[Mechanism of Gardeniae Fructus in ameliorating rheumatoid arthritis based on metabolomics and intestinal microbiota].

Rheumatoid arthritis(RA), a chronic autoimmune disease, is featured by persistent joint inflammation. The development of RA is associated with the disturbance of endogenous metabolites and intestinal microbiota. Gardeniae Fructus(GF), one of the commonly used medicinal food in China, is usually prescribed for the prevention and treatment of jaundice, inflammation, ache, fever, and skin ulcers. GF exerts an effect on ameliorating RA, the mechanism of which remains to be studied. In this study, ultra-perfor-mance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)-based serum non-target metabolomics and 16S rDNA high-throughput sequencing were employed to elucidate the mechanism of GF in ameliorating RA induced by complete Freund's adjuvant in rats. The results showed that GF alleviated the pathological conditions in adjuvant arthritis(AA) rats. The low-and high-dose GF lo-wered the serum levels of interleukin(IL)-6, tumor necrosis factor-α(TNF-α), IL-1ß, and prostaglandin E2 in the rats(P<0.05, P<0.01). Pathways involved in metabolomics were mainly α-linolenic acid metabolism and glycerophospholipid metabolism. The results of 16S rDNA sequencing showed that the Streptococcus, Facklamia, Klebsiella, Enterococcus, and Kosakonia were the critical gut microorganisms for GF to treat AA in rats. Spearman correlation analysis showed that the three differential metabolites PE-NMe[18:1(9Z)/20:0], PC[20:1(11Z)/18:3(6Z,9Z,12Z)], and PC[20:0/18:4(6Z,9Z,12Z,15Z)] were correlated with the differential bacteria. In conclusion, GF may ameliorate RA by regulating the composition of intestinal microbiota, α-linolenic acid metabolism, and glycerophospholipid metabolism. The findings provide new ideas and data for elucidating the mechanism of GF in relieving RA.

[Research progress on precious Tibetan medicine formula in prevention and treatment of central nervous system diseases].

Precious Tibetan medicine formula is a characteristic type of medicine commonly used in the clinical treatment of central nervous system diseases. Through the summary of modern research on the precious Tibetan medicine formulas such as Ratnasampil, Ershiwuwei Zhenzhu Pills, Ershiwewei Shanhu Pills, and Ruyi Zhenbao Pills, it is found that they have obvious advantages in the treatment of stroke, Alzheimer's disease, epilepsy, angioneurotic headache, and vascular dementia. Modern pharmacological studies have shown that the mechanisms of precious Tibetan medicine formulas in improving central nervous system diseases are that they promote microcirculation of brain tissue, regulate the permeability of the blood-brain barrier, alleviate inflammation, relieve oxidative stress damage, and inhibit nerve cell apoptosis. This review summarizes the clinical and pharmacological studies on precious Tibetan medicine formulas in prevention and treatment of central nervous system diseases, aiming to provide a reference for future in-depth research and innovative discovery of Tibetan medicine against central nervous diseases.

[Mechanism of Tibetan medicine Ershiwuwei Songshi Pills in regulating intestinal flora and improving non-alcoholic steatohepatitis].

This study aimed to investigate the effect of Tibetan medicine Ershiwuwei Songshi Pills(ESP) on the intestinal flora of non-alcoholic steatohepatitis(NASH) mice. Forty-eight male C57 BL/6 mice were randomly divided into the control group, model(methionine-choline-deficient, MCD) group, high-(0.8 g·kg~(-1)), medium-(0.4 g·kg~(-1)), and low-dose(0.2 g·kg~(-1)) ESP groups, and pioglitazone(PGZ, 10 mg·kg~(-1)) group, with eight mice in each group. Mice in the control group were fed with normal diet, while those in the remaining five groups with MCD diet for five weeks for inducing NASH. During modeling, they were gavaged with the corresponding drugs. The changes in body mass, daily water intake, and daily food intake were recorded. At the end of the experiment, the liver tissues were collected and stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by oil red O staining for observing fat accumulation in the liver. The levels of serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) and triglyceride(TG) in liver tissue were measured. The changes in intestinal flora of mice were determined using 16 S rRNA high-throughput sequencing technology. The results showed that compared with the model group, the high-, medium-and low-dose ESP groups and the PGZ group exhibited significantly lowered AST and ALT in serum and TG in liver tissues and alleviated hepatocellular steatosis and fat accumulation in the liver. As demonstrated by 16 S rRNA sequencing, the abundance index and diversity of intestinal flora decreased in the model group, while those increased in the ESP groups. Besides, the Firmicutes to Bacteroidetes ratio decreased at the phylum level. In the alteration of the composition of intestinal flora, ESP reduced the abundance of Erysipelotrichia and Faecalibaculum but increased the abundance of Desulfovibrionaceae, Rikenellaceae, Lachnospiraceae, and Ruminococcaceae. This study has revealed that ESP has a protective effect against NASH induced by MCD diet, which may be related to its regulation of the changes in intestinal flora, alteration of the composition of intestinal flora, and inhibition of the intestinal dysbiosis.

[Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways].

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1ß), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

[Ershiwuwei Shanhu Pills regulate Akt/mTOR/GSK-3ß signaling pathway to alleviate Alzheimer's disease mice].

The present study investigated the mechanism of the Tibetan patent medicine Ershiwuwei Shanhu Pills(ESP) in alleviating Alzheimer's disease in mice via Akt/mTOR/GSK-3ß signaling pathway. BALB/c mice were randomly assigned into a blank control group, a model group, low(200 mg·kg~(-1)), medium(400 mg·kg~(-1)) and high(800 mg·kg~(-1)) dose groups of ESP, and donepezil hydrochloride group. Except the blank control group, the other groups were given 20 mg·kg~(-1) aluminum chloride by gavage and 120 mg·kg~(-1) D-galactose by intraperitoneal injection for 56 days to establish Alzheimer's disease model. Morris water maze was used to detect the learning and memory ability of mice. The level of p-tau protein in mouse hippocampus and the levels of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in hippocampus and serum were detected. Hematoxylin-eosin staining and Nissl staining were performed for the pathological observation of whole brain in mice. TdT-mediated dUTP nick-end labeling(TUNEL) staining was employed for the observation of apoptosis in mouse cortex. Western blot was adopted to detect the protein levels of p-mTOR, p-Akt, and GSK-3ß in the hippocampus. Compared with the model group, the ESP groups showcased alleviated pathological damage of the whole brain, decreased TUNEL positive cells, reduced level of p-tau protein in hippocampus, and risen SOD, CAT, and T-AOC levels and declined MDA level in hippocampus and serum. Furthermore, the ESP groups had up-regulated protein levels of p-mTOR and p-Akt while down-regulated protein level of GSK-3ß in hippocampus. Therefore, ESP can alleviate the learning and memory decline and oxidative damage in mice with Alzheimer's disease induced by D-galactose combined with aluminum chloride, which may be related to Akt/mTOR/GSK-3ß signaling pathway.

[Mechanism of Tibetan medicine Ershiwuwei Shanhu Pills on scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway].

This study aims to investigate the mechanism of the Tibetan medicine Ershiwuwei Shanhu Pills(ESP) in improving scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway. ICR mice were randomized into blank group, model group, low-dose(200 mg·kg~(-1)), medium-dose(400 mg·kg~(-1)), and high-dose(800 mg·kg~(-1)) ESP groups, and donepezil hydrochloride group. The learning and memory impairment was induced in mice by intraperitoneal injection of scopola-mine. The learning and memory abilities of mice were detected by Morris water maze test, and the damage of hippocampal neurons and cortical neurons was detected based on Nissl staining. The expression of neuron specific nuclear protein(NeuN) in hippocampus and cortex of mice was determined by immunofluorescence assay, and the content of acetylcholine(Ach) and the activity of acetylcholines-terase(AchE) in hippocampus of mice by kits. Moreover, the content of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in serum of mice was detected. The content of Kelch-like ECH-associated protein 1(Keap1), nuclear factor erythroid 2-related factor 2(Nrf2), and heme oxygenase 1(HO-1) in hippocampus was determined by Western blot. The results showed that there were significant differences in the trajectory map of mice among different groups in the behavioral experiment. Moreover, the latency of ESP groups decreased significantly compared with that in the model group. The hippocampal neurons in the high-dose ESP group were significantly more than those in the model group and the cortical neurons in the high-dose and medium-dose ESP groups were significantly more than those in the model group. The expression of NeuN in the model group was significantly decreased compared with that in the blank group, and the expression in the ESP groups was significantly higher than that in the model group. The AchE activity and MDA level were significantly decreased, and Ach content and levels of SOD, CAT, and T-AOC in the ESP groups were significantly increased in the ESP groups compared with those in the model group. The expression of Keap1 in the model group was significantly increased compared with that in the blank group, and the Keap1 expression increased insignificantly in ESP groups compared with that in the model group. The expression of Nrf2 and HO-1 was significantly lower in the model group than in the blank group, and the expression was significantly higher in the medium-dose ESP group than in the model group. In conclusion, ESP protected mice against the scopolamine-induced learning and memory impairment by regulating the Keap1/Nrf2/HO-1 signaling pathway.

