Study of the mechanism underlying the anti-inflammatory effect of Miao medicine comprising raw and processed Radix Wikstroemia indica using the "sweat soaking method".

ETHNOPHARMACOLOGICAL RELEVANCE: To explore the differences in the anti-inflammatory efficacy and mechanisms of the Miao medicine, both raw and after processing, using the "sweat soaking method" of Radix Wikstroemia indica (RWI). AIM OF THE STUDY: The purpose of this study was to explore the differences in the anti-inflammatory efficacy and mechanism of action before and after the processing of the Miao medicine (RWI) using the "sweat soaking method." MATERIALS AND METHODS: Network pharmacology technology was used to construct the "drug-component target-pathway-disease" network, and the main anti-inflammatory pathways of RWI were identified. Rat models of collagen-induced arthritis were established. The changes in body weight, swelling rate of the foot pad and ankle joint, arthritis index, thymus index, spleen index, pathological changes of the ankle joint, and the content of inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-10, TNF-α, and NO) were used as indices to evaluate the effect of RWI on rats with collagen-induced arthritis before and after its processing. Plasma and urine samples were collected from the rats, and the potential biomarkers of, and metabolic pathways underlying the anti-inflammatory effects of RWI before and after processing were identified using 1H-Nuclear magnetic resonance metabolomics combined with a multivariate statistical analysis. RESULTS: Eleven key anti-inflammatory targets of IL6, IL-1ß, TNF, ALB, AKT1, IFNG, INS, STAT3, EGFR, TP53, and SRC were identified by network pharmacology. The PI3K-Akt signaling pathway, steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, tryptophan metabolism, and other pathways were mainly involved in these effects. Pharmacodynamic studies found that both raw and processed RWI products downregulated inflammatory factors in rats with collagen-induced arthritis and alleviated the pathological changes. A total of 41 potential pathways for the anti-inflammatory effects of raw RWI products and 36 potential pathways for the anti-inflammatory effects of processed RWI products were identified by plasma and urine metabolomics. The common pathways of network pharmacology and metabolomics were steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. CONCLUSIONS: The anti-inflammatory effect of RWI was mainly related to the regulation of steroid hormone biosynthesis, arginine biosynthesis, arginine and proline metabolism, and tryptophan metabolism. Finally, the "sweat soaking method" enhanced the anti-inflammatory effect of RWI.

Efficacy and Safety of the Chinese Herbal Compound TJAOA101 in Treating Diminished Ovarian Reserve: A Protocol for Multicenter, Prospective, and Pre-Post Study.

OBJECTIVE: Diminished ovarian reserve (DOR) can lead to early menopause, poor fecundity, and an increased risk of disorders such as osteoporosis, cardiovascular disease, and cognitive impairment, seriously affecting the physical and mental health of women. There is still no safe and effective strategy or method to combat DOR. We have developed a novel Chinese herbal formula, Tongji anti-ovarian aging 101 (TJAOA101), to treat DOR. However, its safety and efficacy need to be further validated. METHODS: In this prospective and pre-post clinical trial, 100 eligible patients aged 18-45 diagnosed with DOR will be recruited. All participants receive TJAOA101 twice a day for 3 months. Then, comparisons before and after treatment will be analyzed, and the outcomes, including anti-mullerian hormone (AMH) and follicle-stimulating hormone (FSH) levels and the antral follicle count (AFC), the recovery rate of menopause, and the Kupperman index (KMI), will be assessed at baseline, every month during medication (the intervention period), and 1, 3 months after medication (the follow-up period). Assessments for adverse events will be performed during the intervention and follow-up periods. CONCLUSION: A multicenter, prospective study will be conducted to further confirm the safety and efficacy of TJAOA101 in treating DOR and to provide new therapeutic strategies for improving the quality of life in DOR patients.

1H-NMR-based metabolic profiling of rat urine to assess the toxicity-attenuating effect of the sweat-soaking method on Radix Wikstroemia indica.

Radix Wikstroemia indica (L.) C.A. Mey. (RWI) is a toxic medicinal species primarily present in the Miao area of China. The toxicity of RWI is effectively reduced whilst maintaining the therapeutic effect when processed using the 'sweat-soaking method', which is a common method of Traditional Chinese Medicine preparation. However, there is a lack of scientific and medical evidence to explain the potential mechanisms by which the toxicity of RWI is reduced after preparation using this method, and the endogenous systemic metabolic effect of RWI remains uncertain. The aim of the present study was to explore the endogetnous metabolic alterations caused by RWI and to examine the possibility of reducing the toxicity of RWI using the sweat-soaking method using proton nuclear magnetic resonance (NMR) metabolomic analysis in rats. Principal Component Analysis, Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal PLS-DA were used to assess individual proton NMR spectra. A total of 34 metabolic products were altered after delivering raw RWI, and 32 endogenous metabolites were induced by processed RWI. The metabolic pathways that lead to a significant impact on energy and carbohydrate, amino acid, organic acids and lipid metabolism following raw and processed RWI use were identified. The mitochondria of hepatic and renal tubules of rats were injured in the raw RWI group, whereas the processed product reduced or interfered with energy substrate, carbohydrate and amino acid metabolism, whilst reducing the levels of metabolic markers of hepatotoxicity and nephrotoxicity, without causing damage to the mitochondria. Our previous study showed that the median lethal dose (LD50) value of raw RWI was 4.05 g/kg in rats after oral administration; however, the LD50 value of the processed RWI could not be measured. The maximum tolerated dose and minimum lethal dose were 20 and 30 g/kg for the processed RWI, respectively, corresponding to 109 and 164 times the clinical daily dose (0.029 g/kg). Thus, the sweat-soaking method reduced the toxicity of RWI. Moreover, after processing, the toxic component YH-10 was converted into a YH-10 + OH compound, reducing the content of the toxic YH-10 by 48%, whilst also reducing the contents of the toxic components YH-12 and YH-15 by 44 and 65%, respectively. In conclusion, the present study showed that the sweat-soaking method reduced the toxicity of RWI, as evidenced by the reduction of the levels of metabolic markers and the activity of metabolic pathways, thus providing a basis for processing of RWI for clinical use.

