Progress of Clinical Research on the Treatment of Spinal Disorders by the Tongdu Method

Spinal disorders are diseases caused by congenital, degenerative, postural errors, trauma, and external evils that result in lesions of bones, discs, ligaments, and muscles, thus compressing the nerves. Spine-related diseases include scoliosis, intervertebral disc herniation, intervertebral stenosis, spinal fracture, ankylosing spondylitis, spinal cord injury, spinal tumour, etc., which seriously affect people’s daily life. This paper summarises the relevant studies on the treatment of spinal diseases by Tongzhu therapy. The mechanism of action of Tongzhu therapy in treating spinal diseases is closely related to the control of inflammatory factors, the improvement of local microcirculation, analgesia, and the promotion of nerve repair. This paper describes the application and modern mechanism of Tongzhu therapy in the treatment of spinal diseases, which will provide a direction for future research on Tongzhu therapy.

Identification of Qishen Oral Solution with TLC and HPLC

[Objective] This study aimed to establish the standards for quality control of Qishen oral solution. [Method] Astragalus membranaceus, Atractylodes macro- cephala Koidz. and Glycyrrhiza uralensis in the preparation were identified by using thin layer chromatography （TLC）, and Codonopsis pilosula was identified by using high-performance liquid chromatography （HPLC）. [Result] By using the developed TLC system, experimental solution and reference solution showed clear spots, while negative control presented no interference. By using the developed HPLC system, the chromatographic peak of Iobetyolin was detected in experimental solution. [Con- clusion] The developed TLC and HPLC systems presented high specificity and good repeatability for identification of these four components and could be used in the quality control of Qishen oral solution.

Qishen capsule safely boosts cardiac function and angiogenesis via the MEK/ERK pathway in a rat myocardial infarction model

Background Qishen(QS) capsules, a Traditional Chinese Medicine, has been widely used to treat coronary heart disease in China. However, evidence of its effectiveness remains unclear. Methods To explore whether QS has cardioprotective efficacy and/or promotes angiogenesis after myocardial infarction (MI), we performed experiments in a preclinical rat MI model. One month after left anterior descending coronary artery ligation, the rats received either QS solution (0.4 g/kg/day) or the same volume of saline by intragastric injection for four weeks. Results Echocardiographic and hemodynamic analyses demonstrated relatively preserved cardiac function in MI rats administered QS. Indeed, QS treatment was associated with reduced infarct scar size and heart weight index, and these beneficial effects were responsible for enhancing angiogenesis. Mechanistically, QS treatment increased phosphorylation of protein kinase B (Akt) and downregulated phosphorylation of mitogen-activated protein kinase/extracellular-regulated kinase (MEK/ERK). Conclusions QS therapy can improve the cardiac function of rats after MI by an underlying mechanism involving increased angiogenesis, at least partially via activation of the Akt signaling pathway and inhibition of MEK/ERK phosphorylation.

Effect of Qishen Yiqi dripping pills combined with trimetazidine on the treatment of chronic heart failure: A meta-analysis

Objective:To systematically evaluate the safety and effectiveness of Qishen Yiqi dripping pills combined with trimetazidine in the treatment of chronic heart failure.Methods:A total of four English and Chinese electronic databases were searched:CNKI,VIP,CBM,PUBMED.Cochrane bias risk tools were used to assess the methodological quality of qualified studies.Meta-analysis was conducted by Review Manager 5.3.A total of 9 articles were included,with a total sample size of 855 cases,443 cases in the observation group and 412 cases in the treatment group.Results:Qishen Yiqi dripping pills combined with trimetazidine in the treatment of chronic heart failure has a total effective rate(RR=1.22,95%CI[1.15-1.30];p<0.00001),LVEF(MD=6.03,95%CI[5.39,6.67],P<0.00001),BNP(MD=-101.87,95%CI[-109.90,-93.83],P<0.00001),E/A(MD=-4.32,95%CI[-5.70,-2.93],P<0.00001),SYP(MD=-10.32,95%CI[-13.32,-7.32],P<0.00001),LVESD(MD=-5.50,95%CI[-6.03,-4.96],P<0.00001),6-MWT(MD=110.13,95%CI[96.89,123.36],P<0.00001)Adverse reactions occurred less frequently and did not affect treatment.Conclusion:This study shows that QYDP combined with TMZ can improve various indicators of CHF patients.However,due to the small sample size and the generally low quality of research,a more rigorous and reasonably designed RCT is needed to confirm these findings.

