Arginine Biosynthesis Pathway Found to Play a Key Role in the Neuroprotective Effect of Liu-Wei-Luo-Bi (LWLB) Granules in Diabetic db/db Mice with Peripheral Neuropathy Using an Untargeted Metabolomics Strategy.

Aim: Liu-Wei-Luo-Bi (LWLB) granules was a Chinese compound prescription for treating diabetic peripheral neuropathy (DPN). The aim of this study was to investigate the effect of LWLB granules on diabetic mice with peripheral neuropathy and to elucidate the potential mechanism based on an untargeted metabolomics approach. Methods: One hundred forty db/db mice were randomly divided into seven groups: the Control group, DPN group, Mudan (MD) granules group, Epalrestat (Epa) group, and the LWLB low, medium, or high dose (LW-l, LW-m, or LW-h) group. After 12 weeks of treatment, body weight, blood glucose, mechanical pain threshold, motor conduction velocity (MCV), sensory conduction velocity (SCV), and Pathological Organization of the Sciatic and Caudal Nerves in mice were measured. Serum samples were collected for untargeted metabolomics analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and multivariate statistics. Disease-related pathways were screened out with function enrichment analyses of candidate biomarkers. Results: LWLB granules can improve the peripheral neuropathy of type 2 diabetic mice with peripheral nerve conduction disorders, mainly through significantly improving the nerve conduction velocity (P < 0.05) and lowering the mechanical pain threshold (P < 0.05). A total of 43 metabolites were identified as potential biomarkers related to the therapeutic effect of LWLB granules. Fifty, 4, and 26; 23, 4, and 22; and 24, 1, and 16 biomarkers were discovered in the LW-l, LW-m, and LW-h groups at the 4th, 6th, and 12th weeks, respectively. Five, three, seven, five, and four metabolic pathways were found in MD, Epa, LW-l, LW-m, and LW-h groups, respectively. The arginine biosynthesis pathway is the overlapping pathway in LW-l, LW-m, and LW-h groups. Conclusion: LWLB granules have an obvious neuroprotective effect on diabetic peripheral neuropathy, and the metabolism mechanism of LWLB is mainly related to the arginine biosynthesis pathway on diabetic db/db mice with peripheral neuropathy.

Ginsenoside Rb1 induces autophagic lipid degradation via miR-128 targeting TFEB.

In recent years, the effect of lipid metabolism on health has attracted more and more attention. Ginseng is a traditional Chinese herbal medicine in China and is widely used as food in Asia. Ginsenoside Rb1 (Gs-Rb1) is the most abundant ingredient in ginsenoside, which has a variety of biological activities. In this study, we found that Gs-Rb1 can reduce lipid accumulation in mice and HepG2 cells induced by a high-fat diet (HFD) and palmitic acid (PA). At the same time, we also found that Gs-Rb1 could stimulate the autophagic flux of HFD-fed mice and PA-treated HepG2 cells, and it is further verified by adding the autophagy activator rapamycin (Rapa) and autophagy inhibitor chloroquine (CQ). Furthermore, we found that Gs-Rb1 promoted the nucleus translocation of the transcription factor EB (TFEB) and the target role of miR-128, thus stimulating autophagic flux. Therefore, our results showed that Gs-Rb1 enhanced the transcription of TFEB and its downstream lysosome-related genes by inhibiting miR-128, improved the degradation ability of lysosomes to autophagosomes, and then promoted autophagic lipid degradation.

Alliin alleviates LPS-induced pyroptosis via promoting mitophagy in THP-1 macrophages and mice.

Pyroptosis is a new type of programmed cell death associated with inflammation. Excessive pyroptosis can cause body damage. Alliin is an organosulfur compound extracted from garlic, bearing anti-oxidation and anti-inflammatory properties. In this study, we revealed that alliin alleviated LPS-induced macrophage pyroptosis by detecting PI staining, IL-1ß and IL-18 release in vitro and in vivo. In the study of mechanism, we found that alliin might reduce the activation of NLRP3 inflammosome by decreasing intracellular ROS generation. Subsequently, we detected the effect of alliin on mitophagy which degraded damaged mitochondria. The results showed that alliin promoted PINK 1/Parkin-mediated mitophagy. After adding the mitophagy inhibitor CsA, the alleviating effect of alliin on mitochondrial damage and mitochondrial ROS were reversed and the relieving effect of alliin on LPS-induced pyroptosis was inhibited. These results suggested that alliin might reduce intracellular ROS production by promoting mitophagy, thus alleviating LPS-induced macrophages pyroptosis. Our study provides a new perspective and theoretical basis for alliin to alleviate pyroptosis which could further induce body damage.