Reverse tracing anti-thrombotic active ingredients from dried Rehmannia Radix based on multidimensional spectrum-effect relationship analysis of steaming and drying for nine cycles.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) and modern pharmacodynamics, dried Rehmannia Radix (DRR) possesses prominent anti-thrombotic activity that decreases after processing by nine steaming and drying cycles to develop processed Rehmannia Radix (PRR). Due to the complexity of the DRR components, the chemical mechanism leading to efficacy changes of DRR caused by processing is still unclear. AIM OF STUDY: This study aimed to trace the anti-thrombotic active compounds of DRR and different degrees of processed RR (PRR) and to evaluate the synergistic effects among different active components. MATERIALS AND METHODS: The anti-thrombotic active chemical fraction of DRR extracts was evaluated. Targeted fractions of the processed products of RR were prepared at different processing stages. The changes in monosaccharides, oligosaccharides and secondary metabolites during processing were characterized by multidimensional high-performance liquid chromatography (HPLC). The anti-thrombotic effects of targeted fractions of different RR samples were evaluated by analyzing the length of tail thrombus (LT) and serum biochemical indicators in carrageenan-induced tail-thrombus mice. The spectrum-effect relationships were investigated by partial least squares regression (PLSR) analysis and gray correlation analysis (GRA). Finally, the active compounds were screened by spectrum-effect relationship analysis and validated in vivo, and their synergistic effects were determined by Webb's fraction multiplication method. RESULTS: Six ingredients highly associated with anti-thrombotic activities were screened out by the spectrum-effect relationship analysis, of which oligosaccharides (stachyose, sucrose and raffinose) and iridoid glycosides (catalpol, leonuride and melitoside) possessed a synergistic effect on tumor necrosis factors (TNF-α), interleukin 1ß (IL-1ß) and plasminogen activator inhibitor 1 (PAI-1)/tissue-type plasminogen activator (t-PA) ratio in vivo with synergistic coefficient (SC) > 1. CONCLUSION: The main material basis of the anti-thrombotic activities of DRR is oligosaccharide components of stachyose, raffinose and sucrose, iridoid glycosides components of catalpol, leonuride and melittoside. The two kinds of components exert synergistic anti-thrombotic effects by inhibiting the expression of inflammatory factors and regulating the balance of the fibrinolysis system.

Synergistic nourishing 'Yin' effect of iridoid and phenylpropanoid glycosides from Radix Scrophulariae in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Scrophulariae (RS), is a renowned traditional Chinese medicine used as nourishing 'Yin'. The iridoid glycosides (IG) and phenylpropanoid glycosides (PG) are main chemical constituents in RS. However, there had been no pharmacological experiment studies of synergy between IG and PG. Due to the constituents interactions, exploring their synergy profile is of great important for explaining the essence of nourishing 'Yin' efficacy of RS. AIM OF STUDY: The present study was undertaken to evaluate synergistic nourishing 'Yin' effect of IG and PG from RS in vivo and in vitro through their immunoregulation and antioxidant activities. MATERIALS AND METHODS: In this study, IG and PG fractions in RS were isolated and identified by High Performance Liquid Chromatography coupled with tandem quadrupole time-of-flight Mass Spectrometry (HPLC-Q-TOF-MS). The synergistic nourishing 'Yin' effect of two fractions were investigated in vivo and in vitro with thyroxine-induced 'Yin' deficiency (YD) mice model and primary splenic lymphocyte, respectively. The exterior syndrome signs and serologic and cellular biomarkers changes were detected. Then, the synergistic coefficient (SC) of IG and PG on every pharmacodynamics index were calculated by Webb method. RESULTS: Compared with model and mono-therapy group (IG or PG group), IG combined with PG group significantly ameliorated YD by exerting immunoregulation and antioxidant effects. Based on the SC, IG and PG possessed a synergistic effect on heart rate, average speed, upright times, spleen index, LPO, SOD, IL-6, Na+-K+-ATP enzyme in vivo, and cAMP/cGMP, IFN-γ/IL-10, and MDA in vitro with SC > 1. CONCLUSIONS: The nourishing 'Yin' benefits were clearly produced when IG and PG were used in combination, which provided the scientific evidence of multiple-components and multiple-approach synergistic effect of Chinese traditional herbal medicine to control and management of diseases.

