BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide. However, to date, there is no ideal therapy for this disease. AIM To study the effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice. METHODS Twenty-four male C57BL/6 mice were randomized into three groups of eight. The control group (CON) was allowed ad libitum access to a normal chow diet. The high fat diet group (FAT) and Si-Ni-San group (SNS) were allowed ad libitum access to a high fat diet. The SNS group was intragastrically administered Si-Ni-San freeze-dried powder (5.0 g/kg) once daily, and the CON and FAT groups were intragastrically administered distilled water. After 12 wk, body weight, liver index, visceral fat index, serum alanine aminotransferase (ALT), portal lipopoly-saccharide (LPS), liver tumor necrosis factor (TNF)-α and liver triglycerides were measured. Intestinal microbiota were analyzed using a 16S r DNA sequencing technique. RESULTS Compared with the FAT group, the SNS group exhibited decreased body weight, liver index, visceral fat index, serum ALT, portal LPS, liver TNF-α and liver triglycerides ( P < 0.05). Intestinal microbiota analysis showed that the SNS group had different bacterial composition and function compared with the FAT group. In particular, Oscillospira genus was a bacterial biomarker of SNS group samples. CONCLUSION The beneficial effects of Si-Ni-San freeze-dried powder on high fat diet-induced NAFLD in mice may be associated with its anti-inflammatory and changing intestinal microbiota effects.
Quality control of animal-derived traditional Chinese medicines has improved dramatically as proteomics research advanced in the past few decades. However, it remains challenging to identify quality attributes with routine proteomics approaches since protein with fibrinolytic activity is rarely reported in pheretima, a typical animal-derived traditional medicine. A novel strategy based on bioinformatics combined with parallel reaction monitoring (PRM) was developed here to rapidly discover the marker peptides associated with a fibrinolytic effect. Potential marker peptides were found by lumbrokinase sequences’ alignment and in silico digestion. The fibrinogen zymography was used to visually identify fibrinolytic proteins in pheretima. As a result, it was found that the fibrinolytic activity varied among different portions of pheretima. Fibrinolytic proteins were distributed regionally in the anterior and anterior-mid portion and there was no significant fibrinogenolytic activity observed in the mid-posterior and posterior portion. Finally, PRM experiments were deployed to validate and quantify selected marker peptides and a total of 11 peptides were identified as marker peptides, which could be potentially used in quality control of pheretima. This strategy provides a robust workflow to benefit the quality control of other animal-derived traditional medicines.
Pheretima vulgaris has been prescribed for the treatment of cardiovascular diseases in China for several hundred years in the form of dried powder in the clinic. However, the peptides with the potential antithrombotic activity of this source have never been reported. The total active proteins from Pheretima vulgaris were hydrolyzed by eight different commercial proteases and the alcalase hydrolysate showed the strongest thrombolytic activity. Four original thrombolytic peptides were isolated and characterized using bioactivity‐directed fractionation of the active hydrolysate. The amino acid sequences were identified as HEPLPEP (m/z 818.40076), EYPLPEP (m/z 844.39648), LGEPSVP (m/z 698.39648), and LLAPP (m/z 510.28043) by nanoLC‐ESI‐Orbitrap mass spectrometry with PEAKS software. HEPLPEP and EYPLPEP, containing the common ‐PLPEP residue, showed superior thrombolytic activity in plasmin assay and fibrinogen‐thrombin time assay. This research confirmed that Pheretima vulgaris was a potential source of active peptides with thrombolytic activities and provided novel candidates for the thrombolytic agents. Practical applications Thrombosis has become the leading cause of mortality as it was the common underlying pathology of cardiovascular diseases, such as ischemic heart disease, and stroke. The demand for thrombolytics has increased gradually as the incidence trends of thrombosis‐related diseases raise with the aging of the population. Four novel thrombolytic peptides were characterized from Pheretima vulgaris proteins hydrolysates, among which HEPLPEP and EYPLPEP could prevent the formation of thrombus and degrade existing thrombus in vitro. These peptides are promising to be meritorious templates for developing thrombolytic agents. The structure–function relationship of peptides resulting from the presence of specific residues in these sequences may contribute to extending the knowledge about their thrombolytic activity, which may be useful in designing novel thrombolytic agents. The present research based on a bioactivity‐directed isolation strategy could also be applied to other animal‐derived traditional Chinese medicines with proteins or peptides as their function basis.
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