Huang Gan Formula Alleviates Systemic Inflammation and Uremia in Adenine-Induced Chronic Kidney Disease Rats May Associate with Modification of Gut Microbiota and Colonic Microenvironment.

Purpose: This study aims to investigate the effects of Huang Gan formula (HGF), a Chinese herbal prescription used for chronic kidney disease (CKD), on the regulation of the gut microbiota and colonic microenvironment of CKD. Methods: CKD rats were induced by 150 mg/kg adenine gavage for 4 weeks, then orally treated with or without 3.6 g/kg or 7.2 g/kg of HGF for 8 weeks. The renal function and structure were analyzed by biochemical detection, hematoxylin and eosin, Masson's trichrome, Sirius red and immunochemical staining. Average fecal weight and number in the colon were recorded to assess colonic motility. Further, the changes in the gut microbiota and colonic microenvironment were evaluated by 16S rRNA sequencing, RT-PCR or immunofluorescence. The levels of inflammatory cytokines, uremic toxins, and NF-κB signaling pathway were detected by RT-PCR, ELISA, chloramine-T method or Western blotting. Redundancy analysis biplot and Spearman's rank correlation coefficient were used for correlation analysis. Results: HGF significantly improved renal function and pathological injuries of CKD. HGF could improve gut microbial dysbiosis, protect colonic barrier and promote motility of colonic lumens. Further, HGF inhibited systemic inflammation through a reduction of TNF-α, IL-6, IL-1ß, TGF-ß1, and a suppression of NF-κB signaling pathway. The serum levels of the selected uremic toxins were also reduced by HGF treatment. Spearman correlation analysis suggested that high-dose HGF inhibited the overgrowth of bacteria that were positively correlated with inflammatory factors (eg, TNF-α) and uremic toxins (eg, indoxyl sulfate), whereas it promoted the proliferation of bacteria belonging to beneficial microbial groups and was positively correlated with the level of IL-10. Conclusion: Our results suggest that HGF can improve adenine-induced CKD via suppressing systemic inflammation and uremia, which may associate with the regulations of the gut microbiota and colonic microenvironment.

Development and validation of an LC-MS/MS method for detection of glycyrol in rat plasma and its application to a pharmacokinetic study.

Licorice (Glycyrrhiza uralensis) is one of the most popular edible and medicinal plants and is widely used in Asia. Glycyrol (GC) is a major coumarin present in licorice that exhibits various biological activities. We aimed to develop a highly sensitive and rapid liquid chromatography coupled with mass spectrometry method for the quantitative determination analysis of GC in rat plasma. GC showed linear calibration ranges of 1-100 and 50-2,000 ng/ml with correlation coefficients >0.99. The average extraction recovery ranged from 113.26 to 114.84%, and the relative standard deviation of internal standard normalized matrix factors ranged from 6.36 to 9.46%. The intra-day and inter-day precisions of GC were <15%, and the accuracy ranged from 95.31 to 112.72%. Pharmacokinetic studies showed that GC was distributed in the body with a volume of distribution of 9.06 L/kg, and the initial plasma concentration was 3275.11 ng/ml. The area under the plasma concentration vs. time curve was 479.25 ng h/ml. It was rapidly eliminated with a terminal elimination half-life of 1.47 h and a clearance rate of 4.24 L/h/kg. The pharmacokinetic results can help us to better understand the pharmacological effects of GC in the body.

Herb-Drug Interaction Between Xiyanping Injection and Lopinavir/Ritonavir, Two Agents Used in COVID-19 Pharmacotherapy.