Effects of Qishen Ultrafine Powder on Immune Efficacy in Chickens

[Objective] This paper aimed to observe the effects of Qishen ultrafine powder on immune efficacy of infectious bursal disease vaccine in chickens. [ Method] 360 1-day-old chickens were randomly divided into six groups equally, and group I -V were vaccinated with IBD live virus vaccine on the 21 th and 35th day, respectively. From 14th to 21th day, group I - Ⅲ were added with 1%, 0.5% of Qishen ultrafine powder and 2% of Qishan powder in basel diet, respectively, group IV was given Yupingfeng oral liquid and group VI was as a control group without vaccination. Be- fore the expedment （14-day-old chickens as DO ） and 7 （ D7 ）, 14 （ D14 ）, 21 （ D21 ）, 28 （ D26 ）, 35 （ D36 ）, 42 （ D42 ） days after the experiment, the dynamic change of serum IBD specific antibody was determined, respectively. On D7, D21 and D36, the dynamic change of peripheral T lymphocyte proliferation and IL-2 in serum were determined, respectively. The weight change was also observed. [Results] Compared with the immune control group, the immune effects of IBD vaccine of each administration group were significantly improved, and there was no significant difference between each other. [ Condusion] Qishen ultrafine powder could increase immune function of chickens at lower dosage.

Danlu Tongdu tablets treat lumbar spinal stenosis through reducing reactive oxygen species and apoptosis by regulating CDK2/CDK4/CDKN1A expression

Lumbar spinal stenosis is caused by the compression of the nerve root or cauda equina nerve by stenosis of the lumbar spinal canal or intervertebral foramen,and is manifested as chronic low back and leg pain.Danlu Tongdu(DLTD)tablets can relieve chronic pain caused by lumbar spinal stenosis,but the molecular mechanism remains largely unknown.In this study,the potential molecular mechanism of DLTD tablets in the treatment of lumbar spinal stenosis was first predicted by the network pharmacology method.Results showed that DLTD functions in regulating anti-oxidative,apoptosis,and inflammation signaling pathways.Furthermore,the flow cytometry results showed that DLTD tablets efficiently reduced reactive oxygen species content and inhibited rat neural stem cell apoptosis induced by hydrogen peroxide.DLTD also inhibited the mitochondrial membrane potential damage induced by hydrogen peroxide.Elisa analysis showed that DLTD induced cell cycle-related protein,CDK2 and CDK4,and reduced CDKN1A protein expression level.Taken together,our study provided new insights of DLTD in treating lumbar spinal stenosis through reducing reactive oxygen species content,decreasing apoptosis by inhibiting CDKN1A and promoting CDK2 and CDK4 expression levels.

Effect of Qishen decoction on dedifferentiation of sinusoidal endothelial cells by autophagy

Objective:To observe the effect of Qishen decoction on dedifferentiation and autophagy of liver sinusoid endothelial cells(LSEC);Methods:LSEC were randomly divided into the control group,model group,Qishen Decoction low,medium,high dose group,and inhibitor group.The model was induced by 100μg/ml oxidized low-density lipoprotein(oxLDL)for 24 hours,and the corresponding drugs or medicated serum were given for intervention.The expression levels of VEGFR2 and ET1 were detected by RT-qPCR and immunofluorescence staining,the ultrastructure of LSEC was detected by transmission electron microscopy,the content of NO was detected by ELISA,the expression levels of autophagy related proteins(LC3BI,LC3BⅡand p62)and endothelial function related proteins(eNOS and p-eNOS)were detected by western blot;Results:The results of transmission electron microscopy showed that Qishen decoction medicated serum could increase the number of fenestra and autophagy in LSEC cells,and inhibit the formation of basement membrane under endothelium.Compared with the model group,Qishen decoction medicated serum could significantly up-regulate the expression level of VEGFR2 mRNA and protein in LSEC,down regulate the expression level of ET1 mRNA and protein,the difference was statistically significant(P<0.05).In addition,Qishen decoction medicated serum could significantly increase the expression of LC3BII,p-eNOS,eNOS protein and the ratio of LC3BII/LC3BI,p-eNOS/eNOS,and reduce the expression of LC3BI and p62 protein in LSEC,which is statistically significant compared with the model group(P<0.05).Conclusion:Qishen decoction can inhibit the dedifferentiation of LSEC by promoting the autophagy level of LSEC,and then play an anti-fibrosis role.

