), serum levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and soluble TNF receptor-1 (sTNF-R1) were observed in the two groups.@*RESULTS@#①Compared before treatment, the FACT-F score was decreased after treatment in the observation group (<0.05), while there was no significant difference in the control group (<0.05). The change of the FACT-F score in the observation group was larger than that in the control group (<0.05). ②In the observation group, scores of physiological and psychological dimension were decreased (<0.05), score of social support dimension was increased after the treatment (<0.05). The score changes of physiological, psychological and social support dimension in the observation group were larger than those in the control group (all <0.05). ③After treatment, the serum levels of IL-6, TNF-α and sTNF-R1 were decreased in the observation group (<0.05), while the serum levels of CPR and IL-6 were increased in the control group (<0.05). The serum levels of CPR, IL-6 and TNF-α in the observation were lower than those in the control group (<0.05).@*CONCLUSION@#①Acupuncture can improve the related symptoms of depression, weakness and headache in patients with CRF, strengthen their cognition of the support from society and family, and boost the confidence in curing the disease. ②Acupuncture can effectively down-regulate serum levels of the relative inflammatory factors, which may be its possible mechanism on treating CRF.

Comparasion on Effect of Xuefu Zhuyutang and Modified Erchentang on Nonalcoholic Fatty Liver in Mice / 中国实验方剂学杂志

Objective:To compare the effect and mechanisms of modified Erchentang and Xuefu Zhuyutang on high-fat diet-induced apolipoprotein-E knockout (ApoE-/-) mice nonalcoholic fatty liver disease (NAFLD). Method:Ten C57/BL6J mice were taken as normal control group and fed with normal feed. Totally 30 ApoE-/- mice were fed with high-fat diet to establish a disease model for 4 weeks. After 4 weeks, the 30 ApoE-/- mice were divided into model group, Xuefu Zhuyutang group (hereinafter referred to as Huoxue group) and modified Erchentang group (hereinafter referred to as Huatan group) by random number table method, with 10 in each group. The normal group and the model group were intragastrically administered with normal saline. The drug-administered group was intragastrically administered at a dosage that was ten times of the adult dose, once a day, for 8 weeks. Serum and liver were collected after the end of the 12-week experiment. The serum lipid and liver function levels of each group were measured, and the liver pathological morphology was observed. Protein and mRNA expressions of liver inflammatory mediators interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), monocyte chemotactic factor-1 (MCP-1) were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot. Result:The results of serum lipids and liver function showed that compared with the normal group, serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the model group were significantly increased, while serum high-density lipoprotein (HDL) was significantly reduced (P<0.01). Compared with the model group, serum TG ，LDL and ALT were significantly decreased, HDL was significantly increased in the Huoxue group (P<0.05). The serum levels of TC, TG， LDL, AST and ALT in the Huatan group were significantly decreased，HDL was significantly increased (P<0.05，P<0.01), and TG was decreased. The mice serum HDL in the Huatan group was higher than that in the Huoxue group. The serum ALT in the Huoxue group was lower than that in the Huatan group. The pathological observation showed that compared with the normal group, hepatocytes in the model group had severe steatosis with many lipid droplet vacuoles, suggesting that the mouse NAFLD model was successful. Compared with the model group, each administration group alleviated hepatocyte steatosis, with no significant difference between the two administration groups. Western blot and Real-time PCR results showed that compared with the normal group, protein and mRNA expression levels of TNF-α, IL-1β, MCP-1, and MMP-9 in the model group were significantly increased (P<0.05，P<0.01). Compared with the model group, the Huoxue group significantly down-regulated the expressions of IL-1β, MCP-1 protein and MCP-1 mRNA(P<0.05，P<0.01). The Huatan group significantly reduced the expressions of IL-1β, TNF-α, MMP-9, MCP-1 protein, TNF-α and MMP-9 mRNA(P<0.05，P<0.01). Conclusion:Modified Erchentang and Xuefu Zhuyutang can alleviate the therapeutic effect of NAFLD mice to a certain extent, modified Erchentang has a better therapeutic effect.

