[Effects and mechanisms of Gegen Qinlian Decoction in inhibiting M1 polarization of macrophages under inflammatory hypoxia microenvironment].

To explore the effect and mechanism of Gegen Qinlian Decoction(GQD) in inhibiting M1 polarization of macrophages under inflammatory hypoxia by simulating intestinal hypoxia microenvironment in vitro. A tri-gas incubator was used to simulate normal physiological hypoxia of the colon and inflammatory hypoxia microenvironments of ulcerative colitis(UC). RAW264.7 macrophages were divided into 18.5% O_(2 )(normoxia group), 4% O_2(physiological hypoxia group), and 1% O_2(inflammatory hypoxia group), and they were induced by lipopolysaccharide(LPS) for 24 h. M1 polarization was detected by flow cytometry. Under the condition of 1% inflammatory hypoxia, they were divided into blank group, model group, and GQD-containing serum low, medium, and high dose groups. Flow cytometry was used to detect M1 polarization marker CD86, and ELISA was used to detect the expression of tumor necrosis factor-α(TNF-α) and interleukin-1ß(IL-1ß) in cell supernatant. The mRNA expression of hypoxia-inducible factor-1α(HIF-1α), TNF-α, and IL-1ß was detected by qRT-PCR. Western blot was used to detect the expression of HIF-1α/nuclear transcription factor-κB(NF-κB) signaling pathway-related proteins. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The results showed that the positive rate of CD86 in the 1% O_2 group was the highest. Under the condition of 1% inflammatory hypoxia, compared with the blank group, the expression of CD86, TNF-α, IL-1ß, and HIF-1α in the model group increased. Compared with the model group, each group of GQD could reduce the expression of CD86, TNF-α, IL-1ß, and HIF-1α. Compared with the blank group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/ß, and p-IκBα in the model group increased. Compared with the model group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/ß, and p-IκBα in GQD groups was significantly decreased. Compared with the blank group, NF-κB p65 in the model group entered the nucleus significantly. Compared with the model group, the nuclear expression of NF-κB p65 was decreased in each GQD group. Studies have shown that GQD may protect the intestine by down-regulating the HIF-1α/NF-κB signaling pathway to inhibit M1 polarization of macrophages and secretion of related inflammatory factors under 1% inflammatory hypoxia.

Timing of Surgery and Treatment Options for Congenital Ptosis in Children: A Narrative Review of the Literature.

Congenital ptosis has varying degrees of impact on the visual development or psychological health of a child depending on its severity. Some controversies and misconceptions remain regarding the management of congenital ptosis in children. Particularly, the accurate diagnosis of the severity of congenital ptosis in younger children, assessment of the visual developmental status of the child, optimal timing of surgery, and treatment choice are still issues in clinical practice that need to be explored. This report presents a comprehensive review of these aspects of the correction of congenital ptosis to provide a valuable reference for clinical practice. Our review shows that currently used surgical procedures for ptosis may result in over- or under-correction to varying degrees. The differences may be due to the physical condition and age of the child and the degree of cooperation during preoperative examination and assessment, resulting in inaccurate results. Alternatively, intraoperative swelling and bleeding may lead to errors in the values recorded by the surgeon. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

[Metabolomic study on urine of chronic inflammation rats treated with Buyang Huanwu Decoction based on UPLC-Q-TOF-MS].

