Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups， namely Huanglian Wendantang-containing serum group and blank serum group， and given 7.8 g·kg-1·d-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance （IR）， and they were randomly divided into control group， model group， metformin hydrochloride group， blank serum group， and Huanglian Wendantang-containing serum high-， medium-， and low-dose groups. After 24 h of cultivation， the cells of each group were treated with insulin for 15 min at concentration of 1×10-7 mol·L-1， and the cell supernatant was collected. The glucose oxidase （GOD-POD） kit was used to determine the glucose content of each group， and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation， thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group， model group， and Huanglian Wendantang-containing serum group. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in each group were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA and protein expression levels of NOD like receptor protein 3 （NLRP3） in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. In terms of the mechanism， HepG2 cells were randomly divided into control group， empty vector group， NLRP3 overexpression group， empty vector + IR group， empty vector + IR + Huanglian Wendantang-containing serum group， NLRP3 overexpression + IR group， and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells， and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， and NLRP3. Immunofluorescence assay was used to detect NLRP3， GSDMD， and Caspase -1 expressions. ResultAs compared with the control group， the glucose consumption in the model group was significantly decreased （P<0.01）. As compared with the model group， the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group （P< 0.01）. As compared with the control group， the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased （P<0.05， P<0.01）. Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β， IL-18， and NLRP3 mRNA and protein expressions as compared with the model group （P<0.05， P<0.01）. Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group （P<0.01）， and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3， Caspase-1， and GSDMD as compared with the empty vector + IR group （P<0.05， P<0.01）. Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group （P<0.05， P<0.01）. ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway， which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus （T2DM）.

Mechanism of Huanglian Wendantang in Improving IR-HepG2 Based on NLRP3/Caspase-1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups， namely Huanglian Wendantang-containing serum group and blank serum group， and given 7.8 g·kg-1·d-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance （IR）， and they were randomly divided into control group， model group， metformin hydrochloride group， blank serum group， and Huanglian Wendantang-containing serum high-， medium-， and low-dose groups. After 24 h of cultivation， the cells of each group were treated with insulin for 15 min at concentration of 1×10-7 mol·L-1， and the cell supernatant was collected. The glucose oxidase （GOD-POD） kit was used to determine the glucose content of each group， and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation， thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group， model group， and Huanglian Wendantang-containing serum group. The levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in each group were determined by enzyme-linked immunosorbent assay （ELISA）， and the mRNA and protein expression levels of NOD like receptor protein 3 （NLRP3） in each group were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. In terms of the mechanism， HepG2 cells were randomly divided into control group， empty vector group， NLRP3 overexpression group， empty vector + IR group， empty vector + IR + Huanglian Wendantang-containing serum group， NLRP3 overexpression + IR group， and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells， and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， and NLRP3. Immunofluorescence assay was used to detect NLRP3， GSDMD， and Caspase -1 expressions. ResultAs compared with the control group， the glucose consumption in the model group was significantly decreased （P<0.01）. As compared with the model group， the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group （P< 0.01）. As compared with the control group， the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased （P<0.05， P<0.01）. Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β， IL-18， and NLRP3 mRNA and protein expressions as compared with the model group （P<0.05， P<0.01）. Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group （P<0.01）， and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3， Caspase-1， and GSDMD as compared with the empty vector + IR group （P<0.05， P<0.01）. Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group （P<0.05， P<0.01）. ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway， which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus （T2DM）.

Effect of Huanglian Wendantang on Inflammatory Response and Hepatocyte Burn Death in IGT Rats / 中国实验方剂学杂志