Effect of Modified Taohe Chengqitang Combined with Colon Hydrotherapy on Inflammatory Factors and Insulin Resistance in Patients with Severe Nonalcoholic Fatty Liver Disease / 中国实验方剂学杂志

Objective::To define the clinical efficacy of modified Taohe Chengqitang combined with colon hydrotherapy in patients with severe nonalcoholic fatty liver disease (NAFLD) accompanied by phlegm-heat stagnation syndrome and its mechanism. Method::Totally 100 patients with severe NAFLD by phlegm-heat stagnation syndrome were enrolled in the study.They were all given Shanzha Xiaozhi capsule.According to the random number table, the patients were randomly divided into the observation group (50 patients, colon hydrotherapy combined with traditional Chinese medicine) and the control group (50 patients, Shanzha Xiaozhi capsule alone). The observation period was 4 weeks.The therapeutic effect of colon hydrotherapy was verified through determinations of the liver function, blood lipid, insulin resistance index (IRI), controlled attenuation parameter (CAP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) before and after treatment.The mechanism of colon hydrotherapy combined with modified Taohe Chengqitang was preliminarily analyzed based on changes of IR, TNF-α and IL-6. Result::Alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptadase (γ-GT), total cholesterol (TCH), triglyceride (TG), fasting plasma glucose (FPG), fasting insulins (FINS), IRI, CAP, TNF-αand IL-6 of NAFLD patients in both of two groups were significantly lower than those before treatment (P<0.01). ALT, AST, γ-GT, TCH, TG, FPG, IRI, CAP, TNF-α and IL-6 in observation group were significantly lower than those in the control group after treatment (P<0.01). FINS in observation group was significantly lower than that in the control group (P<0.05). Conclusion::Colon hydrotherapy combined with modified Taohe Chengqitang is an effective method for treating NAFLD accompanied by phlegm-heat stagnation syndrome.Its mechanism may be mainly correlated with the reduction of IRI, serum TNF-α and IL-6.The course of colon hydrotherapy, the therapeutic mechanism and the long-term efficacy need to be further studied in the future.

Effect of Huoxue Ointment on Osteoarthritis Caused by Knee Joint Injury in Rabbits / 中国实验方剂学杂志

Objective:To investigate the efficacy and anti-inflammatory and analgesic effect of Huoxue ointment on osteoarthritis of the knee caused by knee joint injury in rabbits. Method:A total of 60 big ear white rabbits were selected, with half male and half female. Ten rabbits were randomly selected by weight and set as normal control group. For the remaining rabbits, the injury of articular cartilage and anterior cruciate ligament were caused in the femoral ankle joint surface of the right hind knee joint, so as to establish an animal model of knee osteoarthritis in rabbits. For the normal control group, the skin was cut open and immediately sutured. Two weeks after the operation, rabbits were randomly divided into model control group according to body weight，positive voltaren group (1 cm·kg-1·d-1), and high-dose (6 g·kg-1·d-1), medium-dose (3 g·kg-1·d-1) and low-dose(1.5 g·kg-1·d-1) Huoxue ointment groups, with 10 animals in each group. Interleukinin-1β （IL-1β） in rabbit articular cavity fluid and peripheral blood were measured, contents of tumor necrosis factor-α （TNF-α） and free radical nitric oxide（NO）, rabbit articular cartilage was examined by histopathology, in order to study the efficacy of Huoxue ointment on rabbit knee arthritis. The model of increased permeability of capillaries in abdominal cavity of mice induced by acetic acid and the model of foot swelling of rats induced by carrageenan were used to determine the absorbance of mice peritoneal fluid and the rat foot swelling value, and study the anti-inflammatory effect of huoxue ointment. A total of 120 SPF ICR mice and 60 SD rats were selected, with half male and half female. The mice were randomly divided according to body weight into model control groups, positive Voltaren group (3 cm·kg-1·d-1), and high-dose (16 g·kg-1·d-1), medium-dose (8 g·kg-1·d-1), low-dose (4 g·kg-1·d-1) Huoxue ointment groups，with 12 animals in each group. The rats were randomly divided into the model control group, positive voltaren group (2 cm·kg-1·d-1), and high-dose (8 g·kg-1·d-1), medium-dose (4 g·kg-1·d-1), low-dose (2 g·kg-1·d-1) Huoxue ointment groups，with 10 animals in each group. Result:The tested dose of Huoxue ointment，the levels of IL-1β, TNF-α and NO in rabbit serum and joint fluid were decreased to varying degrees, and the effect was significant in the high-dose group (P<0.05). According to the pathological examination, the tested dose of Huoxue ointment, knee cartilage and synovial tissue lesions were significantly lower than those of the model control group (P<0.05)，the value of peritoneal fluid concentration and the foot swelling in medium and high-dose Huoxue ointment groups were significantly lower than those in the model control group (P<0.05). The analgesic effect of tail shaking in mice, and the pain threshold rate of the high-dose Huoxue ointment group were significantly higher than those of the model control group (P<0.05)， the analgesic effect lasted for 4 hours, the foot tenderness of rats in the large and medium-dose Huoxue ointment groups was significantly higher than that in the model control group (P<0.05). Conclusion:Huoxue ointment has obvious joint repair and anti-inflammatory and analgesic effects on osteoarthritis of knee caused by knee joint injury in rabbits.