Integrative Analysis of the Gut Microbiota and Metabolome in Obese Mice with Electroacupuncture by 16S rRNA Gene Sequencing and HPLC-MS-based Metabolic Profiling.

Acupuncture has been used to treat numerous diseases such as obesity in China for thousands of years. Several mechanisms of acupuncture on obesity have been surveyed based on metabolomics, but the effects of acupuncture on the alterations in the gut flora are still unclear. In this study, an integrated approach based on 16S rRNA gene sequencing combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS) metabolic profiling was conducted to investigate the effects of acupuncture on high-fat-diet-induced obesity through the regulation of the relative abundances of gut microbiota and their relationships with biomarker candidates. A total of 10 significantly altered bacterial genera and 11 metabolites were recognized, which recovered to normal levels after electroacupuncture treatment. The relative abundances of the bacterial families Muribaculaceae,Lachnospiraceae,Desulfovibrionaceae,Helicobacteraceae, Prevotellaceae,Ruminococcaceae,Rikenellaceae,Deferribacteraceae,Bacteroidaceae andTannerellaceaewere remarkedly changed among the three groups. Potential biomarkers, including LysoPC(0:0/16:0) ([Formula: see text]1),PC(0:0/18:0) ([Formula: see text]2),Cholic acid([Formula: see text]3),LysoPC(16:0) ([Formula: see text]4), 3[Formula: see text],6[Formula: see text],7[Formula: see text]-Trihydroxy-5[Formula: see text]-cholanoic acid([Formula: see text]5), 5beta-Cyprinolsulfate([Formula: see text]6),PC(18:0/0:0) ([Formula: see text]7), 1-Nitro-5-hydroxy-6-glutathionyl-5,6-dihydronaphthalene([Formula: see text]8),Glycocholic acid([Formula: see text]9),[Formula: see text]-Arginine([Formula: see text]10) andGulonic acid([Formula: see text]11), were involved in several metabolic pathways, such as the glycerophospholipid metabolism and primary bile acid biosynthesis. Interestingly, there was a strong correlation between the perturbed gut flora in Bilophila and Bifidobacterium and the altered intestinal metabolite of 3[Formula: see text],6[Formula: see text],7[Formula: see text]-Trihydroxy-5[Formula: see text]-cholanoic acid and Cholanoic acid and [Formula: see text]-Arginine. This finding suggested that the effects of electroacupuncture might change the proportions of Bilophila and Bifidobacterium by regulating the constituents of the functional metabolite of 3[Formula: see text],6[Formula: see text],7[Formula: see text]-Trihydroxy-5[Formula: see text]-cholanoic acid and Cholanoic acid and [Formula: see text]-Arginine. These results indicated that the effects of electroacupuncture focused on custom metabolic pathways as well as depend on the changes in the gut microbiota in obesity. These findings suggest that the 16S rRNA gene sequencing and HPLC-MS-based metabolomics approach can be applied to comprehensively assess the effects of traditional Chinese medicines.

A new method for detecting Na+ , K+ -ATPase activity by ICP-MS: Quantitative analysis on the inhibitory effect of rhein on Na+ , K+ -ATPase activity by ICP-MS in HCT116 cells.

Rhein is an active component from Chinese herbal medicine. It can cause diarrhea by inhibiting Na+ , K+ -ATPase activity on intestinal epithelial cells, thus decreasing the re-absorption of Na+ from intestinal tract to blood. However, when this Na+ , K+ -ATPase inhibition was quantitated by a colorimetric method that measures ATPase-catalyzed release of inorganic phosphorus, the data obtained were inconsistent and showed great variation. We developed a novel method using inductively coupled plasma mass spectrometry (ICP-MS) to quantitate the amount of intracellular Rb+ . This method largely mimics the 86 RbCl tracer flux assay, but it uses non-radioactive RbCl as a flux substrate. The results demonstrated that this method has better precision and accuracy than the conventional colorimetric method. More importantly, this method is free from radioactive substances, which is expected to make it safer and more convenient than the radioactive 86 RbCl tracer flux method. In conclusion, the ICP-MS method for Na+ , K+ -ATPase activity determination is novel and accurate. It can also provide a reference for studying the transport of other metal ions across membranes under biological conditions.

Analysis of the chemical composition changes of Gardeniae Fructus before and after processing based on ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Gardeniae Fructus, the dry fruit of Gardenia jasminoides Ellis, has been widely used for the treatment of different diseases. Although four types of processed Gardeniae Fructus products, characterized by differing effects, are available for clinical use, little is known regarding the respective processing mechanisms. In this study, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with multivariate statistical analysis was applied to characterize the chemical profiles of the differently processed Gardeniae Fructus products and to determine differences in their chemical compositions, thereby enabling us to identify those active compounds associated with the observed clinical effects. A total of 125 compounds were accordingly identified, among which, 56 were established as primary contributors to the significant differences (P < 0.01) between crude and processed Gardeniae Fructus, based on t-test analysis. Furthermore, the potential mechanisms underlying the chemical transformations that occurred during processing were discussed. The findings of this study may not only contribute to the more effective quality control of Gardeniae Fructus but also provide basic information for elucidating the mechanisms underlying the changes in chemical constituents in response to processing, and provide a basis for further investigations of Gardeniae Fructus processing mechanisms.