Use of Qishen granule for the treatment of heart failure: A systematic review and meta-analysis of animal studies

Objective:To evaluate the effect of Qishen granule(QSG)on heart failure(HF)and the potential mechanisms involved in animal models.Methods:Studies of QSG in animals with HF were identified by searches of eight databases up to August 1,2019.Two authors independently reviewed each study and the Collaborative Approach to Metaanalysis and Review of Animal Data from Experimental Studies 10-item checklist was used for quality assessment.The primary outcomes were indicators of left ventricular ejection fraction and/or left ventricular fractional shortening.The secondary outcome measures were ventricular structure,hemodynamics,myocardial injury-related indicators,and the mechanisms whereby QSG ameliorates HF.A metaanalysis was performed and all the data were analyzed using RevMan 5.3 software.Results:Thirteen studies containing a total of 240 animals met the criteria for the meta-analysis.The quality score of the studies ranged from three to six points.The meta-analysis showed a significant effect of QSG to improve cardiac function,inhibit ventricular remodeling,improve circulation,and attenuate myocardial injury.The possible mechanisms was identified to be suppression of the renin-angiotensinaldosterone system,which has anti-fibrotic,anti-apoptotic,anti-inflammatory,and anti-oxidative effects in the myocardium,the prevention of calcium overload,and the promotion of myocardial energy metabolism.Conclusion:Our review shows that QSG has a cardioprotective effect in animal models of CHF.However,due to the relatively poor quality of the included studies and the differences between humans and animal models,the results should be interpreted with caution.

Tongdu Tiaoshen Acupuncture Method for Promoting Hippocampal Nerve Repair and Regeneration and Improving Learning and Memory Dysfunction in Stroke Model Rats

[Objectives]To evaluate the effects of Tongdu Tiaoshen Acupuncture method on improving learning and memory function and hippocampal nerve regeneration and repair after stroke.[Methods]Animal models of cognitive impairment in the recovery phase of cerebral infarction in rats were prepared,and rats were randomly divided into control group,model group,donepezil group,and acupuncture group.Morris water maze experiment was carried out to evaluate learning and memory ability.Nissl staining,BrdU and DCX immunofluorescence staining were used to observe the number and morphological structure of neuronal pyramidal and granular cells in the dentate gyrus(DG)area of the hippocampus,as well as the proliferation and differentiation of neural progenitor cells.TUNEL method was used to detect cell apoptosis.[Results]Before intervention,the scores and ELP of the model group,donepezil group and acupuncture group were significantly higher than those of the blank group(P<0.05);after the intervention,the neurological scores and ELP of the blank group and the model group were not significantly different from those before the intervention(P<0.05);the donepezil group and acupuncture group were significantly reduced,there was no difference between the donepezil group and acupuncture group(P>0.05),but it was still significantly higher than the blank group(P<0.05).The results of BrdU and DCX immunofluorescence staining showed that there were only a few BrdU positive cells and DCX protein in the hippocampal DG area of mice in the blank group,and the positive cells in the model group were significantly increased(P<0.05);donepezil group and acupuncture group showed significant increase compared with the model group(P<0.05);compared with the blank group,the number of neurons in the hippocampal DG area of each group was significantly reduced(P<0.01),and the neuronal apoptosis index was significantly increased(P<0.01);compared with the blank group,the number of neurons in the hippocampal DG area of each group was significantly reduced(P<0.01),and the neuronal apoptosis index was significantly increased(P<0.01).The number of neurons in the hippocampal DG area of rats in the acupuncture group and donepezil group was significantly increased(P<0.01),and the neuronal apoptosis index was significantly decreased(P<0.01).[Conclusions]Tongdu Tiaoshen Acupuncture method can improve the learning and memory function of stroke model rats by promoting the regeneration and repair of hippocampal nerve in rats with cerebral infarction.