Regulatory Effect of Qiwei Baizhusan on Liver Tissue Insulin PI3K/Akt Signal Pathway in Diabetic Mice / 中国实验方剂学杂志

Objective:To study the effect of Qiwei Baizhusan (QWBZS) on liver insulin phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt） signal pathway of diabetic mice induced by high-fat diet and streptozotocin (STZ). Method:The methods of network pharmacology and animal experiments were used to study the hypoglycemic effect of QWBZS. Active chemical components of the drug and disease targets selected through public databases were used to construct the protein-protein interaction network (PPIN), and gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomics(KEGG) enrichment analysis was performed to identify relevant signal pathways in vivo. In the pharmacological experiment, the diabetic mice model was established through intraperitoneal injection with 80 mg·kg-1·d-1 STZ high-glucose, high-fat diet. The mice were divided into normal group (normal saline), model group (normal saline) and QWBZS group (18.7 g·kg-1·d-1). After 28 days, the hypoglycemic effect of the drug and its effect on serum total cholesterol (T-CHO), fasting insulin (FINS) and serum tumor necrosis factor-α (TNF-α) were determined. Western blot and Real-time fluorescence quantitative PCR (Real-time PCR) were used to detect protein and mRNA expressions of insulin receptor (IR), insulin receptor substrate-1 (IRS-1)，phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in liver tissues． Result:A total of 36 active components in this drug were identified by network pharmacology. KEGG analysis suggested that QWBZS might play a role in reducing blood glucose by regulating PI3K Akt signal pathway. Compared with the model group, the levels of blood glucose, serum T-Cho and TNF-α of the intervention group were significantly lower (P<0.01), while the FINS of the intervention group was significantly higher (P<0.01). Protein and mRNA expressions of IR，IRS-1，PI3K and Akt in liver tissues of mice in QWBZS treatment group increased markedly (P<0.05，P<0.01). Conclusion:QWBZS could regulate the levels of blood glucose, TNF-α, T-CHO, and FINS in the serum of diabetic mice induced by high-fat diet and STZ. It can improve PI3K/Akt signal pathway of diabetic mice by regulating protein and mRNA expressions of IR，IRS-1，PI3K and Akt/PKB.

Effect of Plantaginis Semen on Inflammatory Cytokines and mRNA and Protein Expressions of AQP4 in Colon Tissue of Diarrhea Rats / 中国实验方剂学杂志

Objective:To observe Plantaginis Semen's mechanism in treating diarrhea by observing the effect on inflammatory factors in serum and mRNA and protein expressions of aquaporin4 (AQP4) in colon tissue of diarrhea rats. Method:Senne Folium was orally administered to duplicate diarrhea rats. Sixty male SD rats were randomly divided into normal group, model group, hydrochlorothiazide group (9 mg·kg-1), and low, middle, and high-dose Plantaginis Semen groups (0.95, 1.9, 3.8 g·kg-1). Senne Folium (20 mL·kg-1) was intragastrically administered in 5 groups in the morning, except for normal group that was orally given the same dose of distilled water. In the afternoon, each treatment group was orally given the corresponding drugs, while normal group and model group were orally given the same dose of distilled water. The loose stool rate, average degree of loose stool, and diarrhea index were compared according to fecal traits and stool times after 14 days of treatment. The serum and colon tissue were collected to detect the contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) in serum. Hematoxylin-eosin (HE) staining was used to observe the pathological morphological changes of colon tissue, and quantiative Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to detect the mRNA and protein expressions of AQP4 in colon tissue. Result:In the model group, the loose stool rate, average degree of loose stool, and diarrhea index were significantly increased (P<0.01), apoptosis and necrosis were observed in the epidermal cells of colonic mucosa, telangiectasia and congestion in lamina propria were obvious, and a few neutrophils were infiltrated, and the contents of TNF-α, IL-6 and CRP in serum increased (P<0.05, P<0.01), the mRNA and protein expressions of AQP4 significantly decreased (P<0.01). Compared with the model group, the loose stool rate, average degree of loose stool, and diarrhea index were significantly decreased in low, middle, and high-dose Plantaginis Semen groups (P<0.01), the apoptosis and necrosis of epidermal cells, telangiectasia and hyperemia and neutrophil infiltration in colonic mucosa were obviously improved, and the contents of TNF-α and CRP in serum significantly decreased (P<0.05, P<0.01), the mRNA and protein expressions of AQP4 increased （P<0.05, P<0.01）. Conclusion:Plantaginis Semen has a better antidiarrheal effect, and its mechanism may be related to inhibition of inflammatory reaction, repair of pathological damage of colonic mucosa, up-regulation of AQP4 expression and promotion of water and fluid metabolism.

