Ameliorative effect of cheqianzi decoction on hyperuricemia and kidney injury and underlying mechanism in rats.

Cheqianzi Decoction (CQD) is a Traditional Chinese Medicine (TCM) formula comprising four herbs and is recorded in the Ancient Materia Medica "Shengji Zonglu". Individually, these four herbs have been shown to reduce uric acid (UA) levels, to treat hyperuricemia (HUA), and alleviate kidney damage. However, the therapeutic efficacy of the CQD and related mechanism are not yet clear. In this study, high performance liquid chromatography (HPLC) analysis confirmed that the contents of the chemical components of the four herbal medicines were in accordance with the provisions of the Chinese Pharmacopoeia. A total of 99 potential targets were identified in the network pharmacology analysis of CQD, indicating its involvement in the regulation of inflammatory and apoptotic signaling pathways, and potential value for treating HUA and alleviating kidney injury. In vivo pharmacodynamic studies showed that compared with the Model group, significantly decreased levels of serum uric acid (SUA), serum creatinine (SCr), blood urea nitrogen (BUN) (all P < 0.05), and inflammatory factors (P < 0.01) were detected in the CQD group. Quantitative real-time PCR and Western blot analyses showed that compared with the Model group, adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) expression in the CQD group was significantly upregulated (P < 0.01) at both the mRNA and protein levels, while mRNA expression of Caspase3 and NOD-like receptor family member 3 (NLRP3) (P < 0.05) and protein expression of NLRP3 (P < 0.01) were significantly downregulated. In conclusion, CQD promotes UA excretion by activating ABCG2, and induces inflammasome NLRP3-mediated reduction in inflammatory and apoptotic factors to achieve renal protection. Thus, our findings indicate the therapeutic potential of CQD in HUA with kidney injury.

Differences in p38-STAT3-S100A11 signaling after the administration of aristolochic acid I and IVa may account for the disparity in their nephrotoxicity.

BACKGROUND: The safety of herbs containing aristolochic acids (AAs) has become a widespread concern. Previous reports indicate that AAs are highly nephrotoxic and carcinogenic, although there are more than 170 analogues of aristolochic acid. Not all AAs have the same degree of nephrotoxicity or carcinogenicity. Previous studies have found that aristolochic acid IVa (AA-IVa), the principal component of AAs within members of the Aristolochiaceae family, especially Asarum, a commonly used herb in China, has essentially no significant nephrotoxicity. However, several studies, including ours, have shown that aristolochic acid I (AA-I) is clearly nephrotoxic. PURPOSE: The focus of the study was to elucidate the molecular mechanism responsible for the difference in nephrotoxicity between the AA-I and AA-IVa. STUDY DESIGN/METHOD: Mice were administered with AA-I or AA-IVa for 22 weeks through the oral route, followed by a 50-week recovery time. The kidney tissues of mice were extracted at the end of 22 weeks. Pathological examination and proteomic detection (tandem mass tagging (TMT) and phosphorylated proteomics) were performed on the kidney tissue to investigate the key signaling pathways and targets of AAs-induced renal interstitial fibrosis (RIF). The key signaling pathways and targets were verified by Western blot (WB), siRNA transfection, and luciferase assays. RESULTS: AA-I caused severe nephrotoxicity, high mortality, and extensive RIF. However, the same AA-IVa dosage exhibited almost no nephrotoxicity and does not trigger RIF. The activation of the p38-STAT3-S100A11 signaling pathway and upregulated expression of α smooth muscle actin (α-SMA) and Bcl2-associated agonist of cell death (Bad) proteins could be the molecular mechanism underlying AA-I-induced nephrotoxicity. On the other hand, AA-IVa did not regulate the activation of the p38-STAT3-S100A11 signaling pathway and had relatively little effect on the expression of α-SMA and Bad. Consequently, the difference in the regulation of p38-STAT3-S100A11 pathway, α-SMA, and Bad proteins between AA-I and AA-IVa may be responsible for the divergence in their level of nephrotoxicity. CONCLUSION: This is the first study to reveal the molecular mechanism underlying the difference in nephrotoxicity between AA-I and AA-IVa. Whether STAT3 is activated or not may be the key factor leading to the difference in nephrotoxicity between AA-I and AA-IVa.