Objective:To investigate whether there is inflammatory reaction and cell pyroptosis in impaired glucose tolerance （IGT） rats induced by high-fat diet and the intervention effect of Huanglian Wendantang. Method:Healthy male SD rats were fed with 45% fat content diet for 20 weeks to replicate the IGT model. The rats in line with the model establish criteria were randomly divided into 3 groups， with 10 rats in each group， another 10 rats were selected as the blank control group. Huanglian Wendantang group was given 7.8 g·kg^-1·d^-1 compound decoction of Huanglian Wendantang， and the positive control group was given 0.05 g·kg^-1·d^-1 aqueous solution of metformin hydrochloride， with the dose volume of 10 mL·kg^-1·d^-1. The blank group and the model group were given the same volume of distilled water. After continuous intragastric administration for 4 weeks， the body weight， body length and abdominal circumferences were measured， the Lee's obesity index was calculated， and the levels of fasting plasma glucose （FPG） and 2-hour plasma glucose （2 h PG） were detected in each group. Serum interleukin-6 （IL-6） content was detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of nuclear factor kappa B （NF-<italic>κ</italic>B）， cysteinyl aspartate specific proteinase-1 （Caspase-1） and NOD-like receptor protein 3 （NLRP3） in liver tissues were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Immunofluorescence was used to detect the expression of Caspase-1 and NLRP3 in rat liver. Hematoxylin and eosin （HE） staining was used to observe the morphology of liver tissue. Result:Compared with blank group， the body weight， abdominal circumference， body length and Lee's index in model group were significantly increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， the levels of FPG and 2 h PG were significantly increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， serum IL-6 content and NF-<italic>κ</italic>B， Caspase-1 and NLRP3 gene and protein expressions in liver tissue were significantly increased （<italic>P</italic><0.01）， immunofluorescence technique showed that Caspase-1 and NLRP3 expressions increased obviously in IGT rat model’s liver tissues， and inflammatory cells infiltration was observed obviously in HE stained rat liver tissue model. Compared with model group， Huanglian Wendantang and metformin hydrochloride groups could effectively reduce the body weight of IGT rats （<italic>P</italic><0.01） and abdominal obesity， and correct the levels of FPG and 2 h PG （<italic>P</italic><0.01）， while effectively reducing serum IL-6 content and gene and protein expressions of NF-<italic>κ</italic>B， Caspase-1 and NLRP3 in liver tissues of IGT rats， and alleviating inflammatory cell infiltration. Conclusion:Inflammatory response exists in IGT model rats induced by high-fat diet. Huanglian Wendantang can obviously reduce its inflammatory response and alleviate liver cell pyroptosis.

Efficacy of Modified Huanglian Wendantang and Shaofu Zhuyutang Against Ovulation Disorder in Combined Phlegm and Stasis-Induced Polycystic Ovary Syndrome / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of modified Huanglian Wendantang and Shaofu Zhuyutang in the treatment of ovulation disorder in patients with polycystic ovary syndrome （PCOS） due to the combined phlegm and stasis-induced and its influence on chronic inflammation. Method:According to the random number table， 100 patients were divided into a control group （50 cases） and an observation group （50 cases）. Apart from lifestyle intervention and oral administration of clomiphene citrate （CC） capsules to induce ovulation， patients in the control group further received Guizhi Fulingwan， 6 g/time， 2 times/day， while those in the observation group were treated with the modified Huanglian Wendantang and Shaofu Zhuyutang， 1 dose/day， for six menstrual cycles. The ovulation， endometrial thickness， proportion of type A endometrium， as well as the pulsatility index （PI） and resistance index （RI） of uterine artery were monitored before and after treatment. The fasting blood glucose （FBG）， fasting insulin （FINS）， luteinizing hormone （LH）， follicle stimulating hormone （FSH）， serum testosterone （T）， estradiol （E₂）， dehydroepiandrosterone sulfate （DHEAS）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， high-sensitivity C-reactive protein （hs-CRP）， adiponectin （APN）， and interleukin-6 （IL-6） levels before and after treatment were detected， followed by the calculation of homeostasis model assessment-insulin resistance （HOMA-IR） value and the evaluation of ovarian volume and combined phlegm and stasis-induced syndrome score. Result:The overall response rate of the observation group was （44/47） 93.62%， which was higher than （36/46） 78.26% of the control group （<italic>χ</italic>²=4.802， <italic>P</italic><0.05）. The ovulation rate in the observation group was （199/264） 75.38%， higher than （173/272） 63.60% in the control group （<italic>χ</italic>²=8.714， <italic>P</italic><0.01）. The clinical pregnancy rate of the observation group was （11/47） 23.40%， higher than （5/46） 10.87% of the control group， but the difference was not statistically significant （<italic>χ</italic>²=2.564， <italic>P</italic>>0.05）. Compared with the control group， the observation group exhibited reduced PI， RI， LH， T， DHEAS， FINS， FPG， HOMA-IR， TNF-<italic>α</italic>， hs-CRP， and IL-6 （<italic>P</italic><0.01）， but elevated E₂， FSH， and APN （<italic>P</italic><0.01）. Besides， the bilateral ovarian volume and combined phlegm and stasis-induced syndrome score of the observation group were smaller than those of the control group （<italic>P</italic><0.01）， while the endometrial thickness and proportion of type A endometrium were higher （<italic>P</italic><0.01）. Conclusion:On the basis of CC treatment， the modified Huanglian Wendantang and Shaofu Zhuyutang alleviates the ovulation disorder in PCOS patients of combined phlegm and stasis-induced syndrome and regulates IR and chronic inflammation， thus creating a favorable condition for clinical pregnancy， which is worthy of further research.