Dexamethasone protects the glycocalyx on the kidney microvascular endothelium during severe acute pancreatitis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Objective: This study demonstrated that dexamethasone (DEX) protects the endothelial glycocalyx from damage induced by the inflammatory stimulus tumor necrosis factor-α (TNF-α) during severe acute pancreatitis (SAP), and improves the renal microcirculation. Methods: Ninety mice were evenly divided into 3 groups (Sham, SAP, and SAP+DEX). The SAP mice model was established by ligature of pancreatic duct and intraperitoneal injection of cerulein. Renal perfusion and function, and morphological changes of the glycocalyx were evaluated by laser Doppler velocimetry, electron microscopy, and histopathology (hematoxylin and eosin (H&E) staining), respectively. Serum levels of syndecan-1 and TNF-α were assessed by enzyme-linked immunosorbent assay (ELISA). The protective effects of dexamethasone on the glycocalyx and renal microcirculation were evaluated. Results: Significantly high levels of serum TNF-α were detected 3 h after the onset of SAP. These levels might induce degradation of the glycocalyx and kidney hypoperfusion, resulting in kidney microcirculation dysfunction. The application of dexamethasone reduced the degradation of the glycocalyx and improved perfusion of kidney. Conclusions: Dexamethasone protects the endothelial glycocalyx from inflammatory degradation possibly initiated by TNF-α during SAP. This is might be a significant discovery that helps to prevent tissue edema and hypoperfusion in the future.

Scalp-acupuncture improves neurological function by regulating expression of IL-10 mRNA, IL-6 mRNA and TNF-α of parahippocampal gyrus in cerebral ischemic rats / 针刺研究

OBJECTIVE: To observe the effect of scalp-acupuncture intervention on the expression of Interleukin (IL)-10 mRNA, IL-6 mRNA and tumor necrosis factor (TNF) - α in the parahippocampal gyrus of cerebral ischemia (CI) rats, so as to explore its molecular mechanisms underlying improvement of CI. METHODS: A total of 64 male SD rats were randomized into normal control, model, medication and scalp-acupuncture groups (n＝16 rats in each group). The focal CI model was established by middle cerebral artery occlusion. Intraperitoneal injection of Ammonium Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS6), followed by twirling the needles at 100 cycles/min for 1 min, once again every 10 min during 20 min' needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－4 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schabitz's (2004) methods, respectively. The expression levels of IL-10 mRNA and IL-6 mRNA were detected by fluorescence quantitative PCR, and the expression of TNF-α was detected by immunohistochemistry. RESULTS: After modeling, the neurologic deficit and neurological function scores and the expression levels of IL-10 mRNA, IL-6 mRNA and TNF-α protein in the parahippocampus were significantly increased in the model group than in the normal control group (P<0.01). Following the intervention, the neurologic deficit and neurological function scores as well as IL-6 mRNA and TNF-α protein expression were significantly down-regulated in both scalp-acupuncture and medication groups (P<0.05), and the expression of IL-10 mRNA was obviously increased (P<0.05) relevant to the model group. CONCLUSION: Scalp-acupuncture can improve neurologic function in CI rats, which is related to its effects in up-regulating the expression of IL-10, then down-regulating the expression of IL-6 and TNF-α (reducing inflammatory response) in the parahippocampal gyrus.