The global epidemic outbreak of the coronavirus disease 2019 (COVID-19), which exhibits high infectivity, resulted in thousands of deaths due to the lack of specific drugs. Certain traditional Chinese medicines (TCMs), such as Xiyanping injection (XYPI), have exhibited remarkable benefits against COVID-19. Although TCM combined with Western medicine is considered an effective approach for the treatment of COVID-19, the combination may result in potential herb-drug interactions in the clinical setting. The present study aims to verify the effect of XYPI on the oral pharmacokinetics of lopinavir (LPV)/ritonavir (RTV) using an in vivo rat model and in vitro incubation model of human liver microsomes. After being pretreated with an intravenous dose of XYPI (52.5 mg/kg) for one day and for seven consecutive days, the rats received an oral dose of LPV/RTV (42:10.5 mg/kg). Except for the t1/2 of LPV is significantly prolonged from 4.66 to 7.18 h (p < 0.05) after seven consecutive days pretreatment, the pretreatment resulted in only a slight change in the other pharmacokinetic parameters of LPV. However, the pharmacokinetic parameters of RTV were significantly changed after pretreatment with XYPI, particularly in treatment for seven consecutive days, the AUC0-∞ of RTV was significantly shifted from 0.69 to 2.72 h µg/mL (p < 0.05) and the CL exhibited a tendency to decrease from 2.71 L/h to 0.94 L/h (p < 0.05), and the t1/2 of RTV prolonged from 3.70 to 5.51 h (p < 0.05), in comparison with the corresponding parameters in untreated rats. After administration of XYPI, the expression of Cyp3a1 protein was no significant changed in rats. The in vitro incubation study showed XYPI noncompetitively inhibited human CYP3A4 with an apparent Ki value of 0.54 mg/ml in a time-dependent manner. Our study demonstrated that XYPI affects the pharmacokinetics of LPV/RTV by inhibiting CYP3A4 activity. On the basis of this data, patients and clinicians can take precautions to avoid potential drug-interaction risks in COVID-19 treatment.

Effect of total glucosides of paeony and Tripterygium wilfordii polyglycosides on erythrocyte methotrexate polyglutamates in rats, analysed using ultra-high-performance liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The aim of the study was to explore the effect of total glucosides of paeony (TGP) and Tripterygium wilfordii polyglycosides (TWP) on erythrocyte methotrexate polyglutamates (MTXPGs), the metabolites of methotrexate (MTX). METHODS: An ultra-high-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method was developed to determine MTXPGs. The effects of MTXPGs were analysed using 24 male Sprague-Dawley rats that were randomly divided into the MTX alone, MTX-TGP combined, and MTX-TWP combined groups. Rats were administered MTX at a dose of 0.9 mg/kg once a week, TGP at 0.054 g/kg and TWP at 1.8 mg/kg three times a day. Venous blood (1.0 ml) was collected at weeks 2, 4, 6, 9, 12 and 15 and then analysed using the developed UPLC-MS/MS method. KEY FINDINGS: Specificity, linear range, inter-and intra-day precision, recovery, matrix effect and stability of MTXPGs met the standard regulations. This method was successfully used for the detection of MTXPGs. After administration of MTX alone, erythrocyte MTXPGs increased and accumulated in a time- and dose-dependent manner. Compared to MTX alone, the combination with TGP significantly decreased the content of total MTXPGs and short-chain MTXPGs (Methotrexate [MTX/MTXPG1] and 4-amino-10-methylpteroyldiglutamic acid [MTXPG2], P < 0.05), but had no significant effect on long-chain MTXPGs (4-amino-10-methylpteroyltriglutamic acid [MTXPG3], P > 0.05) and very long-chain MTXPGs (4-amino-10-methylpteroyltetraglutamic acid [MTXPG4] and 4-amino-10-methylpteroylpentaglutamic acid [MTXPG5], P > 0.05) at week 15. The combination of MTX with TWP had no significant effect on the content of total MTXPGs, short-chain MTXPGs and long-chain MTXPGs (P > 0.05), but it significantly decreased the content of very long-chain MTXPGs (P < 0.05) at week 15. CONCLUSIONS: The UPLC-MS/MS method was successfully used to determine MTXPGs in rat erythrocytes. TGP and TWP in combination with MTX affected the production of MTXPGs of different chain lengths in erythrocytes.

Protective effects of Paeoniflorin against AOPP-induced oxidative injury in HUVECs by blocking the ROS-HIF-1α/VEGF pathway.

