Protective effects of Huang-Qi-Ge-Gen decoction against diabetic liver injury through regulating PI3K/AKT/Nrf2 pathway and metabolic profiling.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Qi-Ge-Gen decoction (HGD) is a traditional Chinese medicine prescription that has been used for centuries to treat "Xiaoke" (the name of diabetes mellitus in ancient China). However, the ameliorating effects of HGD on diabetic liver injury (DLI) and its mechanisms are not yet fully understood. AIM OF THE STUDY: To elucidate the ameliorative effect of HGD on DLI and explore its material basis and potential hepatoprotective mechanism. MATERIALS AND METHODS: A diabetic mice model was induced by feeding a high-fat diet and injecting intraperitoneally with streptozotocin (40 mg kg-1) for five days. After the animals were in confirmed diabetic condition, they were given HGD (3 or 12 g kg-1, i. g.) for 14 weeks. The effectiveness of HGD in treating DLI mice was evaluated by monitoring blood glucose and blood lipid levels, liver function, and pathological conditions. Furthermore, UPLC-MS/MS was used to identify the chemical component profile in HGD and absorption components in HGD-treated plasma. Network pharmacology and molecular docking were performed to predict the potential pathway of HGD intervention in DLI. Then, the results of network pharmacology were validated by examining biochemical parameters and using western blotting. Lastly, urine metabolites were analyzed by metabolomics strategy to explore the effect of HGD on the metabolic profile of DLI mice. RESULTS: HGD exerted therapeutic potential against the disorders of glucose metabolism and lipid metabolism, liver dysfunction, liver steatosis, and fibrosis in a DLI model mice induced by HFD/STZ. A total of 108 chemical components in HGD and 18 absorption components in HGD-treated plasma were preliminarily identified. Network pharmacology and molecular docking results of the absorbed components in plasma indicated PI3K/AKT as a potential pathway for HGD to intervene in DLI mice. Further experiments verified that HGD markedly reduced liver oxidative stress in DLI mice by modulating the PI3K/AKT/Nrf2 signaling pathway. Moreover, 19 differential metabolites between normal and DLI mice were detected in urine, and seven metabolites could be significantly modulated back by HGD. CONCLUSIONS: HGD could ameliorate diabetic liver injury by modulating the PI3K/AKT/Nrf2 signaling pathway and urinary metabolic profile.

Beneficial effects of ginsenosides on diabetic nephropathy: A systematical review and meta-analysis of preclinical evidence.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng is one of the most widely used herbs in the world for the treatment of various diseases, and ginsenoside is the representative bioactive component in ginseng. There have been many in vivo studies on ginsenoside for the treatment of diabetic nephropathy (DN), the most common diabetic microvascular complication and the main cause of diabetic morbidity and mortality. AIM OF THE STUDY: The purpose of this study is to evaluate the efficacy of ginsenosides on DN by preclinical evidence and meta-analysis. Meanwhile, the main possible action mechanisms of ginsenosides against DN were also summarized. MATERIALS AND METHODS: We systematically searched PubMed, WOS, Embase, Cochrane, WanFang, Cqvip, CNKI and CBM databases from January 1, 2000, to November 15, 2021, to evaluate the animal experiments of ginsenosides for the treatment of DN. Finally, 30 animal experiments were included. Twelve outcome measures, including renal function indicators (24-h urine protein, serum creatinine, urea nitrogen, creatinine clearance, uric acid, urinary albumin to creatinine ratio), oxidative stress biomarkers (GPX, MDA, SOD), inflammatory factors (IL-1, IL-6, TNF-α) were obtained by using RevMan 5.4 software for meta-analysis. RESULTS: The results showed that except for no significant difference in CCr, other indicators such as 24h UP, SCr, blood urea nitrogen, uric acid and UACR were significantly decreased. It showed that ginsenoside could improve renal function in diabetes. Meanwhile ginsenoside significantly up-regulated antioxidant enzymes SOD and GPX, down-regulated MDA and inflammatory factors IL-1, IL-6 and TNF-α, indicating that ginsenoside may have antioxidant and anti-inflammatory effects. CONCLUSION: Ginsenoside can protect against the renal failure in diabetes through anti-inflammation, anti-oxidation, anti-renal fibrosis, anti-apoptosis/pyroptosis, regulation of blood glucose/lipid metabolism, etc. Which provides preclinical evidence for the application of ginsenoside in the treatment of DN.