Effect of Modified Si Junzitang on Angiogenesis of H22 Tumor-bearing Mice / 中国实验方剂学杂志

Objective:To investigate the effect of modified Si Junzitang on angiogenesis in transplanted tumor of H22 tumor-bearing mice. Method:The effect of modified Si Junzitang on tumor inhibition and growth of peripheral blood vessels in tumor-bearing mice was observed by tumorigenesis experiment in mice. Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) were used to detect the distribution of blood vessels and the expression of vascular endothelial markers (CD31) in tumor-bearing mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2) and tumor necrosis factor-α (TNF-α) in tumor tissue. Result:The inhibition rates of modified Sijunzi Tang in low-dose group (ig, 11.83 mg·kg-1·d-1), middle-dose group (ig, 23.66 mg·kg-1·d-1) and high-dose group (ig, 47.32 mg·kg-1·d-1) were 29.97%, 59.80%and 82.34%, respectively. Compared with the model group, the average tumor weight was lower in middle and high-dose groups, with statistically significant differences (PPPα in middle and high-dose modified Si Junzitang groups were lower than those in the model group (PConclusion:Modified Si Junzitang can inhibit the tumor growth of H22 tumor-bearing mice and the angiogenesis of transplanted tumors, which may be related to the reduction of TNF-α, VEGF and VEGFR2 expression levels.

Effect of Modified Erchentang on PPARγ Expression in Lung Tissue of Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective: To explore the effect of modified Erchentang on peroxisome proliferator-activated receptor gamma (PPARγ) protein and gene expressions in lung tissue of chronic obstructive pulmonary disease (COPD) rat model, and the expressions of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α) in serum, lung homogenate and bronchoalveolar lavage fluid. Method: Seventy SD rats were randomly divided into seven groups:normal group, model group, high, medium and low-dose modified Erchentang groups (40, 20, 10 g · kg-1 · d-1), Xiaokechuan group (5 g · kg-1 · d-1), and Erchentang group (5 g · kg-1 · d-1). The rat COPD model was established through smoking and intratracheal instillation of lipopolysaccharide (LPS). After successful modeling, the treatment group was given drug by gavage, while the normal group and the model group were given the same amount of saline. The concentrations of IL-6, IL-10 and TNF-α in serum, lung homogenate and bronchoalveolar lavage fluid(BALF) of rats were measured by enzyme-linked immunosorbent assay (ELISA). Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expression of peroxisome proliferator-activated receptor gamma (PPARγ), and immunohistochemistry (IHC) and Western blot were used to detect the expression of PPARγ in lung tissue. Result: Compared with the normal group, the levels of IL-6 and TNF-α in serum, lung homogenate and BALF of the model group rats increased significantly (PPγ mRNA in lung tissue of rats in model group were significantly decreased (Pγ protein was significantly inhibited(Pα in serum, lung homogenate and BALF of each treatment group decreased to varying degrees(Pα in the middle-dose Erchentang group were particularly significant. The PPARγ mRNA and protein expressions in lung tissue of rats in each treatment group were increased to varying degrees (PConclusion: Modified Erchentang may improve pulmonary inflammation and pulmonary function in COPD rats by increasing the expression of PPARγ and the content of IL-10 and decreasing the contents of IL-6 and TNF-α.

Effect and Mechanism of Mori Folium on Insulin Resistance in 3T3-L1 Cells / 中国实验方剂学杂志

Objective: To observe effect of Mori Folium-containing serum on glucose consumption and cell activity of fat cell line 3T3-L1 insulin resistance (IR) model, in order to screen out the optimal concentration of drug-containing serum, detect effect of Mori Folium on the content of inflammatory factors, and explore the possible mechanism. Method: 3T3-L1 preadipocytes in logarithmic growth phase were selected, and induced with 10 mg·L-1 insulin (Ins), 0.25 mmol·L-1 dexamethasone (DEX) and 0.5 mmol·L-1 3-isobutyl-methylxanthine(IBMX) for 48 h and then with 10 mg·L-1 Ins for 48 h. After the cells were differentiated into mature adipocytes, they were induced with 1 μmol·L-1 DEX for 96 h to establish IR model. Glucose content in the supernatant of cells was detected by glucose oxidase after serum containing Mori Folium cultured for 12,24,36, 72 h. Methyl-thiazdyl-tetrazolium(MTT) was used to detect the effect of serum containing Mori Folium on IR cells activity. The content of tumor necrosis factor-α(TNF-α) was determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, the effects of inflammatory factors on the expressions of insulin signaling pathway proteins insulin receptor (InsR), insulin receptor substrate (IRS), p-IRS1 and glucose transporter 4 (GLUT4) were determined by Western blot. Result: Serum containing Mori Folium could significantly increase the glucose consumption rate and cell activity of IR cells (Pα (PPPConclusion: Mori Folium can significantly improve IR status of 3T3-L1 cells, and its mechanism may be related to inhibiting TNF-α and promoting the expressions of insulin signaling pathway proteins.