A theoretical study of miRNA155-DNA methylation mediated mitokATP regulating mitochondrial autophagy in the improvement of myocardial ischemia-reperfusion injury by Qishen Yiqi Dropping Pills

In the state of acute myocardial ischemia,miRNA expression can regulate related genes and proteins,reduce myocardial cell damage,and thus play a protective role in the myocardium.However,the specific mechanism still needs to be further explored.Recent studies have found that the opening of the mitoKATP channel can regulate mitochondrial autophagy,and the initiation of miRNA-DNA methylation plays a regulatory role in inducing cell autophagy.The applicant research team previously found that Qishen Yiqi Dropping Pills could significantly improve myocardial ischemia by mediating MitokATP channels to regulate mitochondrial autophagy.,and animal experiments have confirmed that miR-155 plays a significant role in the aspect of autophagy regulates,inflammatory reaction and Vascular smooth muscle cell migration.Therefore,the applicant innovatively proposed that Qishen Yiqi Dropping Pills can regulate miRNA155-DNA methylation to mediate the opening of mitoKATP,thereby regulating mitochondrial autophagy and improving myocardial ischemia.In this paper,the association between mitochondrial autophagy and oxidative stress injury after myocardial ischemia was described,and the possible mechanism of Qisen Yiqi dropping pills regulating mitochondrial autophagy by regulating miRNA155-DNA methylation to mediate MitokATP to improve myocardial ischemia reperfusion injury was discussed,so as to provide theoretical ideas for related research.

Effects of Qishen Yiqi dripping pills-containing serum on KATP channel opening and PI3K/AKT signaling pathway in hypoxic/reoxygenated H9C2 cardiocytes

Objective:To investigate the effects of Qishen Yiqi dropping pills serum on KATP channel opening and PI3K/AKT signaling pathway of hypoxic/reoxygenated H9C2 cardiocytes.Methods:H9C2 cardiocytes cultured in vitro were randomly divided into five groups,A:H9C2 cell group B:H9C2 cells+H2O2 model group C:H9C2 cells+H2O2 model+Qishen Yiqi group D:H9C2 cells+H2O2 model+Qishen Yiqi+wort group E:H9C2 cells+H2O2 model+Qishenyiqi+5-HD group,the drug intervention is according to the corresponding conditions.CCK-8 method was used to detect the cell activity of each group;Western blot was used to detect the expression of AKT and P-Akt proteins in myocardial cells in each group.The current was recorded by the standard patch clamp whole cell recording method,and the current was collected and analyzed by Pclamp6.0 software.Results:CCK-8 test results showed that compared with group A,the activity of myocardial cells in group B was significantly decreased,and the difference was statistically significant(P<0.01);compared with group B,the difference in group C was statistically significant(P<0.01);compared with C,cardiomyocyte activity in D and E group were significantly decreased,and the difference was statistically significant(P<0.05);WB results showed that compared with A,p-Akt protein expression in B,C,D and E groups were significantly decreased,and the difference was statistically significant(P<0.01);compared with group B,p-Akt protein expression in C,D and E group were significantly increased,and the difference was statistically significant(P<0.01),but there was no significant difference in AKT expression among groups(P>0.05);The results of whole cell patch clamp experiment showed that the outward current of B was significantly increased compared with that of A,and the difference between groups was statistically significant(P<0.01);compared with group B,cardiomyocytes in group C further increased the outward current,and the difference between groups was statistically significant(P<0.01);compared with C,the current of D and E group were significantly decreased,with statistical significance between groups(P<0.01).Conclusion:QishenYiqi dropping pills can protect cardiomyocytes by activating p-Akt protein expression and KATP channel opening in H9C2 cardiomyocytes.