Involvement of p38 MAPK/cPLA2 and arachidonic acid metabolic pathway in Shengmai injection-induced pseudo-allergic reactions.

ETHNOPHARMACOLOGICAL RELEVANCE: Adverse reactions to traditional Chinese medicine injections involve pseudo-allergic reactions (PARs). However, in clinical practice, "immediate allergic reactions" and PARs in response to these injections are not often differentiated. AIM OF THE STUDY: This study aimed to clarify the type of reactions produced by Shengmai injections (SMI) and elucidate the possible mechanism. MATERIALS AND METHODS: A mouse model was used to evaluate vascular permeability. Metabolomic and arachidonic acid metabolite (AAM) analyses were performed using UPLC-MS/MS, and the p38 MAPK/cPLA2 pathway was detected by western blotting. RESULTS: The first exposure to intravenous SMI rapidly and dose-dependently induced edema and exudative reactions in the ears and lungs. These reactions were not IgE-dependent and were likely to be PARs. Metabolomic analysis showed that endogenous substances were perturbed in SMI-treated mice, in which the arachidonic acid (AA) metabolic pathway was the most affected. SMI substantially increased the levels of AAMs in lung, including prostaglandins (PGs), leukotrienes (LTs), and hydroxy-eicosatetraenoic acids (HETEs). The p38 MAPK/cPLA2 signaling pathway was activated after a single SMI dose. Inhibitors of cyclooxygenase-2 and 5-lipoxygenase enzymes reduced exudation and inflammation in the ears and lungs of mice. CONCLUSION: Production of inflammatory factors that increase vascular permeability may result in SMI-induced PARs, and p38 MAPK/cPLA2 signaling pathway and downstream AA metabolic pathway are involved in the reactions.

Huangqin Tang Interference With Colitis Associated Colorectal Cancer Through Regulation of Epithelial Mesenchymal Transition and Cell Cycle.

Background: Although the exact molecular mechanisms of colitis-associated colorectal cancer are not fully understood, the chronic inflammation was positively correlated with tumorigenesis. The traditional Chinese medicine botanical formulation Huangqin Tang has significant anti-inflammatory effects. We investigated whether HQT can ameliorate the progression of inflammation to cancer through its anti-inflammatory effects by using relevant predictions and experiments. Methods: We used the azoxymethane/dextran sodium sulfate method to induce the mice colitis-associated colorectal cancer model. After preventive administration of Huangqin Tang to the mice model, colonic tissues were taken for quantitative proteomic analysis of tandem mass tags, and the proteomic results were then experimentally validated using the molecular biology approach. Results: Proteomic screening revealed that the effect of the mechanism of Huangqin-Tang on the colitis-associated colorectal cancer mice model may be related to infinite replication which demonstrated abnormal G1/S checkpoint and epithelial mesenchymal transition acceleration. The levels of inflammatory factors such as interleukin-1α, interleukin-1ß, interleukin-6, and tumor necrosis factor-α were significantly reduced in colitis-associated colorectal cancer mice treated with Huangqin Tang; the aberrant expression of G1/S checkpoint-associated sites of cell cycle protein-dependent kinase 4, D1-type cyclins, and dysregulation of related sites of the WNT pathway which are most related to the acceleration of the epithelial mesenchymal transition process including WNT3A, ß-catenin, E-cadherin, and glycogen synthase kinase 3ß has been improved. Conclusion: Reducing inflammation and thus inhibiting the progression of colitis-associated colorectal cancer by using Huangqin-Tang is effective, and the mechanism of action may be related to the inhibition of uncontrolled proliferation during tumorigenesis. In the follow-up, we will conduct a more in-depth study on the relevant mechanism of action.