Protective Effect and Mechanism of Sodium Ferulate on Cerebral Ischemia-reperfusion Inflammatory Injury in Rats / 中国实验方剂学杂志

Objective: To investigate the protective effect of sodium ferulate on cerebral ischemia-reperfusion injury in rats and to explore its possible mechanism.Method: The cerebral ischemia reperfusion injury model was established by middle cerebral artery occlusion (MCAO) in SD male rats. 36 modeled rats with neurologic damage were randomly divided into 4 groups:model group, low,medium,high-dose sodium ferulate groups (25,50,100 mg·kg-1).Another nine rats were selected as a sham operation group.Neurological function was assessed by neurological scoring system in rats.Hematoxylin-eosin (HE) staining was performed to observe the pathological changes of the rats' brain. The levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum and brain tissues were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the protein expression of nucleus and cytoplasm nuclear factor-kappa B p65 (NF-κB p65) in brain tissues.Result: As compared with normal group, neurological deficit score was increased; the neuronal necrosis and inflammatory cell number were present;the serum and brain tissue levels of TNF-α, IL-1β and IL-6 were increased; nucleus/cytoplasm NF-κB p65 protein expression ratio was increased significantly in model group (PPPα, IL-1β and IL-6(PPκB p65 protein(PPConclusion: Sodium ferulate protects the brain against focal cerebral ischemia reperfusion injury, and the mechanism may be related to inhibiting nuclear translocation of NF-κB p65 protein to alleviate inflammatory response.

Effect of acupuncture on the expressions of TNF-α and IFN-γ in patients with premature ovarian failure / 中国针灸

OBJECTIVE@#To explore the clinical effect of acupuncture and the potential effect mechanism in patients with premature ovarian failure.@*METHODS@#A total of 104 patients with premature ovarian failure were randomized into an acupuncture group and a western medication group, 52 cases in each one. In the western medication group, the conjugated estrogens tablets were prescribed for oral administration, 0.625 mg each time, once a day, consecutively for 21 days. On the 16th day of medication with conjugated estrogens tablets, the oral administration of medroxyprogesterone acetate tablets were supplemented, 10 mg each time, once a day, consecutively for 5 days, and then, these two kinds of western medication were discontinued for 1 week. A total of 3 cycles were required in treatment with 28 days as an artificial cycle. In the acupuncture group, acupuncture was applied. Two groups of acupoints were selected. The first group of acupoints were stimulated before ovulation and the acupoints were Guanyuan (CV 4), Guilai (ST 29), Taichong (LR 3), Taixi (KI 3), Xuehai (SP 10), Sanyinjiao (SP 6), Zigong (EX-CA 1), Yinlingquan (SP 9), Zusanli (ST 36), Shuidao (ST 28), Dahe (KI 12) and Tianshu (ST 25). The second group of acupoints were stimulated after ovulation and the acupoints included Ciliao (BL 32), Shiqizhui (EX-B 8), Ganshu (BL 18), Shenshu (BL 23), Geshu (BL 17) and Pishu (BL 20). The therapeutic effect was observed and compared in the patients between the two groups, as well as the expressions of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) and the levels of serum luteinizing hormone (LH), follicule stimulating hormone (FSH) and estradiol (E) before and after treatment.@*RESULTS@#The total effective rate was 90.4% (47/52) in the acupuncture group, higher than 67.3% (35/62) in the western medication group (<0.05). After treatment, the expressions of IFN-γ and TNF-α in the acupuncture group were obviously lower than the western medication group (<0.05). Except for serum LH after treatment, at the end of treatment and in 30 days and 90 days after treatment, the levels of serum E in the acupuncture group were higher obviously than the western medication group and the levels of serum LH and FSH were lower obviously than the western medication group (all <0.05).@*CONCLUSION@#Acupuncture promotes the regular menstruation, effectively regulates the levels of serum LH, FSH and E and improves the pituitary gland and the ovary endocrine in the patients with premature ovarian failure. Such effect may be related to the the improvements in the expressions of IFN-γ and TNF-α, the inhibition of the apoptosis of ovarian granulosa cells, the recovery of ovarian function and the enhancement of reserve capacity.