Effect of Huanglian Wendantang on Classical Pathway of Skeletal Muscle Pyroptosis in IGT Rats / 中国实验方剂学杂志

Objective:To explore the effects of Huanglian Wendantang （HLWDT） on pyroptosis of skeletal muscle in rats with impaired glucose tolerance （IGT） and to explain the mechanism based on NOD-like receptor protein 3 （NLRP3）/cysteine aspartate-specific protease-1 （Caspase-1）/gasdermin D （GSDMD）/interleukin-1<italic>β </italic>（IL-1<italic>β</italic>）/IL-18 signaling pathway. Method:The SD male rats were fed with 45% high-fat diet for 20 weeks to induce the IGT model. After modeling， the rats were randomly divided into a blank group， a model group， a positive control group （metformin hydrochloride， 0.05 g·kg^-1d^-1）， and an HLWDT （7.8 g·kg^-1d^-1） group based on the body weight of rats. The blank group and the model group were fed with the same volume of distilled water. The dose for each group was set as 10 mL·kg^-1d^-1. After four weeks of continuous gavage， blood was collected and serum was separated. The skeletal muscles of rats were stored in liquid nitrogen. Subsequently， serum IL-1<italic>β</italic> and IL-18 were detected by the enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of NLRP3， Caspase-1， and GSDMD were detected by real-time quantitative PCR （Real-time PCR） and Western blot， respectively. The expression of GSDMD， IL-1<italic>β</italic>， and IL-18 proteins in skeletal muscle tissues was detected by immunofluorescence. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of skeletal muscles. Result:Compared with the blank group， the model group showed increased IL-1<italic>β</italic> and IL-18 in serum， and NLRP3， Caspase-1， and GSDMD gene and protein expression in skeletal muscle tissues （<italic>P</italic><0.01）. Immunofluorescence assay showed that GSDMD， IL-18， and IL-1<italic>β </italic>protein expression in skeletal muscle tissues of the model group was significantly elevated （<italic>P</italic><0.01）. HE staining showed obvious pathological changes in skeletal muscles. Compared with the model group， the HLWDT group and the positive control group could decrease IL-1<italic>β</italic> and IL-18 in serum and NLRP3， Caspase-1， and GSDMD gene and protein expression in skeletal muscle tissues （<italic>P</italic><0.01）. In addition， immunofluorescence assay revealed that HLWDT could reduce protein expression levels of GSDMD， IL-1<italic>β</italic>， and IL-18 in skeletal muscles of IGT rats （<italic>P</italic><0.01）. The results of HE staining showed that HLWDT could improve the pathological changes of skeletal muscles in IGT rats<bold>.</bold> Conclusion:HLWDT can inhibit skeletal muscle pyroptosis of IGT rats， and the mechanism may be closely related to NLRP3/Caspase-1/GSDMD/IL-18/IL-1<italic>β</italic> signaling pathway.