Comparisons of antithrombosis, hematopoietic effects and chemical profiles of dried and rice wine-processed Rehmanniae Radix extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), Rehmanniae Radix (RR, derived from the root of Rehmannia glutinosa (Gaertn.) DC.) is commonly used as natural medicine for thousands of years, two types including the dried and rice-wine processed RR were used for different clinical purposes respectively, which were the typical case that pharmaceutical effect changed by processing in TCM. AIM OF STUDY: The goal of this study was to investigate the differences in the antithrombosis and hematopoietic effects of extracts of dried and processed RR (DRR and PRR) in vivo, and to explore the chemical basis underlying changes of medicinal properties caused by processing. MATERIALS AND METHODS: The aqueous extracts of DRR and PRR were prepared. Protective effect of varying doses of different extracts were investigated in type-I carrageenan induced mice tail thrombosis and cyclophosphamide induced myelosuppression model. The chemical composition of DRR and PRR extracts were determined by High Performance Liquid Chromatography coupled with tandem quadrupole time-of-flight Mass Spectrometry (HPLC/Q-TOF-MS). RESULTS: In antithrombosis activity tests, PRR possessed less ameliorated effects than DRR in the model mouse on body temperature, tail thrombus length and blood flow. Both DRR and PRR had no significant influence on prothrombin time (PT) and activated partial thromboplastin time (APTT), only high dose DRR could decrease the content of fibrinogen (FIB) in plasma. Histological examination of lung tissue suggested that thrombosis was significantly improved in DRR-H group. For myelosuppression model, only PRR could improve peripheral hemogram, both DRR and PRR had hematopoietic effects as demonstrated by their abilities to ameliorate the bone marrow nucleated cells (BMNC) and pathology of bone marrow tissue. The hematopoietic effects of PRR were significantly more potent than that of DRR at the concentration of 9 g/kg. By comparing the chemical composition, we found that iridoid glycosides were decreased and furfural derivatives increased in DRR after processing which may be the chemical mechanism contribute to the differences in efficacy. CONCLUSIONS: According to the results of this research, processing with rice wine for nine cycles significantly reduced antithrombotic effects and enhanced the hematopoietic effects of DRR as demonstrated in model mice. It can scientifically explain the different effect among two types of RR in clinical through the diverse method of processing and usage. Meanwhile, the predicted activity compounds from two types of RR can be potential candidates for the treatment of thrombosis and anemia.

Anti-HBV Activities of Three Compounds Extracted and Purified from Herpetospermum Seeds.

The goal of this research was to evaluate the anti-hepatitis B virus (HBV) activities of three compounds extracted and purified from Herpetospermum seeds (HS) on HepG2.2.15 cells. Herpetin (HPT), herpetone (HPO), and herpetfluorenone (HPF) were isolated from HS and identified using HR-ESI-MS and NMR. Different concentrations of the drugs were added to the HepG2.2.15 cells. Cell toxicity was observed with an MTT assay, cell culture supernatants were collected, and HBsAg and HBeAg were detected by ELISA. The content of HBV DNA was determined via quantitative polymerase chain reaction (PCR) with fluorescent probes. The 50% toxicity concentration (TC50) of HPF was 531.48 µg/mL, suggesting that this species is less toxic than HPT and HPO. HPT and HPF showed more potent antiviral activities than HPO. The 50% inhibition concentration (IC50) values of HPF on HBsAg and HBeAg were 176.99 and 134.53 µg/mL, respectively, and the corresponding therapeutic index (TI) values were 2.66 and 3.49, respectively. HPT and HPF were shown to significantly reduce the level of HBV DNA in the HepG2.2.15 culture medium compared to the negative control. This initial investigation of the anti-HBV constituents of HS yielded three compounds that revealed a synergistic effect of multiple components in the ethnopharmacological use of HS.

[Preparation of herpetin lyophilized liposome and evaluation on its safety and pharmacodynamics].

In this study, the herpetin (HPT) lyophilized liposome was prepared, and its saftey and pharmacodynamics were evaluated. HPT lyophilized liposome was prepared by thin-film ultrasonication method. The lyoprotectant was optimized using particle size and encapsulation efficiency as indexes. Then, the influencing factors of HPT lyophilized liposome were investigated. In addition, preliminary safety and therapy efficiency of HPT lyophilized liposome to liver injury induced by CCl4 in the mice. The optimal lyoprotectant was 5% sucrose plus 5% lactose and the dispersed HPT lyophilized liposomes were spherical with the mean diameter of (107.0 ± 1.2) nm and the mean encapsulation efficiency of (99.7 ± 0.50)%. The lyophilized powder was sensitive to temperature, humidity and illumination. None of hemolysis, hemagglutination and vein irritation was observed after intravenous injection of HPT lyophilized liposomes into rabbits. HPT lyophilized liposome showed obviously therapy efficiency to liver injury induced by CCl4 in the mice. The improvements of ALT, AST and ALP were better than that in HPT free drug. The obtained HPT lyophilized liposome met the standard of CP with fine particle size and encapsulation efficiency after dispersion. The HPT lyophilized liposome showed good safety and enhanced the treatment efficacy of HPT. The HPT lyophilized liposome should be stored in low temperature, sealed condition far away from light.