Qualitative analysis and differentiation of ginkgo cultivars based on UHPLC-QTOF-MS/MS with the characteristic ion and neutral loss strategy combined with chemometric methods.

The identification of chemical constituents can assist the discovery of active ingredients and can differentiate herbs with multiple cultivars. In this study, a diagnostic ion and neutral loss filtering strategy was developed for the qualitative analysis of ginkgo leaf. The strategy is based on an ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. A large number of (110) of GL compounds were identified, including 8 potentially novel compounds and 42 previously unreported GL constituents. Moreover, 64 available compounds in 48 GL cultivars were analyzed via a combined multicomponent quantitative analysis and statistical analysis. The distribution of the 64 compounds among different cultivars was clarified in a principal component analysis and hot map visualization. Via a variable-importance-for-prediction score analysis, ten main differential compounds were found among the different cultivars. Collectively, these results indicated the usefulness of our approach in chemical profiling and discrimination of herbs with multiple botanical origins. This strategy can also help chemists rapidly identify novel compounds from a complex matrix.

Quantitative Determination and Toxicity Evaluation of Aristolochic Acid Analogues in Asarum heterotropoides F. Schmidt (Xixin) and Traditional Chinese Patent Medicines.

Asarum (Xixin), which contains analogues of aristolochic acid (AA), is the only species of the genus Aristolochia included in the Chinese Pharmacopoeia 2020. However, the contents and nephrotoxic effects of AA analogs in Asarum (Xixin) and its formulations have not been clarified. An automatic, effective solid phase extraction process and UPLC-MS/MS method were established for the pretreatment and quantitative detection of AA analogues in commercially available traditional Chinese patent medicines. The cytotoxicity and DNA damage induced by five analogues of AA were evaluated by CCK8 using human kidney cells (HK-2) and comet assays. HPLC was used to detect the analogues of AA in Asarum heterotropoides F. Schmidt (Xixin). The results showed that the contents of AA I, AA II, and AA IIIa were below the detection limit, while AA IVa and AL I presented relatively high contents of Asarum heterotropoides F. Schmidt (Xixin), within the range of 66.50-121.03 µg/g and 19.73-43.75 µg/g, respectively. The levels of AA analogues were in the nanogram-per-gram level in the main traditional Chinese patent medicines. AA I and AL I exhibited relatively high cytotoxicity at 48 h in CCK8 assays, while AA II, AA IIIa, and AA IVa showed weak cytotoxicity even at 800-1,000 µM. AA I induced significant pathological alterations and direct DNA damage at 40 mg/kg and 20 mg/kg, respectively. No distinct nephrotoxicity or hepatotoxicity was observed in mice treated with AA II, AA IIIa, AA IVa, or AL I at 40 mg/kg in this study. Consumption of Asarum heterotropoides F. Schmidt (Xixin) with controlled doses and periods is relatively safe as the contents of AA analogues in Asarum heterotropoides F. Schmidt (Xixin) and its formulations were far below those causing acute toxicity in this study. But, the long-term toxicity of Asarum heterotropoides F. Schmidt (Xixin) still needs further study.

Effect of Huangqin Tang on Urine Metabolic Profile in Rats with Ulcerative Colitis Based on UPLC-Q-Exactive Orbitrap MS.

As a classic prescription, Huangqin Tang (HQT) has been widely applied to treat ulcerative colitis (UC), although its pharmacological mechanisms are not clear. In this study, urine metabolomics was first analysed to explore the therapeutic mechanisms of HQT in UC rats induced by TNBS. We identified 28 potential biomarkers affected by HQT that might cause changes in urine metabolism in UC rats, mapped the network of metabolic pathways, and revealed how HQT affects metabolism of UC rats. The results showed that UC affects amino acid metabolism and biosynthesis of unsaturated fatty acids and impairs the tricarboxylic acid cycle (TCA cycle). UC induced inflammatory and gastrointestinal reactions by inhibiting the transport of fatty acids and disrupting amino acid metabolism. HQT plays key roles via regulating the level of biomarkers in the metabolism of amino acids, lipids, and so on, normalizing metabolic disorders. In addition, histopathology and other bioinformatics analysis further confirm that HQT altered UC rat physiology and pathology, ultimately affecting metabolic function of UC rats.