Astragalus Salvia Granules to Benefit the Qi (Qishen Yiqi Keli) protects H9C2 cardiomyocytes by suppressing oxidative stress

Objective:To investigate the effects of Qishen Yiqi Keli(QSYQ;Astragalus Salvia Granules to Benefit the Qi)on ROS scavenging and inhibition of NADPH oxidase in an effort to identify new natural antioxidants.Methods:A total of 23 representative components in QSYQ were investigated,their effects on hydrogen peroxide induced ROS were assayed by dichlorofluorescein assay.Lucigenin chemiluminescence was adopted to test the effect on NADPH oxidase activity in H9C2 cardiomyocyte cells,and pyrogallol autoxidation was adopted to test the superoxide scavenging capacity.Results:Nine compounds in QSYQ could significantly inhibited H2O2-induced ROS increase compared with the model group(P<.05),including tanshinone I,salvianolic acid A,danshinone IIA,and cryptotanshinone from salvia root,luteolin from salvia root,harpagoside,harpagide,and angoroside C from scrophularia root.Nine compounds in QSYQ could significantly inhibited the production of
O2in H9C2 cell.Tanshinone I showed a lowest IC50 value 0.07 mM,the other including salvianolic acid A,cryptotanshinone,and salvianolic acid B,luteolin,isochlorogenic acid C,astragaloside IV,and glycyrrhetinic acid.Licochalcone A inhibited the autoxidation of pyrogallol at a low concentration,and tanshinone I showed no significant inhibitory effect from 2 mM to 20 mM.Conclusion:QSYQ had significant effects on ROS scavenging and inhibition of NADPH oxidase.Tanshinone I and salvianolic acid A are potential NADPH oxidase inhibitors.

Network pharmacology research and experimental validation of Qishen decoction in the treatment of non-alcoholic fatty liver disease

Objective:To screen the main active components of Qishen decoction by network pharmacology and predict the target of its treatment for nonalcoholic fatty liver disease(NAFLD),and to verify it by experiments.Methods:The main active components of Qishen decoction and the disease target of NAFLD were screened through the database;the drug disease target network and PPI were constructed by the software of Cytoscape and string database;the enrichment of go and KEGG were analyzed by the database of DAVID;HE staining,red oil O staining,serum biochemical index and Western blot were used to verify the effect mechanism of Qishen Decoction on NAFLD.Results:A total of 207 active compounds and 95 drug-disease-targets of Qishen Decoction were selected in this study.The results of KEGG enrichment analysis showed that the mechanism of Qishen decoction in the treatment of NAFLD involved adipocytokines,insulin signaling pathway,fatty acid biosynthesis,etc.The results showed that Qishen decoction could significantly reduce the liver NAS score and oil red O staining area of NAFLD rats(P<0.05).At meanwhile,Qishen decoction significantly reduced the levels of serum glucose,insulin,HOMA-IR,TC,TG,AST and ALT in NAFLD rats(P<0.05).In addition,Qishen decoction can significantly up regulate the expression of p-INSRβin liver tissue,down regulate the expression of SREBP-1c,Fas and p-ACC(P<0.05).Conclusion:Qishen decoction can improve the insulin resistance of NAFLD rats through insulin signaling pathway,so as to improve NAFLD fat deposition and liver injury.In addition,Qishen decoction can also achieve the therapeutic effect of NAFLD through multiple channels and targets.

Qishen decoction suppresses liver fibrosis by downregulating PI3K/Akt/mTOR signal pathway