Ginsenoside Rb1 alleviates liver injury induced by 3-chloro-1,2-propanediol by stimulating autophagic flux.

In recent years, foodborne pollutants have become a hot issue in the field of food safety. 3-chloro-1,2-propanediol (3-MCPD) is a widely existing food contaminant. In our previous study, it was confirmed that 3-MCPD can block autophagic flux by inhibiting lysosomal function, thus causing liver injury. Ginseng is a traditional Chinese herbal medicine that contains a variety of bioactive ingredients, among which ginsenoside Rb1 (Gs-Rb1) is the most abundant. In this study, we aim to use Gs-Rb1 to improve 3-MCPD-induced autophagic flux blockage to alleviate liver injury. First, a nontoxic dose of Gs-Rb1 was identified by screening with the MTT method in which Gs-Rb1was added to HepG2 cells and co-treated with 3-MCPD. We found that Gs-Rb1 effectively enhanced the cell activity inhibited by 3-MCPD. Meanwhile, apoptosis data showed that Gs-Rb1 significantly alleviated the apoptosis of HepG2 cells induced by 3-MCPD. Subsequently, we found that Gs-Rb1 could alleviate autophagic flux blockage caused by 3-MCPD in a dose-dependent manner by detecting autophagy-related protein levels and transfecting mRFP-GFP-LC3 adenovirus. On this basis, we used Western blotting and qPCR to explore whether miR-128 was involved in the alleviation effect of Gs-Rb1 on autophagic flux blockade induced by 3-MCPD. The results showed that Gs-Rb1 inhibited the expression of miR-128 and promoted the nuclear expression and target gene transcription of TFEB. Finally, the findings were confirmed by using a hsa-miR-128 inhibitor and mimic. We found that hsa-miR-128 inhibitor alleviated the autophagic flux blockage and apoptosis caused by 3-MCPD and Gs-Rb1 also had a certain alleviation effect on the autophagic flux blockage and apoptosis caused by hsa-miR-128 mimic. This study elaborated the mechanism by which Gs-Rb1 alleviates hepatotoxicity induced by foodborne 3-MCPD by stimulating autophagic flux via miR-128-targeted TFEB, which provides a reliable theoretical basis and target for the use of natural substances to reduce the harm of food processing pollutants on the human body. PRACTICAL APPLICATION: We found that natural ginsenoside Rb1 can alleviate liver injury induced by 3-MCPD(a toxic substance found in foods such as refined vegetable oil, soy sauce, and baby milk powder), which is conducive to the development and utilization of ginseng and has practical significance for the prevention of foodborne liver injury.

[Core prescription excavation of launched Chinese patent medicines for treating different syndromes of angina pectoris].

To analyze the medication features and regularity of prescriptions of traditional Chinese medicines in treating patients with coronary heart disease angina pectoris based on the launched Chinese patent medicines. In the article,we collected all of the launched Chinese patent medicines for treating coronary heart disease and angina pectoris from the Chinese patent medicine value assessment information database,and set up a medical record normalized database,then carried out the classification of syndromes. The medication features and prescription rules for angina pectoris were analyzed by frequency statistics and association rules(IBM SPSS Modeler 14. 1 Premiums software,Apriori algorithm). Finally,a total of 170 prescriptions were selected,and 197 Chinese herbs were included,involving to totally 11 syndrome types,in which blood stasis syndrome,Qi stagnation and blood stasis syndrome,Qi deficiency and blood stasis syndrome,Qi-Yin deficiency and blood stasis syndrome were the main syndrome types. The frequency of single-herb medicines for the four main syndrome types,the combination of commonly used medicines,and the core prescriptions were summarized. After comparing the core prescriptions of the four syndrome types,we could analyze the medication features and prescription rules. In conclusion,the therapeutic principle is blood-activating and stasis-dissolving,and consideration was also given to promoting Qi,invigorating Qi and resuscitation and invigorating Qi-Yin. The main medicines include Danshen(Salvia Miltiorrhizae Radix et Rhizoma) and Chuan-xiong(Chuanxiong Rhizoma). According to different types of syndromes,Chinese herbal medicines are added,such as Jiangxiang(Dalbergiae Odoriferae Lignum), Chishao(Paeoniaeradix Rubra), Sanqi(Notoginseng Radix et Rhizoma), Honghua(Carthami Flos),Bingpian(Borneolum Syntheticum),Renshen(Ginseng Radix et Rhizoma). Frequency statistics and association rules are combined to explore the medication features and core prescriptions,which provide ideas for the treatment of angina pectoris and the development of new drugs.