Screening bioactive components of Glycyrrhiza uralensis Fisch. with isolated perfused lung extraction and HPLC-ESI-MSn analysis.

The isolated perfused rat lung (IPL), coupled with high performance liquid chromatography\tandem mass spectrometry analysis (HPLC-ESI-MSn), has been developed as a tool for screening bioactive components in Glycyrrhiza uralensis Fisch. (GU). First, IPL was perfused with the water extract of GU (EGU), the bioactive components in the EGU would selectively combine to the receptors or channels of lung. By changing the pH of perfused solution, the combined components were eluated and then detected by HPLC-ESI-MSn. Four compounds were detected in the desorption eluate of IPL, among these compounds, liquiritin (1), ononin (2) and glycyrrhizic acid (4) were identified by comparing with the chromatography of the standards, while licorice-saponin G2 (3) were determined by analysis of the structure clearage characterization of mass spectrometry. Then, due to the lack of compound 3 sample, compounds 1, 2 and 4 with respective concentrations of 50 µM, 5 µM, 500 nM, 50 nM and 5 nM were applied to evaluate the protective effect of pulmonary epithelial cells (PEC, A549 cell) injury induced by lipopolysaccharide (LPS) for anti-inflammatory activity assessment. The results showed that except the 5 nM group of compound 1, 5 nM and 50 nM groups of compound 2, all other groups could remarkably inhibit the PEC injury (vs LPS group, 2-500 nM groups: p < 0.05; other groups: p < 0.01), all compound showed the dose-dependent effect. In conclusion, IPL coupled with HPLC-ESI-MSn was successfully used to screen the anti-inflammatory components of GU for the first time. The application of IPL coupled with HPLC-ESI-MSn for screening bioactive components of TCMs is rapid, convenient and reliable, and the isolated perfused technology could be extended to isolated heart, liver, kidney, and so on.

The C-terminal tails of 4,4'-diphenylmethane-bis(methyl) carbamate are essential for binding to receptor for advanced glycation end products to attenuate advanced glycation end products-induced inflammation and apoptosis responses in human umbilical vein endothelial cells.

OBJECTIVES: A novel compound 4,4'-diphenylmethane-bis(methyl) carbamate (CM1) was shown to possess preventive activity on AGEs-induced human umbilical vein endothelial cells (HUVECs) damage via binding to RAGE. However, the underlying structural basis of CM1 on binding to RAGE was not fully understood. METHODS: In the present study, CM1 analogues were designed and synthesized to compare the activity differences on inhibiting AGEs-induced inflammatory response including TGF-ß1, RAGE protein expression in HUVECs, and macrophages migration and adhesion to HUVECs. In addition, the cell viability and anti-apoptosis activities of CM1 analogues were also examined. KEY FINDINGS: These results indicated that CM1 had higher activities on preventing AGEs-induced HUVECs damage (inflammation, cell viability and apoptosis) than other analogues. The bioaffinity assay was conducted by CMC and demonstrated that the IC50 and dissociation equilibrium constants (Kd) of CM1 were lower whereas the Bmax was higher than other analogues. The incubation of RAGE protein with CM1 analogues by equilibrium dialysis method showed CM1 had a stronger binding rate than other CM1 analogues. CONCLUSION: Our findings suggested that the C-terminal tails (methoxycarbonyl groups) of CM1 were the active groups for binding to RAGE and then led to the attenuation on RAGE-mediated endothelial dysfunction.

Calycosin entered HUVECs and ameliorated AGEs-promoted cell apoptosis via the Bcl-2 pathway.

Endothelial cell (EC) apoptosis plays a pivotal role in the progression of diabetic complications. Abundant studies have demonstrated the pivotal role of advanced glycation end products (AGEs) on the development of diabetes. The aim of the present study was to investigate the effect of calycosin, a phytoestrogen, on AGEs-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Fluorescence polarization and fluorescence absorption assays indicated that calycosin interacted with AGEs in a time-dependent manner. Further studies found that calycosin entered the cells as detected by HPLC. The MTT method demonstrated that calycosin ameliorated AGEs-induced HUVEC apoptosis in a dose-dependent manner, and statistical significance was observed at 1 × 10(-8) M of calycosin; this behavior was further demonstrated by acridine orange/ethidium bromide staining in that the presence of calycosin dramatically reduced AGEs-induced red staining in HUVECs. Further studies found that pre-incubation with calycosin at 1 × 10(-8) M dramatically increased anti-apoptotic Bcl-2 while decreased pro-apoptotic Bax and Bad expressions as detected by immunocytochemistry, and the effect of calycosin on rebalancing the ratio of Bcl-2/Bax was more significant than that of its glycoside, calycosin-7-O-ß-D-glucopyranoside (CG). Furthermore, calycosin slightly reversed AGEs-induced cell oxidative stress at 1 × 10(-8) M, but its antioxidative stress effect was less significant than that of CG. The present study strongly indicates that calycosin can enter the cell and modulate endothelial cell dysfunction by ameliorating AGEs-induced cell apoptosis.