The study investigated the effect of Buyang Huanwu Decoction(BYHWD) on endogenous biomarkers in the urine of rats with chronic inflammation induced by lipopolysaccharide(LPS) using ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS), aiming to elucidate the molecular mechanism underlying the therapeutic effect of BYHWD on chronic inflammation from a metabolomics perspective. Male SD rats were randomly divided into a normal group, a model group, and low-, medium-, and high-dose BYHWD groups(7.5, 15, and 30 g·kg~(-1)). The model group and BYHWD groups received tail intravenous injection of LPS(200 µg·kg~(-1)) on the first day of each week, followed by oral administration of BYHWD once a day for four consecutive weeks. Urine samples were collected at the end of the administration period, and UPLC-Q-TOF-MS was used to analyze the metabolic profiles of the rat urine in each group. Multivariate statistical analysis methods such as principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to analyze the effect of BYHWD on endogenous metabolites. One-way ANOVA and variable importance for the projection(VIP) were used to screen for potential biomarkers related to chronic inflammation. The identified biomarkers were subjected to pathway and enrichment analysis using MetaboAnalyst 5.0. A total of 25 potential biomarkers were screened and identified in the rat urine in this experiment. Compared with the normal group, the model group showed significant increases in the levels of 14 substances(P<0.05) and significant decreases in the levels of 11 substances(P<0.05). BYHWD was able to effectively reverse the trend of most endogenous biomarkers. Compared with the model group, BYHWD significantly down-regulated 13 biomarkers(P<0.05) and up-regulated 10 biomarkers(P<0.05). The metabolic products were mainly related to the biosynthesis of pantothenic acid and coenzyme A, tryptophan metabolism, retinol metabolism, and propionate metabolism. BYHWD has therapeutic effect on chronic inflammation induced by LPS, which may be related to its ability to improve the levels of endogenous metabolites, enhance the body's anti-inflammatory and antioxidant capabilities, and restore normal metabolic activity.

[Study on mechanisms of anti-Alzheimer's disease action of absorbed components of Gardeniae Fructus based on network pharmacology].

Gardeniae Fructus has the traditional effects of promoting intelligence and inducing resuscitation, but its mechanism is unclear. In this study, the relationship between Gardeniae Fructus's traditional effect of promoting intelligence and inducing resuscitation and anti-Alzheimer's disease effect was taken as the starting point to investigate the anti-Alzheimer's disease mechanism of the major absorbed components in Gardeniae Fructus by the network pharmacology method. The network pharmacology research model of "absorbed composition-target-pathway-disease" was adopted. In this study, the active components screening and target prediction technology were used to determine the active components and targets of Gardeniae Fructus in treatment of Alzheimer's disease. The enrichment pathway and biological process of Gardeniae Fructus were studied by using the bioinformatics annotation database(DAVID), and the results of molecular docking validation network analysis were used to elaborate the mechanism of Gardeniae Fructus in treatment of Alzheimer's disease. It was found that 35 absorbed components of Gardeniae Fructus not only regulated 48 targets such as cholines-terase(BCHE) and carbonic anhydrase 2(CA2), but also affected 11 biological processes(e.g. transcription factor activity, nuclear receptor activity, steroid hormone receptor activity, amide binding and peptide binding) and 7 metabolic pathways(MAPK signaling pathway, Alzheimer disease and estrogen signaling pathway, etc.). Molecular docking results showed that more than 60% of the active components could be well docked with key targets, and the relevant literature also showed that the active components could inhibit the MAPK1 expression of key targets, indicating a high reliability of results. These results indicated that Gardeniae Fructus may play its anti-Alzheimer's disease action via a "multi-ingredients-multi-targets and multi-pathways" mode, providing a scientific basis for further drug research and development.

[Research on network pharmacology of Mongolian medicine Cymbaria in treatment of type 2 diabetes].

The network pharmacology was used to explore the potential active ingredients and action mechanisms of Mongolian medicine Cymbaria in the treatment of type 2 diabetes. According to the literatures collected, Cymbaria component database was established to define important active ingredients and key targets for the anti-hyperglycemic effect to predict action mechanism by active ingredient screening and target prediction techniques. Molecular docking predicted binding activity of main active components with key targets in Cymbaria, then verified the action mechanism in vitro. The Cymbaria component database contained 177 chemical components, 90 chemical structures were confirmed, including 34 chemical components with effective targets. According to the prediction results from network pharmacology, 61 biological processes were significantly affected, such as fatty acid metabolism including PPARs signaling pathway, protein kinase activity and insulin signal pathway. Moreover, the key target proteins were Akt1 and TNFα and quercetin, luteolin and catalpol were the main active ingredients of Cymbaria. Molecular docking prediction showed that luteolin, quercetin and catalpol had a strong binding activity with Akt1; luteolin had strong binding activity but quercetin and catalpol had a certain binding activity with TNFα. Furthermore, catalpol showed hypoglycemic effects in vitro, which up-regulated p-Akt(Ser473)/Akt, PPARα and PPARδ levels and reduced FABP4 expression to regulate glycose and lipid metabolism for improving insulin sensitivity. The network pharmacology predicted that the hypoglycemic effect of Cymbaria was mainly related to anti-inflammatory and lipid regulation with a multi-component, multi-target manner. It provided a scientific view of hypoglycemic effect and action mechanism of Cymbaria for further study.