BACKGROUND: Paeoniflorin, a monoterpene glycoside, exerts protective vascular effects, showing good antioxidant properties. However, whether Paeoniflorin has protective effect against the oxidative damage induced by advanced oxidation protein products (AOPPs) in Human umbilical vein endothelial cells (HUVECs) is unknown, as is the underlying mechanism. PURPOSE: The present study was designed to investigate the effect of Paeoniflorin on oxidative damage of HUVECs and elucidate its underlying molecular mechanisms. METHODS: The fluorescence intensity of 2', 7'-dichlorofluorescein-diacetate (DCFH-DA) staining was detected for intracellular reactive oxygen species (ROS) production. The increases mitochondrial membrane potential (MMP) was measured via flow cytometry and confocal microscopy using MitoTracker® Deep Red/ MitoTracker® Green staining. The intracellular adenosine triphosphate (ATP) was measured by ATP Determination Kit according to the manufacturer's protocol. Nox2, Nox4, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) p65 expressions were detected by western blot. RESULTS: Our results showed that Paeoniflorin increases MMP and ATP levels of HUVECs induced by AOPPs, and attenuates NF-κB p65 expression on HUVECs might mainly result from its antioxidant capability by suppressing ROS production. Moreover, we also found that Paeoniflorin can suppress HIF-1α and VEGF protein expression through a decrease of ROS production via down-regulation of Nox2/Nox4 expression in HUVECs. AOPP-induced RAGE mRNA up-regulation was blocked by Paeoniflorin treatment in HUVECs. CONCLUSION: Our results provided the first experimental that Paeoniflorin protects against AOPP-induced oxidative damage in HUVECs, mainly through a mechanism involving a decrease in ROS production by the inhibition of Nox2/Nox4 and RAGE expression; restored ATP depletion and mitochondria dysfunction via ROS suppression; and down-regulated HIF-1α/VEGF, possibly via the ROS-NF-κB axis.

Sub-100nm, long tumor retention SN-38-loaded photonic micelles for tri-modal cancer therapy.

The tumor penetration and accumulation of nanoparticle-based drug delivery systems are highly dependent on the particle size. Nanomedicines in the sub-100nm range have been suggested by previous studies to have superior antitumor efficacy on various solid tumors. SN-38 is a very important and highly potent drug for several cancers including colon cancer. However, due to the ultra-flat aromatic structure of SN-38, it is typically very difficult to produce sub-100nm, SN-38-encapsulated nanoparticles without modification of the chemical structure. Here, we report on the successful production of 20-30nm, SN-38-encapsulated photonic micelles for effectively trimodal cancer therapy. Taking advantages of the supramolecular "π-π" stacking and hydrophobicity interaction between SN-38, and a unique class of photonic nanoporphyrin micelles (NPM), the extremely hydrophobic SN-38 was successfully encapsulated into NPM with significantly increased water solubility (up to 500 times). At equivalent dose of drug, photosensitizer and light irradiation, combination therapy with SN-38-encapsulated nanoporphyrin micelles (SN-NPM) enhanced the in vitro antitumor activity by 78 and 350 times over single treatment with SN-38 and phototherapy alone, respectively. Due to the relatively small size, SN-NPM possessed superior long tumor retention time (>5days) and much higher accumulation in tumors than in normal organs, as shown by near-infrared fluorescence (NIRF) imaging. Furthermore, the trimodal therapy (photothermal-, photodynamic- and chemo-therapy) with SN-NPM demonstrated dramatically enhanced in vivo antitumor efficacy over single treatment on nude mice bearing HT-29 colon cancer xenograft. Therefore, these sub-100nm, SN-38-encapsulated photonic micelles show great promise for multimodal cancer therapy.

Huang Gan Formula Eliminates the Oxidative Stress Effects of Advanced Oxidation Protein Products on the Divergent Regulation of the Expression of AGEs Receptors via the JAK2/STAT3 Pathway.