Protection of glycyrrhizic acid against AGEs-induced endothelial dysfunction through inhibiting RAGE/NF-κB pathway activation in human umbilical vein endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice (Glycyrrhiza uralensis roots) is used as a traditional medicine for the treatment of diabetes mellitus and its vascular complications. Glycyrrhizic acid (GA, also known as Glycyrrhizin), a triterpenoid saponin glycoside, is considered to be a bioactive component in Licorice and is beneficial to diabetic vascular complications. AIM OF STUDY: The present study was conducted to evaluate the potential protective activities on AGEs-induced endothelial dysfunction, including anti-apoptosis, antioxidant stress and anti-proinflammatory responses, and explore the underlying mechanism. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated and pre-treated with GA (10(-9)-10(-6)M) or RAGE-Ab (5µg/ml) in the presence or absence of 200µg/ml AGEs. AO/EB fluorescence staining assay was performed to evaluate anti-apoptosis activity. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in cell supernatant were detected by kits while the intracellular reactive oxygen species (ROS) generation was determined by 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) kit. Immunocytochemistry analysis was designed to determine transforming growth factor beta1(TGF-ß1) protein expression while immunofluorescence analysis for RAGE and NF-kB. The protein expressions of TGF-ß1, RAGE and NF-kB were analyzed by Western blot analysis. RESULTS: Pretreatment with GA at a concentration of 10(-8)-10(-6)M significantly reduced the AGEs-induced apoptosis in HUVECs. GA significantly increased antioxidant enzyme SOD activity and decreased peroxide degradation product MDA level in a dose-dependent manner. Furthermore, GA also remarkably inhibited the overgeneration of AGEs-induced ROS. Both immunocytochemistry analysis and western blot analysis showed that GA significantly decreased the protein expression of poinflammatory cytokine TGF-ß1 in a similar manner which RAGE-Ab did. Additionally, AGEs-induced RAGE and NF-kB protein expressions were down-regulated significantly by the pretreatment with GA or RAGE-Ab. CONCLUSION: These findings provide evidences that GA possesses protective activity on AGEs-induced endothelial dysfunction, including anti-apoptosis, anti-inflammation and antioxidant stress, via inhibiting RAGE/NF-kB pathway. GA might be an alternative for the prevention and treatment of diabetic vascular complications in an appropriate dosage.

Pre-column incubation followed by fast liquid chromatography analysis for rapid screening of natural methylglyoxal scavengers directly from herbal medicines: case study of Polygonum cuspidatum.

Methylglyoxal (MGO), a very reactive metabolite of glucose, plays a pivotal role in the pathogenesis of several chronic diseases associated with diabetes, and it has been validated as an attractive target for them. In the present study, a simple and effective method, namely pre-column incubation followed by fast high performance liquid chromatography based on superficially porous particles (shell), coupled with diode array detection and tandem mass spectrometry (UHPLC-DAD-MS(n)), was proposed for rapid and high-throughput screening of natural MGO scavengers directly from the crude extract of Polygonum cuspidatum Sieb. et Zucc, a well-known traditional Chinese medicine which was used for treatment of diabetic complications. The hypothesis is that upon reaction with MGO, the peak areas of components with MGO scavenging potency in the chromatogram will be significantly reduced or disappear, and the structural characterization could be achieved by UHPLC-DAD-MS(n) hyphenated technique. First of all, 12 compounds in P. cuspidatum were well separated within shorter time (~12 min) than previous methods and identified, and two of them, i.e. 3,5,4'-trihydroxystilbene-3-O-(6″-galloyl)-glucoside (3) and emodin-8-O-(6'-malonyl)-glucoside (8) were firstly reported ingredients. After incubation with MGO, four stilbene derivatives were demonstrated to possess potential MGO trapping activities. Furthermore, it was proved that both polydatin (piceid) and resveratrol exhibited effective MGO-trapping capacity by UHPLC analysis, and they could significantly inhibit the formation of advanced glycation end products (AGEs) in the human serum albumin (HSA)-MGO assay, indicating that they were potential candidate agents for delaying and preventing diabetic complications. Additionally, MGO trapping mechanism exploration by UHPLC-MS(n) showed that the positions 2 and 4 of the A ring of stilbene were major active sites for trapping MGO to form both mono- and di-MGO adducts, however, the glucosylation of the hydroxyl group would significantly decrease the trapping efficiency. Collectively, the current work provides a very promising method for rapid discovery of natural MGO scavengers directly from complex matrices such as herbal medicines with huge resources.