Clinical Efficacy of Modified Huanglian Wendantang in Treatment of Newly Diagnosed Type 2 Diabetes Mellitus with Phlegm (Dampness)-Heat Syndrome / 中国实验方剂学杂志

Objective:To observe the efficacy of modified Huanglian Wendantang in treating newly diagnosed type 2 diabetes mellitus （T2DM） with phlegm （dampness）-heat syndrome， in order to study the effect on islet β cell function and adipocytokines. Method:A total of 130 patients were randomly divided into two groups by random number table （65 cases in each group）. The 60 patients in control group completed the treatment （4 patients fell off or lost visit， 2 were eliminated because of breach of plan）， and the 61 patients in observation group completed the treatment （3 patients fell off， 1 were eliminated）. And 20 healthy volunteers were taken as normal control group. Both groups′ patients got lifestyle interventions and metformin hydrochloride tablets （1 tablet/time， 1 time/day during the meal）. In addition， patients in control group got Huazhuo Qingshen Keli in the morning and at night， 5 g/time， 2 times/day， and patients in observation group got modified Huanglian Wendantang， 1 dose/day. And the treatment was lasted for 3 months. Before and after treatment， levels of fasting blood glucose （FBG）， postprandial 2 blood glucose （PBG）， HbA1c and fasting insulin （FINS）， insulin resistance index （HOMA-IR）， insulin sensitivity index （InISI）， islet β cell function index （HOMA-β）， early insulin secretion index （I30/△G30） and late insulin secretion index （AUCI30~I120/G30~G120）， total cholesterol （TC） and triglycerides （TG）， high-density lipoprotein （HDL-C）， low-density lipoprotein （LDL-C）， adiponectin， TNF -α （TNF-α）， resistin and leptin were detected. And syndrome of phlegm （dampness） combined with heat were scored， and the safety was discussed. Result:The total effective rate in observation group was 91.80% （56/61）， which was higher than 78.33% （47/60） in control group （χ2=4.333， P<0.05）. And the score of phlegm （dampness）-heat syndrome was lower than that in control group （P<0.01）， levels of FBG， PBG， HbA1c， HOMA-IR， AUCI30~I120/G30~G120， TC， TG， LDL-C， TNF-α， leptin and resistin were lower than those in control group （P<0.01）， while levels of I30/△G30， HOMA-β， InISI， HDL-C and adiponectin were higher than those in control group （P<0.01）. There was no adverse reaction related to modified Huanglian Wendantang. Conclusion:In addition to treatment with metformin， modified Huanglian Wendantang can effectively control blood glucose and lipid， regulate adipocyte factor， improve early and late phase insulin secretion， improve the function of β cell and insulin sensitivity of islet， improve IR， with a better comprehensive efficacy and a safety in clinical use.

Effect of Huanglian Wendantang in Treatment of Metabolic Syndrome / 中国实验方剂学杂志

Metabolic syndrome (MS) is a pathological condition characterized by central obesity, insulin resistance, hypertension, and hyperlipidemia. With the increase of poor dietary habits and lifestyles in modern society, especially the poor living habits of sedentariness and less movement, the prevalence of MS has increased year by year. According to relevant data, the number of MS patients worldwide will reach about 2.568 billion by 2040, which will seriously endanger human life and health. Huanglian Wendantang, as a famous traditional Chinese medicine prescription for clearing away heat and drying dampness, regulating Qi and resolving phlegm, and benefiting the stomach and gall, has been proved to have significant pharmacological effects in lowering blood fat, reducing blood sugar and resisting inflammation by modern pharmacological studies, and widely used in the treatment of metabolic diseases, cardiovascular diseases and other systemic diseases. In recent years, a large number of studies have proved that Huanglian Wendantang has a significant effect on MS. In terms of clinical efficacy, it could significantly improve the pathological state of obesity, dyslipidemia, abnormal glucose metabolism and hypertension in MS patients. Meanwhile, it could also interfere with the inflammatory state, prethrombotic state, abnormal vascular regulation and other potential risk factors in the body, with a high safety and fewer side effects. In terms of experimental study, it could enhance the insulin sensitivity, and improve the insulin resistance of MS animal models and cell models through interventions in insulin signal transduction, inflammatory response, and antioxidant stress. By retrieving PubMed, CNKI, Weipu, Wanfang and other databases, the author summarized the study reports of Huanglian Wendantang on MS in recent years in three aspects: theoretical study, clinical efficacy study and experimental mechanism study, in the expectation of provide some scientific references for in-depth study of the mechanism of Huanglian Wendantang in treating MS and the development and clinical promotion of the prescription.