Anti-inflammatory effects and mechanism of Plantago asiatica L. and Lonicera japonica Thunb. extracts based on canine and feline kidney cell models

Ethnopharmacological relevance: Stone symptoms are one of the most common health problems in pets. Inflammation in the kidneys causes the pet's urine to form a hard substance that blocks the urinary tract. Plantago asiatica L. and Lonicera japonica Thunb., as traditional Chinese diuretics, have remarkable effects on antiinflammatory and analgesia. However, their mechanism of action remains unclear. Purpose: The alleviating effect of Plantago asiatica and Lonicera japonica extracts upon lipopolysaccharide (LPS)induced inflammation in canine and feline kidney cells was investigated in this work. Materials and methods: Inflammatory factor concentrations and oxidative stress indicators were used to evaluate the inflammatory response. The mechanism by which two extracts reduced inflammation was explored using quantitative real-time polymerase chain reaction (RT-qPCR) and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) metabolome assay. Results: The results demonstrated that Plantago asiatica or Lonicera japonica extract at safe concentration (25-200 mu g/mL for canine kidney cell and 1-20 mu g/mL for feline kidney cell, respectively) could significantly reduce the release of nitric oxide (p <0.05) and oxidative damage (p < 0.05) after LPS stimulation, inhibit the production of inflammatory factors (p < 0.05), and improve cell migration ability (p < 0.05). The RT-qPCR results confirmed that Plantago asiatica and Lonicera japonica extracts significantly reduced the mRNA expressions of TLR4, MyD88, NF-kB, Caspase9 and Bax (p < 0.05), and enhanced the mRNA expression of Bcl-2 (p < 0.05). Non-targeted metabolomics results indicated that the cells treated with two extracts raised the contents of allopurinol, further inhibited uric acid and gout and lowered the contents of adenosine and adenine. Moreover, it was revealed that the Plantago asiatica and Lonicera japonica participated in purine metabolism, glycerophospholipid metabolism, protein digestion and absorption, nucleotide metabolism pathways to alleviate kidney cell inflammation. Conclusions: The interaction mechanism was revealed to reduce the content of inflammatory factors by inhibiting TLR4-MyD88-NF-kB signaling pathway, and participate in purine metabolism to reduce the inflammation of kidney cells. These findings could provide significant insight into alleviating nephritis in canine and feline, and strategies for preventing urinary tract and kidney stones using Plantago asiatica and Lonicera japonica extracts.