Macrophage biospecific extraction and HPLC-ESI-MSn analysis for screening immunological active components in Smilacis Glabrae Rhizoma.

A cell-permeable membrane, as typified by Transwell insert Permeable Supports, permit accurate repeatable invasion assays, has been developed as a tool for screening immunological active components in Smilacis Glabrae Rhizoma (SGR). In this research, components in the water extract of SGR (ESGR) might conjugate with the receptors or other targets on macrophages which invaded Transwell inserts, and then the eluate which contained components biospecific binding to macrophages was identified by HPLC-ESI-MS(n) analysis. Six compounds, which could interact with macrophages, were detected and identified. Among these compounds, taxifolin (2) and astilbin (4) were identified by comparing with the chromatography of standards, while the four others including 5-O-caffeoylshikimic acid (1), neoastilbin (3), neoisoastilbin (5) and isoastilbin (6), were elucidated by their structure clearage characterizations of tandem mass spectrometry. Then compound 1 was isolated and purified from SGR, along with 2 and 4, was applied to the macrophage migration and adhesion assay in HUVEC (Human Umbilical Vein Endothelial Cells) -macrophages co-incultured Transwell system for immunological activity assessment. The results showed that compounds 1, 2 and 4 with concentration of 5µM (H), 500nM (M) and 50nM (L) could remarkably inhibit the macrophage migration and adhesion (Vs AGEs (Advanced Glycation End Produces) group, 1-L, 2-H and 4-L groups: p<0.05; other groups: p<0.01). Moreover, 1 and 4 showed satisfactory dose-effect relationship. In conclusion, the application of macrophage biospecific extraction coupled with HPLC-ESI-MS(n) analysis is a rapid, simple and reliable method for screening immunological active components from Traditional Chinese Medicine.

Development of self-microemulsifying drug delivery system for oral bioavailability enhancement of berberine hydrochloride.

The purpose of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) to improve the oral bioavailability of Berberine hydrochloride (BBH), an important bioactive compound from Chinese Medicines with poor water solubility. Pseudoternary phase diagrams were constructed using oil, surfactant and co-surfactant types to identify the efficient self-microemulsification region. SMEDDS was characterized by morphological observation, droplet size, zeta-potential determination, stability, in vitro release and in vivo bioavailability study. The optimal formulation with the best self-microemulsifying and solubilization ability consisted of 40% (w/w) of ethyl linoleate and oleic acid (2:1), 35% (w/w) Tween-80 and 25% (w/w) glycerol. The SMEDDS of BBH could exhibit good stability. In vitro release test showed a complete release of BBH from SMEDDS was in 5 h. In vivo results indicated that the peak plasma concentration (C(max)) and the area under the curve (AUC(0→12 h)) of SMEDDS of BBH were higher than the commercial tablet by 163.4% and 154.2%, respectively. The relative bioavailability of SMEDDS of BBH was enhanced about 2.42-fold compared with the commercial tablet in rats. The study confirmed that the SMEDDS formulation could be used as a possible alternative to traditional oral formulations of BBH to improve its bioavailability.

The in vitro protective effects of curcumin and demethoxycurcumin in Curcuma longa extract on advanced glycation end products-induced mesangial cell apoptosis and oxidative stress.