Paeonol suppresses lipopolysaccharide-induced inflammatory cytokines in macrophage cells and protects mice from lethal endotoxin shock.

Paeonol (2'-hydroxy-4'-methoxyacetophenone) is the main phenolic compound of the radix of Paeonia suffruticosa which has been used as traditional Chinese medicine. In this study, we primarily investigated the anti-inflammatory effects and the underlying mechanisms of paeonol in RAW macrophage cells; and based on these effects, we assessed the protective effects of paeonol on lipopolysaccharide-induced endotoxemia in mice. The in vitro study showed that paeonol regulated the production of TNF-α, IL-1ß, IL-6, and IL-10 via inactivation of IκBα, ERK1/2, JNK, and p38 MAPK. In mouse model of lipopolysaccharide-induced endotoxemia, pro- and anti-inflammatory cytokines are significantly regulated, and thus the survival rates of lipolysaccharide-challenged mice are improved by paeonol (150, 200, or 250 mg/kg). Therefore, paeonol has a beneficial activity against lipopolysaccharide-induced inflammation in RAW 264.7 cell and mouse models.

Inhibitory effects of salidroside on nitric oxide and prostaglandin E2 production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

The aim of this study was to evaluate the effect of salidroside on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophages and related anti-inflammatory mechanism. PGE2 production was measured by enzyme-linked immunosorbent assay (ELISA); NO production was tested by Griess reagent. Inducible nitric oxidesynthase (iNOS) and COX-2 were determined by RT-PCR and Western blot analysis; IκB and P-IκB protein express were detected by Western blot analysis; cytosolic free Ca²âº ([Ca²âº](i)) was measured by a fluorescent microscope. The data showed salidroside inhibited LPS-induced NO and PGE2 production and reduced iNOS and COX-2 protein expression in RAW 264.7 macrophages. Consistent with these observations, salidroside inhibited LPS-induced cytosolic free Ca²âº concentration ([Ca²âº](i)) elevation. In addition, we further investigated signal transduction mechanisms and found that the activation of NF-κB was suppressed by salidroside in a dose-dependent manner. These results suggest that salidroside suppresses NO and PGE2 production by inhibiting iNOS and COX-2 protein expression, level of [Ca²âº](i), and activation of NF-κB signal transduction pathway.

Suppression of T-cell activation in vitro and in vivo by cordycepin from Cordyceps militaris.

BACKGROUND: In addition to achieving a balance between the positive (controlling rejection) and the negative (infection and malignancy) aspects of drug-induced immunodeficiency, new immunosuppressive combinations must address the issue of nonimmune drug toxicity that may be dose limiting. Cordycepin is a type of adenosine analog extracted from Cordyceps militaris. In the present study, we investigated its immunosuppressive effect on T cell both in vitro and in vivo. METHODS: We evaluated the effects of cordycepin on concanavalin A-induced production of immune mediators in mouse splenocyte by enzyme-linked immunosorbent assay and flow cytometry. Furthermore, using Western blotting, we studied signal transduction mechanisms to determine how cordycepin inhibited T-cell activation in purified mouse T lymphocytes. To confirm the immunosuppressive activity of cordycepin in vivo, we induced the T cell-mediated delayed-type hypersensitivity reaction in a 2,4-dinitro-1-fluorobenzene-induced mouse model. RESULTS: The in vitro results showed that cordycepin markedly suppressed concanavalin A-induced splenocyte proliferation, Th1 and Th2 cytokine production, and the ratio of CD4(+)-to-CD8(+) T cells. The administration of cordycepin in vivo markedly suppressed the T cell-mediated delayed-type hypersensitivity reaction. The data revealed that cordycepin effectively shocked the nuclear factor kappa B and nuclear factor of activated T cells 2 signal transduction pathways but had no effect on the mitogen activated protein kinase signal transduction pathway. CONCLUSIONS: These observations indicated that cordycepin has a potential role in downregulating the immune system and could be developed as a useful immunosuppressive agent for treating undesired immune responses.

Phytochemical composition and toxicity of an antioxidant extract from Pimpinella brachycarpa (Kom.) Nakai.