[Compound traditional Chinese medicine in treatment of diabetes].

Diabetes is a chronic disorder of glucose metabolism characterized by elevated blood glucose levels. With the improvement in living standards and the changes in lifestyle,T2 DM incidence has a dramatic increase in the past decades,bringing a series of public health problems. In the research and development field of diabetic drugs,T1 DM drugs are mainly long-acting sustained-release insulin preparations,whereas T2 DM drugs mainly are based on single target. T2 DM drugs usually have a good anti-hyperglycemic effect,but with side effects for long-term administration. Therefore,the research and development of hypoglycemic drugs focus on formula drugs with multi-component,multi-target and multi-pathway effects. In the similar principle of action,traditional Chinese medicine formula has achieved a good efficacy in the treatment of diabetes,with mild anti-hyperglycemic effects and multiple-component synergistic effects in intervening the pathogenesis of diabetes,including additive effect,synergy effect and toxicity scattering effect. This article mainly reviews clinical trials and mechanisms of action of traditional Chinese medicine formula for the treatment of diabetes,so as to provide a reference to the rational and effective clinical application of traditional Chinese medicine formula in treating diabetes.

[Gegen Qinlian decoction activates PPARγ to ameliorate adipocytic insulin resistance in diabetic SD rats and IR-3T3-L1 adipocytes].

To investigate the effects of Gegen Qinlian decoction(GQD) in improving adipocytic insulin resistance(IR) and explore its related molecular mechanism. Diabetic rats models were induced by high glucose and high-fat diet with a small dose of streptozotocin, and after GQD treatment for 3 months, blood biochemical indexes such as fasting blood-glucose(FBG), insulin, glycosylated serum protein(GSP) and HOMA-IRI were detected and assessed. After the total RNA was extracted from the adipose tissue of diabetic SD rats, PPARγ, ADPN, GLUT4, GLUT2, ACACA and ACACB mRNA expression levels were separately detected by qPCR. Then, stable IR-3T3-L1 adipocyte model was built with 1 µmol•L⁻¹ dexamethasone. After the cell viability was detected by CCK-8 assay, 5%, 10% and 15% GQD-containing serum(GQD-CS) were respectively used to treat IR-3T-L1 adipocytes for 24 h. The contents of glucose, nonesterified fatty acid(NEFA) and adiponectin in cell culture supernatants were separately detected whereas the intracellular triglyceride(TG) contents of IR-3T3-L1 adipocytes were also measured. The ADPN, PPARγ and GLUT4 mRNA and protein expression levels were respectively detected by qPCR and Western blot in IR-3T3-L1 adipocytes. Results showed that GQD significantly decreased fasting blood glucose, insulin and GSP(P<0.01), and down-regulated HOMA-IRI(P<0.05) after the high-fat diet/streptozotocin-induced diabetic SD rats were treated for three months, with a good hypoglycemic effect. Moreover, PPARγ, ADPN, GLUT4, GLUT2, ACACA and ACACB mRNA expression levels were significantly elevated in the adipose tissue of GQD-treated diabetic SD rats. The 5%, 10% and 15% GQD-CS significantly increased glucose consumption of IR-3T3-L1 adipocytes at 24 h treatment(P<0.01), significantly decreased the intracellular TG content (P<0.01), and down-regulated NEFA to a certain extent but not significantly. Moreover, GQD-CS significantly up-regulated GLUT4 and ADPN expression. The results indicated that GQD could activate PPARγ to ameliorate adipocytic insulin resistance in the diabetic SD rats and IR-3T3-L1 adipocytes.

[Effects of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin-resistant adipocytes].