Objective:To observe the effect of Qishen decoction on PI3K/Akt/mTOR signal pathway in rats with liver fibrosis;Methods:40 SD rats were randomly divided into four groups: the control group, the model group, Qishen decoction group, and Colchicine group. Except the control group, the remaining three groups were used to establish liver fibrosis model by intraperitoneal injection of carbon tetrachloride. At the end of modeling, Qishen decoction and colchicine group were given corresponding drug gavage treatment, rats in the model group and the control group were treated with equal volume distilled water for 8 weeks. At the end of the treatment, the blood and liver tissues of rats in each group were collected, the liver function indexes and hydroxyproline content were detected by ELISA, the pathological morphology of liver tissue was detected by HE and Masson staining. Immunohistochemical method was used to detect α-SMA protein expression and Western blot was used to detect the expression of α-SMA, Col-I, Col-Ⅲ and key proteins in PI3K/Akt/mTOR signaling pathway.Results: Compared with the model group, Qishen decoction significantly reduced the levels of AST, ALT, and TBIL in serum, and reduced Hyp content, inflammatory score, fibrosis score, and collagen staining area in liver tissue, differences were statistically significant (P<0.05). At meanwhile, Qishen decoction significantly reduce the expression level of α-SMA, CoL-I and Col-Ⅲ in liver tissue(P<0.05). In addition, compared with the model group, Qishen decoction significantly down-regulated the expressions of p-PI3K, p-Akt and p-mTOR in liver tissue, difference were statistically significant (P<0.05).Conclusion: Qishen decoction suppresses liver fibrosis and inhibits the deposition of collagen in liver tissue by down-regulating PI3K/Akt/mTOR signal pathway.

醒神通督针刺对脑缺血再灌注损伤大鼠自噬的作用研究

目的从自噬探讨醒神通督针刺抗脑缺血再灌注损伤的作用机制。方法雄性SD大鼠随机分为假手术组、脑缺血再灌注模型组(简称模型组)和脑缺血再灌注+醒神通督针刺组(简称针刺组)。采用大脑中动脉线栓法构建脑缺血再灌注损伤模型,针刺组大鼠造模前5 d开始干预,取穴“百会”“四神聪”“足三里”,每日针刺1次,直至再灌注24 h。另外2组大鼠只抓取不针刺。再灌注24 h后采用氯化三苯基四氮唑(triphenyltetrazolium chloride,TTC)染色观察脑梗死面积,透射电镜观察神经元及自噬的亚显微结构,Western blot、免疫荧光检测缺血皮层微管相关蛋白1轻链3(microtubule-associated protein light chain 3B,LC3)、肌球蛋白样BCL2结合蛋白(coiled coil moesin like BCL2 interacting protein,Beclin-1)、自噬相关蛋白(autophagy related protein,Atg)12、蛋白激酶B(protein kinase B,PKB,又称Akt)与磷酸化Akt(phosphorylated Akt,p-Akt)的表达。结果与假手术组比较,模型组大鼠大脑中动脉供血区梗死面积明显增加(P<0.05),且缺血皮层神经元结构严重破坏,自噬明显激活,自噬蛋白Beclin-1、Atg12表达及LC3Ⅱ/I比值显著升高(P<0.05),p-Akt表达下降(P<0.05)。与模型组比较,针刺组大鼠脑梗死面积减少(P<0.05),透射电镜显示神经元结构较为正常,自噬体较少。此外,针刺组自噬蛋白Beclin-1、Atg12表达及LC3Ⅱ/I比值明显降低(P<0.05),p-Akt表达显著上调(P<0.05)。结论针刺具有抗脑缺血再灌注损伤作用,可能通过抑制自噬的活化实现。

Medicated Serum of Qishen Yiqi Pill Affect Vascular Smooth Muscle Cell Proliferation,Cell Cycle,Cyclin D1 and CDK4 Mechanism Research

Observation of stilbene dropping pill and yiqi drug-containing serum influence mechanism of vascular smooth muscle proliferation， cell cycle and Cyclin D1 and CDK4Choose male SD rats were randomly divided into 2 groups， lavage qishen yiqi pill and the gastric saline group，extract the drug-containing serum and normal serum；To set the two groups of serum respectively different concentrations，concentration in different time by CCK8 detection effects on vascular smooth muscle cell proliferation， select best concentration and action time.Flow cytometry instrument and high-throughput screening detect serum medicated effect on vascular smooth muscle cell cycle；Western blot detect the drug-containing serum of cell cycle protein Cyclin D1 and CDK4 expression.Result is 5%， 10% medicated serum inhibits cell proliferation significantly higher than the normal serum concentrations of same within 24 h， 48 h.G1 phase cells 5% medicated serum group was obviously higher than that of 5% in normal group (P<005)， serum and cell proliferation index significantly less than 5% normal serum group (P<005)，At the same time， Cyclin D1 and CDK4 expression significantly less than 5% normal serum group (P<005).Conclusion serum of qishen yiqi pill can inhibit vascular smooth muscle cell proliferation， may be through inhibiting cell cycle protein Cyclin D1 and CDK4 expression， block the cell cycle G1 process is closely related to the role.