Chronic kidney disease (CKD) has a high prevalence and low cure rate and represents a significant health issue. Oxidative stress is common in CKD due to metabolic disorders, inflammation, and impaired renal function changing normal proteins into advanced oxidation protein products (AOPPs). Huang Gan formula (HGF) is a new type of traditional Chinese herbal medicine. Although we previously investigated the protective effects of HGF against oxidative stress, the mechanism of HGF in CKD is still not fully understood. In this study, we used western blotting, quantitative polymerase chain reaction, and biochemical assays to show that HGF significantly decreased AOPP-induced oxidative stress damage. Moreover, the protective effects of HGF might be associated with upregulation of the advanced glycation end product receptor 1 (AGE-R1) and downregulation of the receptor for advance glycation end products (RAGE). Treatment with HGF and the Janus kinase 2 (JAK2) inhibitor, AG4-90, significantly attenuated AOPP-induced JAK2/STAT3 protein levels. These findings indicate that HGF inhibits AOPP-mediated biological responses by inactivating the JAK2/STAT3 pathway. In conclusion, HGF eliminated AOPP-induced effects in human mesangial cells (HMCs) by interrupting JAK2/STAT3 signaling, which altered RAGE/AGE-R1 expression and reduced oxidative stress in CKD.

Protective effect of Huang Gan formula in 5/6 nephrectomized rats by depressing the Wnt/ß-catenin signaling pathway.

Huang Gan formula (HGF) is a new traditional Chinese herbal medicine created according to the basic theory of traditional Chinese medicine. The aim of this study is to evaluate the effects of HGF on chronic kidney disease and determine the mechanisms of action. The extract of HGF was prepared, and qualitative and quantitative determination of phytochemical was performed with quadrupole time-of-flight mass spectrometer and high-performance liquid chromatography. Sprague-Dawley rats (n=72) were submitted to 5/6 nephrectomy (Nx), and then respectively treated with uremic clearance granule, losartan, HGF low dose, HGF middle dose, and HGF high dose once per day for 12 weeks. The sham group of operated rats (n=22) was treated with normal saline or HGF middle dose as a background control group. Blood and urine biochemical parameters, renal tissue morphology, and mRNA and proteins of Wnt/ß-catenin signaling pathways were investigated. The results showed that the quality of the extraction process could be controlled, and a total of eight major compounds were identified and quantified. HGF could decrease the level of serum creatinine, blood urea nitrogen, and urine protein and increase the renal index and creatinine clearance rate in a dose-dependent manner. HGF also remarkably reduced the glomerulosclerosis and tubulointerstitial fibrosis by blocking the Wnt/ß-catenin signaling pathway through inhibiting the Wnt1, ß-catenin, transcription factor 4, and fibronectin 1 expressions, simultaneously measured through mRNA and protein levels in the remnant kidney. These results suggest that extraction of HGF could improve remnant renal function and possibly ameliorate glomerulosclerosis and tubulointerstitial fibrosis by depressing the Wnt/ß-catenin signaling pathway.

[Compound Huang Gan delays chronic renal failure after 5/6 nephrectomy in rats].

OBJECTIVE: To observe the effect of compound Huang Gan in delaying chronic renal failure in rats after 5/6 nephrectomy and explore the possible mechanisms. METHODS: High-performance liquid chromatography was used to was used identify the components of compound Huang Gan extract. Rat models of 5/6 nephrectomy received a 12-week treatment with intragastric administration of Niaoduqing, Cozaar, or compound Huang Gan at low, moderate or high doses (n=10). After the treatments, the rats were sacrificed for detecting Scr, BUN, Ucr and 24h UPr , pathological examination of the renal tissues, and determination of FN, MCP-1, and ICAM-1 expression levels in the renal tissues using RT-PCR and immunohistochemistry. RESULTS: The major chemical components of compound Huang Gan extract included glycyrrhizin (0.61%), paeonol (1.2%), aloe emodin (0.72%), rhein (0.85%), emodin (0.87%), chrysophanol (0.79%) and physcion (0.8%). Treatment with compound Huang Gan at low, moderate and high doses significantly reduced Scr, BUN, Ucr , Ccr and 24 h UPr levels (P(P<0.05), improved interstitial fibrosis and glomerulosclerosis, and reduced FN and ICAM-1 expressions (P(P<0.05) in rats following nephrectomy. CONCLUSIONS: Compound Huang Gan can improve the renal function and lessen glomerulosclerosis and renal interstitial fibrosis to delay the progression of chronic renal failure in rat models of 5/6 nephrectomy.