Adipocytokines are closely associated with insulin resistance (IR) in adipose tissues, and they are more and more seriously taken in the study of the development of diabetes. This experiment was mainly to study the effect of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin resistant adipocytes, and investigate the molecular mechanism of berberine in enhancing insulin sensitization and application advantages of droplet digital PCR (ddPCR). ddPCR absolute quantification analysis was taken in this experiment to simply and intuitively determine the appropriate reference genes. ddPCR and quantitative Real-time PCR (qPCR) were used to compare the effect of different doses of berberine (10, 20, 50, 100 µmol•L⁻¹) on mRNA expression levels of PPARγ, adiponectin, resistin and leptin in IR 3T3-L1adipocytes. Antagonist GW9662 was added to study the inherent correlation between PPARγ and adiponectin mRNA expression levels. ddPCR results showed that the expression level of ß-actin in adipocytes was stable, and suitable as reference gene for normalization of quantitative PCR data. Both of ddPCR and qPCR results showed that, as compared with IR models, the mRNA expression levels of adiponectin were decreased in the treatment with berberine (10, 20, 50, 100 µmol•L⁻¹) in a dose-dependent manner (P<0.01); the expression of PPARγ was decreased by 20, 50, 100 µmol•L⁻¹ berberine in a dose-dependent manner in qPCR assay (P<0.01) and decreased only by 50 and 100 µmol•L⁻¹ berberine in ddPCR assay (P<0.05). PPARγ specific antagonist GW9662 intervention experiment showed that adiponectin gene expression was directly relevant with PPARγ (P<0.05). ddPCR probe assay showed that various doses of berberine could significantly reduce mRNA expression levels of resistin and leptin (P<0.01) in a dose-dependent manner. In conclusion, berberine enhanced insulin sensitization effect not by up-regulating adiponect in expression of transcriptional level in PPARγ-dependent manner, but may by the elevated multimerization of adiponectin in the posttranslational regulation level. Berberine down-regulated the resistin and leptin expression levels, which could alleviate lipolysis and improve IR in adipocytes. ddPCR provided better sensitivity and linear range than qPCR, with obvious technical advantages for the detection of low abundance expression of target genes.

[Kudzu root (Ge-Gen) regulates on glucose and lipid metabolism to ameliorating insulin resistance on 3T3-L1 adipocytes].

This study aimed to explore the mechanism of Chinese traditional medicine, Kudzu root(Chinese name：Ge-Gen; Latin name： Puerariae Lobatae Radix) how to improving insulin resistance (IR) through the regulation of the glucose and lipid metabolism in the IR-3T3-L1 adipocytes. After the 3T3-L1 mouse preadipocytes were differentiated into mature adipocytes, IR model(IR-3T3-L1) was built with 1 µmol•L-1 dexamethasone treatment for 96 h. IR adipocytes were treated with different concentrations (5%,10% and 15%) of Ge-Gen containing serum (GG-CS)for 12 h or 24 h, whereas rosiglitazone group as positive control in this study. The glucose contents in cell culture supernatants were detected by glucose oxidase assay and the intracellular triglyceride (TG) contents were measured by glycerol phosphate oxidase assay respectively.The mRNA expression levels of PPARγ, ADPN, GLUT4, LPL, FABP4 and FASn gene were determined by real-time quantitative PCR(qPCR).Results showed that IR-3T3-L1 adipocytes significantly increased glucose consumption (P<0.01)and decreased TG contents (P<0.01) as compared with the normal control group, the glucose consumption significantly increased with the treatment of GG-CS (P<0.01) by dose-dependent and time-dependent manners,whereas the intracellular TG content was sigificantly decreased (P<0.01) by dose-dependent manner.qPCR analysis revealed that 10% and 15% GG-CS significantly up-regulated the mRNA expression level of PPARγ, ADPN and GLUT4 (P<0.01) with the same dose-dependent manner,whereas the GLUT4 mRNA expression was showed similar expression pattern with the treatment of 10% and 15% GG-CS (P<0.01).We also detected the mRNA expression levels of several important lipid-metabolizing enzymes such as LPL, FASn and FABP4 by PPARγ regulation. 15% GG-CS elevated LPL mRNA expression (P<0.05);10% and 15% GG-CS enhanced the FASn mRNA expression (P<0.01), whereas 5%,10% and 15% GG-CS down-regulated FABP4 mRNA expression (P<0.01). Together, our results indicated that GG could regulate the glucose and lipid metabolism to ameliorate IR with multi-target manners in 3T3-L1 adipocytes.