Pimpinella brachycarpa (Kom.) Nakai (PB) is one of the most favored edible greens grown in Asian regions. In our previously study, we found PB extract had antioxidant effects in vitro. In the present study, an EtOAc soluble extract (PBet) was isolated from PB. Then the antioxidant properties at cellular level, phytochemical composition and toxicity of PBet were examined. The results indicated that PBet (0.5-2mg/mL) could protect Bel-7404 cells from H(2)O(2) induced cell damage through scavenging of intracellular ROS. Moreover, myristic acid, 24ζ-methyl-5α-lanosta-25-one, ß-sitosterol, pregnenolone and ß-daucosterol were firstly isolated from PB. In addition, PBet (0.75g/kg BW, ig) had no acute toxicity and it (0.03-0.12g/kg BW, ig, 7 d) could not influence the rate of bone marrow polychromatic erythrocytes micronucleus and chromosome aberration in KM mice. All above findings suggested that PBet could be considered as a safe functional food with antioxidant activities.

In vitro and in vivo protection provided by pinocembrin against lipopolysaccharide-induced inflammatory responses.

Pinocembrin or 5, 7-dihydroxyflavanone is a flavanone, a type of flavonoid. In the present study, we first assessed the anti-inflammatory effects of pinocembrin in RAW macrophage cells; and based on these effects, we investigated the therapeutic effects of pinocembrin in murine model of endotoxin-induced acute lung injury. We found that in vitro pretreatment with pinocembrin remarkably regulated the production of TNF-α, IL-1ß, IL-6 and IL-10 via inhibiting the phosphorylation of IκBα, ERK1/2, JNK and p38MAPK. In the mouse model of LPS-induced acute lung injury, pinocembrin (20 or 50 mg/kg, i.p.) attenuated the development of pulmonary edema, histological severities, as well as neutrophil, lymphocyte and macrophage infiltration, which were increased by LPS administration. Additionally, TNF-α, IL-1ß and IL-6 concentrations decreased significantly while the concentration of IL-10 was significantly increased after pinocembrin pretreatment. Our results also showed that pinocembrin attenuated LPS-induced lung injury through suppression of IκBα, JNK and p38MAPK activation. These findings suggest that pinocembrin may represent a novel candidate for the modulation of inflammatory responses.

Traditional medicine alpinetin inhibits the inflammatory response in Raw 264.7 cells and mouse models.

Alpinetin, one of the main constituents of the seeds of Alpinia katsumadai Hayata, belonging to flavonoids, has been known to exhibit antibacterial, anti-inflammatory and other important therapeutic activities. The purpose of this study was to investigate the protection of alpinetin on inflammation in Lipopolysaccharide (LPS) stimulated Raw 264.7 cells and LPS induced vivo lung injury model. The effects of alpinetin on pro-inflammatory cytokines and signaling pathways were analyzed by enzyme-linked immunosorbent assay and Western blot. The results showed that alpinetin markedly inhibited the LPS- induced TNF-α, IL-6 and IL-1ß production both in vitro and vivo. Furthermore, alpinetin blocked the phosphorylation of IκBα protein, p65, p38 and extracellular signal-regulated kinase (ERK) in LPS stimulated RAW 264.7 cells. From in vivo study, it was also observed that alpinetin attenuated lung histopathologic changes in mouse models. These results suggest that alpinetin potentially decreases the inflammation in vitro and vivo, and might be a therapeutic agent against inflammatory diseases.

Salidroside attenuates LPS-induced pro-inflammatory cytokine responses and improves survival in murine endotoxemia.

Salidroside is a major component isolated from the Rhodiola rosea. In the present study, we investigated the anti-inflammatory effects of salidroside on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that salidroside reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) secretions. This inspired us to further study the effects of salidroside in vivo. Salidroside significantly attenuated TNF-α, IL-1ß and IL-6 productions in serum from mice challenged with LPS, and consistent with the results in vitro. In the murine model of endotoxemia, mice were treated with salidroside prior to or after LPS challenge. The results showed that salidroside significantly increased mouse survival. Further studies revealed that salidroside could downregulate LPS-induced nuclear transcription factor-қB (NF-қB) DNA-binding activation and ERK/MAPKs signal transduction pathways production in RAW 264.7 macrophages. These observations indicated that salidroside modulated early cytokine responses by blocking NF-қB and ERK/MAPKs activation, and thus, increased mouse survival. These effects of salidroside may be of potential usefulness in the treatment of inflammation-mediated endotoxemia.