Scalp-acupuncture Improves Neurological Function by Regulating Expression of NF-κB p 65 mRNA, IκB mRNA and IL-1 β and TNF-α in Parahippocampal Gyrus of Cerebral Ischemia Rats / 针刺研究

OBJECTIVE: To observe the effect of scalp-acupuncture intervention on the expression of parahippocampal factor-κB p 65 mRNA (NF-κB p 65 mRNA), IκB mRNA, interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in rats with cerebral ischemia (CI), so as to investigate its molecular mechanisms underlying improving CI by reducing inflammatory response. METHODS: A total of 64 SD rats were randomized into normal control, model, medication and scalp-acupuncture groups, with 16 rats in each group. The focal CI model was established by middle cerebral artery occlusion (MCAO). Intraperitoneal injection of Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), followed by twirling the needles at 200 cycles/min for 1 min, once again every 10 min during 30 min's needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－5 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schäbitz's (2004) methods, respectively. The expression levels of NF-κB p 65 mRNA and IκB mRNA in the parahippocampus gyrus tissue were detected by fluorescence quantitative reverse transcription-PCR, and IL-1 β and TNF-α proteins in the parahippocampus gyrus tissue were detected by immunohistochemistry. RESULTS: After modeling, the neurologic deficit and neurological function scores and the expression levels of NF-κB p 65 mRNA, IL-1 β and TNF-α in the parahip-pocampus were significantly increased in the model group than in the normal group (P<0.01), while the expression of IκB mRNA was considerably down-regulated (P<0.01). Following treatment intervention, the neurologic deficit and neurological function scores as well as NF-κB p 65 mRNA, and IL-1 β and TNF-α protein expression were significantly decreased in both scalp-acupuncture and medication groups compared with the model group (P<0.05, P<0.01), and the decreased expression of IκB mRNA was obviously increased (P<0.05)．. CONCLUSION: Scalp-acupuncture can improve neurologic function in cerebral ischemic rats, which is related with its effects in up-regulating the expression of IκB to inhibit the dissociation of NF-κB, then decreasing the expression of IL-1 β and TNF-α (reducing inflammatory response) in the parahippocampal gyrus tissue.

Repression of lung tumor suppressor gene G protein-coupled receptor class C group 5 member A expression by inflammatory signal pathway / 上海交通大学学报（医学版）

Objective: To investigate the regulatory effects of inflammatory signaling pathway on the expression of G protein-coupled receptor class C group 5 member A (GPRC5A). Methods: Nuclear factor κB (NF-κB)-driven luciferase mice were intraperitoneally injected with lipopolysaccharide (LPS) to evaluate the activation of NF-κB in lungs. GPRC5A expression in lungs was assessed by Western blotting in the C57BL/6J mice injected with LPS. In vitro tests, human lung tumor cell lines Calu-1 and H322, and human embryonic kidney cell line HEK293T were administered with tumor necrosis factor α (TNF-α) or transfected with p65 expression plasmid; Western blotting, RT-PCR, luciferase reporter gene experiment and immunofluorescence assay were used to analyze the effect of inflammation on GPRC5A expression. Results: Intraperitoneal injection of LPS induced activation of NF-κB pathway in lung tissues, which suppressed the expression of GPRC5A in mice lungs. In Calu-1 cells, TNF-α treatment greatly suppressed the expression of GPRC5A protein and mRNA. In HEK293T cells, transfection of p65 subunit of NF-κB suppressed the expression of GPRC5A promoter-driven luciferase reporter. The H322 cells transfected with green fluorescent protein-p65 almost did not express GPRC5A. Conclusion: NF-κB pathway acts at the promoter of GPRC5A and suppresses its mRNA and protein expression.