Curcuma longa L. (CLL), a traditional herbal medicine, has been widely used for the prevention of diabetic vascular complications in recent years. However, the protective effects of curcuminoids in CLL on the AGEs-induced damage to mesangial cell are not fully understood. In this present study, dihydroethidium, superoxide dismutase kit, malondialdehyde kit, and acridine orange/ethidium bromide staining methods were used to evaluate the activities of curcumin and demethoxycurcumin (10(-11)-10(-9) M) on AGEs-induced oxidative stress and apoptosis, which were associated with the damage to mesangial cell. The results showed that these two compounds could significantly restore advanced glycation end products (AGEs)-induced apoptosis to normal levels (IC50 = 3.874 × 10(-11) M for curcumin and IC50 = 6.085 × 10(-11) M for demethoxycurcumin) and reduce remarkably reactive oxygen species generation in mesangial cell. Furthermore, curcumin and demethoxycurcumin dramatically elevated AGEs-decreased superoxide dismutase activity while significantly reducing AGEs-increased malondialdehyde content in cell culture supernatant. Our results suggest that both curcumin and demethoxycurcumin have a significant protective potential to the prevention of diabetic nephropathy.

Preventative effects of 4,4'-diphenylmethane-bis(methyl) carbamate isolated from cortex mori on human umbilical vein endothelial cell dysfunction induced by advanced glycation end products.

Advanced glycation end-products (AGEs) have been regarded as an initial motivating factor in the pathogenesis of endothelial dysfunction in diabetic complications. 4,4'-Diphenylmethane-bis(methyl) carbamate (DMPC), a carbamate compound, was isolated from Cortex Mori and its prevention effects against AGEs-induced endothelial dysfunction were studied. 4,4'-Diphenylmethane-bis(methyl) carbamate significantly reduced cell apoptosis to normal level at 10⁻9 mol/L concentration. Advanced glycation end-products up-regulated the expression of Bad and Bax and down-regulated Bcl-2 proteins, and pretreatment with DMPC significantly down-regulated Bad and Bax while up-regulating Bcl-2 expressions. In addition, ICAM (intercellular adhesion molecule)-1 and TGF (transforming growth factor)-ß1 expressions in human umbilical vein endothelial cell (HUVEC) were significantly enhanced by AGEs. More importantly, these increases of ICAM-1 and TGF-ß1 expressions were reduced meaningfully with the pretreatment of DMPC. All the results showed DMPC had prevention effects against the progression of AGE-induced endothelial dysfunction, and this compound might be a promising agent against endothelial dysfunction in diabetic vascular complications.

Isolated perfused lung extraction and HPLC-ESI-MS(n) analysis for predicting bioactive components of Saposhnikoviae Radix.

A novel strategy for predicting bioactive components in traditional Chinese material herb was proposed, using isolated perfused rat lung (IPL) extraction and high performance liquid chromatography\tandem mass spectrometry (HPLC-MS(n)) analysis. The hypothesis is that when the IPL is perfused with the extract of Saposhnikoviae Radix (ESR), the potential bioactive components in the ESR should selectively combine with the receptor or channel of lung, by changing the pH of perfused liquid, the combining components would be eluated and then detected by HPLC-ESI-MS(n). Five compounds were detected in the desorption eluate of IPL; among these compounds, two potential bioactive compounds, prim-O-glucosylcimifugin (2) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (4) were identified by comparing with the chromatography of the standard sample, and three other compounds, i.e. cimifugin (1), 5-O-methylvisamminol (3) and sec-O-glucosylhamaudol (5) were determined by analysis of the structure clearage characterization of mass spectrometry. The application of IPL extraction coupled with HPLC-ESI-MS(n) for predicting potential bioactive components of TCMs is rapid, convenient, operational, economic and reliable.

Inhibitory effects of two major isoflavonoids in Radix Astragali on high glucose-induced mesangial cells proliferation and AGEs-induced endothelial cells apoptosis.

Radix Astragali, the dried roots of Astragalus membranaceus var. mongholicus, is well known to have a protective effect on diabetic nephropathy. However, the effects of isoflavonoids in Radix Astragali on glomerular cells, which play a key role in the development of diabetic vascular complications, remain largely unknown. Thus, the purpose of this study was to investigate in vitro the effect of calycosin and calycosin-7-O-ß-D-glucoside, two major isoflavonoids in Radix Astragali, on high glucose-induced rat mesangial cells proliferation and AGEs-induced human glomerular endothelial cell apoptosis. The results indicated that both calycosin and calycosin-7-O-ß-D-glucoside (10-100 µM) could inhibit high glucose-induced mesangial cell early proliferation. Additionally, AGEs-mediated cell apoptosis was also attenuated by treatment of glomerular endothelial cells with either calycosin or calycosin-7-O-ß-D-glucoside (1-100 µM). Therefore, the results obtained in this study suggest that both calycosin and calycosin-7-O-ß-D-glucoside have a significant therapeutic potential to modulate the development and/or progression of diabetic nephropathy.