Effect of Huangqin Tang on Colonic Gene Expression in Rats with Ulcerative Colitis.

In this study, we explored the pharmacological mechanisms of Huangqin Tang (HQT; a traditional Chinese medicine formula) in ulcerative colitis (UC) and provided evidence for potential roles HQT plays by gene expression profiling. The UC rat model was made via a compound method (trinitrobenzene sulfonic acid plus ethanol). After a ten-day treatment, microarray analysis was performed from the colon segment of the rats. Biological functions and specific signaling pathways were enriched based on differentially expressed genes (DEG), and corresponding gene networks were constructed via Ingenuity Pathway Analysis (IPA). Through the network, we screened the potential "candidate targets," such as ITGB1, FN1, CASP3, and ITGA5 and FABP1, ABCB1, FABP2, and SLC51B. These potential candidate targets were functionally related to immune responses, inflammation, and metabolism. Moreover, HQT significantly decreased serum levels of proinflammatory factors nitrogen monoxide (NO), proinflammatory cytokines interleukin- (IL-) 17, and prostaglandin E2 (PGE2). The degree of HE staining of colonic tissue was severe in the model group but reduced significantly in the HQT group. HQT exhibited protective effects against colon damage by inhibiting the inflammatory response.

Forsythoside A and Forsythoside B Contribute to Shuanghuanglian Injection-Induced Pseudoallergic Reactions through the RhoA/ROCK Signaling Pathway.

In recent years, hypersensitivity reactions to the Shuanghuanglian injection have attracted broad attention. However, the componential chief culprits inducing the reactions and the underlying mechanisms involved have not been completely defined. In this study, we used a combination of approaches based on the mouse model, human umbilical vein endothelial cell monolayer, real-time cellular monitoring, immunoblot analysis, pharmacological inhibition, and molecular docking. We demonstrated that forsythoside A and forsythoside B contributed to Shuanghuanglian injection-induced pseudoallergic reactions through activation of the RhoA/ROCK signaling pathway. Forsythoside A and forsythoside B could trigger dose-dependent vascular leakage in mice. Moreover, forsythoside A and forsythoside B slightly elicited mast cell degranulation. Correspondingly, treatment with forsythoside A and forsythoside B disrupted the endothelial barrier and augmented the expression of GTP-RhoA, p-MYPT1, and p-MLC2 in a concentration-dependent manner. Additionally, the ROCK inhibitor effectively alleviated forsythoside A/forsythoside B-induced hyperpermeability in both the endothelial cells and mice. Similar responses were not observed in the forsythoside E-treated animals and cells. These differences may be related to the potential of the tested compounds to react with RhoA-GTPγS and form stable interactions. This study innovatively revealed that some forsythosides may cause vascular leakage, and therefore, limiting their contents in injections should be considered.

Flavonoid profiling of a traditional Chinese medicine formula of Huangqin Tang using high performance liquid chromatography.

The quality control processes for herbal medicines have been problematic. Flavonoids are the major active components of Huangqin Tang (HQT, a traditional Chinese medicine formula). In this study, we used a combinative method approach consisting of chromatographic fingerprinting (high performance liquid chromatography; HPLC), quantitative methods and a pharmacodynamic evaluation model to analyze the flavonoids of HQT obtained from different sources. Ten batches of HQT were analyzed by the HPLC fingerprinting method and 26 common peaks were detected, of which 23 peaks corresponded with the chemical profile of HQT. In addition, 11 major compounds were identified by LC-MS analysis (liquid chromatography-tandem mass spectrometer; LC-MS (n) ) and quantified by the HPLC quantitative method approach. The studied 10 batches of HQT were found to be homogeneous in their composition with a similarity between 0.990 and 1.000. The distribution of the 11 identified compounds was found to be very similar among the batches. Only slight pharmacodynamic differences were detected between the different batches, confirming the homogeneity of HQT. The results of this study prove that the combination of chromatographic fingerprinting and quantitative analysis can be readily used for comprehensive quality control of herbal medicines.