Mechanism of Qishen Decoction inhibition of macrophage M1 type polarization by targeting TGR5-mediated NLRP3 inflammasome

Objective:To observe the effect of Qishen decoction on TGR5-mediated activation of NLRP3 inflammasome,so as to clarify the molecular mechanism of its inhibition of macrophage M1-type polarisation to ameliorate non-alcoholic steatohepatitis;Methods:Mouse macrophage cell line RAW264.7 was randomly divided into a control group,model group,Qishen decoction group,TGR5 agonist group and Qishen decoction+TGR5 agonist group.Except for the control group,the remaining groups were constructed the macrophage NLRP3 activation model by palmitic acid induction,and the corresponding drugs were given to intervene.ELISA was used to detect the levels of TNF-α,IL-6,IL-1βand CXCL2 in macrophage supernatants,flow cytometry was used to detect the expression levels of macrophage polarisation marker molecules CD86 and iNOS,and Western blot was used to detect the expression of the TGR5/STAT1/STAT6 signaling pathway and the expression of NLRP3 inflammasome-associated proteins,respectively.Results:Compared with the control group,the contents of macrophages TNF-α,IL-6,IL-1β,CXCL2 and the proportion of macrophages with positive expression of CD86 and iNOS were significantly increased in the model group,and the differences were all statistically significant(P<0.01).Compared with the model group,the contents of TNF-α,IL-6,IL-1β,CXCL2 and the proportion of macrophages with positive expression of CD86 and iNOS were significantly decreased in the Qishen decoction group,and the differences were all statistically significant(P<0.01).In addition,the expression of NLRP3 and Pro-IL-1βproteins in the macrophage lysate and the expression of Caspase-1 p10,Caspase-1 p20 and IL-1βp17 proteins in the cell supernatant of the model group were significantly increased when compared with the control group,and the differences were all statistically significant(P<0.01).Compared with the model group,the expression of NLRP3 and Pro-IL-1βproteins in macrophage lysate and the expression of Caspase-1 p10,Caspase-1 p20 and IL-1βp17 proteins in cell supernatant of the Qishen decoction were significantly reduced,and the differences were all statistically significant(P<0.01);Conclusion:Qishen decoction can inhibit the activation of NLRP3 inflammasome in macrophages by inhibiting the TGR5/STAT1/STAT6 signaling pathway,thereby inhibiting macrophage M1 polarization and improving inflammatory response.

Exploration of the molecular mechanism of Qishen decoction in regulating miR-495/FTO pathway mediated macrophage polarization to improve insulin resistance therapy of type 2 diabetes

Objective:To observe the effect of Qishen decoction on macrophage polarization mediated by miR-495/FTO signaling pathway,and to clarify the molecular mechanism of Qishen decoction in improving insulin resistance in the treatment of type 2 diabetes.Methods:THP-1 was induced to differentiate macrophages with phorbol ester.It was divided into the control group,the model group,the Qishen decoction group,the miR-495 inhibitor group,and the Qishen decoction+miR-495 inhibitor group.Except for the control group,the remaining groups were stimulated with 30 mmol/L glucose to construct a macrophage polarization model,and corresponding drugs were given for intervention.Cells were collected from each group for 24 hours and the content of inflammatory factors(IL-6,IL-1β,IL-4,and IL-10)were detected using enzyme-linked immunosorbent assay.The expression of macrophage polarization marker molecules,miR-495,and FTO were detected by flow cytometry,qPCR,and Western blot to detect.Results:Compared with the control group,there was no significant change in the activity of macrophages in the control serum,Qishen decoction containing serum,and miR-495 inhibitor transfected serum,and the difference was not statistically significant(P>0.05).In addition,compared to the control group,the content of IL-6 and IL-1β,the expression levels of CD68,iNOS,COX-2,miR-495,and the ratio of CD68/CD206,were significantly increased(P<0.01).While the content of IL-4 and IL-10,as well as the expression of CD206,Arg-1,YM-1,and FTO were significantly reduced(P<0.01).Compared with the model group,the QiShen decoction significantly reduced the contents of IL-6 and IL-1β,and the expression levels of CD68,iNOS,COX-2,and miR-495,as well as the ratio of CD68/CD206,while the content of IL-4 and IL-10,as well as the expression of CD206,Arg-1,YM-1,and FTO were significantly increased(P<0.01).Conclusion:Qishen decoction upregulate the expression of FTO to promote M2 type polarization of macrophages,thereby inhibiting inflammation and improving insulin resistance by inhibiting the expression of miR-495.