[Effection of Qing-Xuan tablets on behavior pattern and striatal TNF-alpha of Parkinson model mice].

OBJECTIVE: To study the effects of Qing-Xuan tablets (QXT) on behavior pattern and striatal TNF-alpha in mice model of Parkinson's disease (PD). METHODS: The PD models were established by intraperitoneal injection of MPTP (30 mg/kg). 30 C57BL/6J mice were randomly divided into six groups: control group, PD model group, QXT high dosage group, QXT middle dosage group, QXT low dosage group and trihexyphenidyl hydrochloride group. After 7 days of treatment, the behavior pattern of mice were observed, and striatum were seperated to detect the content of TNF-alpha by ELISA. RESULTS: QXT increased the behavior of mice in behavioral tests (open field, pole test, grid test) (P<0.05 or P<0.01) but depressed TNF-alpha activity in striatum (P<0.05). CONCLUSION: QXT can significantly enhance the behavioral activity of mice,and depress TNF-alpha content in striatum,which suggest QXT can effectively relieve the symptom of PD.

[Determination of arsenic content in Changtong oral liquid by inductively coupled plasma- mass spectrometry].

OBJECTIVE: To determine arsenic content in Changtong oral liquid, inductively coupled plasma-mass spectrometry (ICP-MS) was employed, which generated linear calibration curves in the range of 5-25 ng/ml for As (r=0.9998). The average recovery of As was 98.94% (n=5, RSD=2.58%).

Chinese medicine compound Changtong oral liquid on postoperative intestinal adhesions.

AIM: The aim of this study was to observe the effect of a Chinese medicine compound Changtong oral liquid (CT) on tissue plasminogen activity (t-PA), plasminogen activator inhibitor (PAI), TGF-beta1 and hydroxyproline (OHP). METHODS: Two sets of animal experiments were performed in the present study. Forty New Zealand rabbits and 48 Sprague-Dawley (SD) rats were assigned randomly to one of the five groups: sham adhesion, adhesion with saline, adhesion with low dosage of the CT, adhesion with middle dosage of the CT and adhesion with high dosage of the CT. t-PA and PAI activity in plasma, OHP and TGF-beta1 expression in adhesion were investigated. Analysis of variance was used to test differences among groups. RESULTS: CT treatment increased plasma t-PA activity in rabbits but decreased TGF-beta1 activity in rats. The data were expressed from low to high dose respectively as follows: t-PA, 46.1+/-8.6 microkat/L, 59.6+/-10.1 microkat/L, 64.0+/-11.5 microkat/L; TGF-beta1 28+/-7.23%, 31+/-3.05%, 30+/-4.04%. There were significant differences compared with saline-treated animals (t-PA 26.4+/-5.1 microkat/L, TGF-beta1 54+/-5.51%). OHP content in cecum of rabbits from middle and high but not low dose of CT lowered significantly as compared with saline-treated rabbits, 0.3641+/-0.1373, 0.3348+/-0.0321, 0.2757+/-0.0497 mg/g vs 0.4183+/-0.0883 mg/g of protein, P>0.05, P<0.05, P<0.05 respectively. The rabbit plasma PAI activity and OHP content in abdominal wall had no difference in all groups. CONCLUSION: CT treatment significantly enhanced t-PA activity in rabbits, but decreased TGF-beta1 content in rats, OHP content in cecum of rabbits, and failed to affect the activity of PAI and OHP content in abdominal wall in rabbits, compared with saline group. The result suggests that CT could effectively prevent adhesions without interfering wound healing.