[Effect of Huangqin Tang on the function of regulatory TLR4/MyD88 signal pathway in rats with ulcerative colitis].

This study was designed to investigate the effect of Huangqin Tang (HQT) on TLR4/Myd88 pathway and the downstream cytokines in rats with ulcerative colitis (UC) to explore its underlying mechanisms of action. The model of UC rats with cell immunoreactivity was made using a compound method (trinitrobenzene sulfonic acid plus ethanol). Rats were randomly divided into the control group, the model group, the salazosulfapyridine (SASP) group, high, medium and low dose(20, 10, 5 g·kg-1) of HQT groups. After a three-day treatment, production of NO in serum was detected by Griess assay, the levels of interleukin (IL)-4, IL-10, IL-17 and prostaglandin E2（PGE2） in serum were detected by ELISA. After a five-day treatment, the positive protein expressions of COX-2 and iNOS in the colon tissue were determined by ICH method, the protein expressions of TLR4 and MyD88 in colon tissue were determined by Western blot. Compared with the control group, the levels of NO, IL-17, PGE2, the protein expressions of TLR4, MyD88 and the protein positive expressions of COX-2, iNOS were apparently higher in the model group. Compared with model group, the above indexes were significantly improved in the SASP and high-dose HQT groups (P < 0.05). These results show that HQT has a definite effect on UC in rats. Its mechanisms of action may be achieved by inhibiting the activity of TLR4/MyD88/NF-κB signal pathway and down-regulation of NO, IL-17 and PGE2 production.

[Simultaneous determination and quantitation of anthraquinones, lignans and flavonoids in Xiao-Cheng-Qi Tang, Hou-Po-San-Wu Tang and Hou-Po-Da-Huang Tang by HPLC].

A simple and selective HPLC method for simultaneous determination and quantification of anthraquinones, lignans and flavonoids in Xiao-Cheng-Qi Tang (XCQT), Hou-Po-San-Wu Tang (HPSWT) and Hou-Po-Da-Huang Tang (HPDHT) was developed and validated. An Agilent Zorbax SB-C 18 (4.6 mm x 250 mm, 5 µm) column with the mobile phase of acetonitrile and 0.5% acetic acid aqueous solution in gradient elution mode was used. The flow rate was 1.0 mL · min(-1) at 30 °C, and injection volume was 10 µL. The detection wavelength was set at 254 nm and 294 nm simultaneously for the quantitative analysis. The current HPLC assay was validated for linearity, intra-day and inter-day precisions, accuracy, recovery and stability. The method was applied to the content comparison of the gallic acid, cinnamic acid, sennoside A, sennoside B, rhein, emodin, aloe-emodin, chrysophanol, physcion, magnolol, honokiol, narirutin, naringin, hesperidin, neohesperidin, hesperetin, naringenin and nobiletin in XCQT, HPSWT and HPDHT. The good linear equations of eighteen constituents were obtained within the investigated ranges (r > 0.998). The recovery of the method was 94.28%-99.89% and the precision was less than 5%. The sample was stable within 16 h. There were some differences between the contents of anthraquinones, lignans and flavonoids in analogous formulae about XCQT. XCQT contained the greatest abundance of anthraquinones and flavonoid, HPSWT contained the greatest abundance lignans. In conclusion, the methods are simple, low-cost, precise, accurate and reliable for the determination of eighteen constituents in analogous formulae about XCQT, and these results provide methodological support for its quality control.