16S rRNA Technology Approach to Explore Mechanism of Qishen Decoction in Improving Nonalcoholic Fatty Liver Fibrosis

Objective: To explore the mechanism of Qishen Decoction in the treatment of nonalcoholicfatty liver fibrosis (NAFLF) by 16S rRNA technology. Methods: NAFLF rat model was established by intraperitoneal injection of carbon tetrachloride combined with high fat diet. During the modeling period, each group was given corresponding drug intervention treatment for 8 weeks. The changes of liver histopathology, serum liver function, lipid and liver fibrosis were analyzed and compared after treatment in each group. The contents of cecum end were collected and the intestinal flora was sequenced by Illumina Miseq platform. Results: Compared with the model group, Qishen Decoction could significantly improve the pathological changes of liver tissue in NALFL rats, and reduce the NAS score, oil red staining area, collagen staining area, ALT, AST, TC, TG, HA, LN, PIIINP and C-IV levels, with significant differences (P<0.05). In addition, compared with the control group, the intestinal flora abundance and diversity of the rats in the model group were significantly reduced (P<0.05), Qishen decoction could significantly increase the abundance and diversity of the intestinal flora of NAFLF rats (P<0.05), and upregulated the abundance of Bacteroidales_S24-7_group_unclassified, Bifidobacterium, Lactobacillu s, Turicibacter, Parabacteroides, Phascolarctobacterium, Lachnospiraceae_NK4A136_group, Coriobacteriaceae_UCG-002, Parasutterella, Odoribacter, Anaerostipes, Ruminococcaceae_unclassified, Allobaculum, Romboutsia, Holdemanella, and Haem ophilus, the difference was statistically significant (P<0.05). Conclusion: Qishen Decoction inhibits liver fibrosis in NAFLF rats by restore the diversity of intestinal flora and increase the abundance of probiotics in intestinal tract.

Effects of adjuvant Qishen Yiqi Drop Pill therapy on renal function changes and prognosis of patients with early diabetic nephropathy

Objective: To study the effects of adjuvant Qishen Yiqi Drop Pill therapy on renal function changes and prognosis of patients with early diabetic nephropathy. Methods: The patients with early diabetic nephropathy treated in our hospital between February 2015 and April 2017 were chosen and divided into two groups by random number table, observation group received Qishen Yiqi Drop Pill combined with conventional therapy and control group accepted conventional therapy. The renal function indexes, cytokine contents and oxygen free radical generation were compared before treatment and 3 months after treatment. Results:Urine UREA levels as well as serum β2 microglobulin (β2-MG), cystatin C (CysC), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), stromal cell-derived factor-1 (SDF-1), interleukin-17 (IL-17), transforming growth factor-β1 (TGF-β1), malondialdehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α) contents of both groups were significantly lower while superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) contents were significantly higher after treatment, and urine UREA level as well as serum β2-MG, CysC, TNF-α, IL-6, SDF-1, IL-17, TGF-β1, MDA and 8-iso-PGF2α contents of observation group after treatment was significantly lower than those of control group while SOD and T-AOC contents were significantly higher than those of control group. Conclusion: Adjuvant Qishen Yiqi Drop Pill therapy can improve the renal function and reduce the inflammatory response and oxidative stress response in early diabetic nephropathy.