Systematic reevaluation on the antipyretic, analgesic effects, and uncover the mechanisms of Chaiqin Qingning capsule, a traditional Chinese medicine, via establishing lipopolysaccharide - induced fever models, physical and chemical - stimuli pain models.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaiqin Qingning capsule (CQQNC), a traditional Chinese patent medicine, has been extensively used in alleviating fever and throat pain caused by respiratory tract infections, without obvious adverse reactions. However, there still lack of systematically basic reevaluation in its antipyretic, analgesic effects, and potential mechanisms unveiled.

AIM OF THE STUDY: This studies aimed to systematically reevaluate the antipyretic and analgesic effects of CQQNC, and reveal its underlying mechanisms using LPS-induced fever models in vitro and in vivo, and physical and chemical-stimulated pain animal models, respectively.

MATERIALS AND METHODS: Firstly, systematic reevaluation on the antipyretic effects of CQQNC was evaluated in vivo and in vitro using LPS-induced fever models in rats and RAW264.7 cells. Briefly, body temperature was monitored per hour after intraperitoneal injection of LPS (100 μg/kg) in rats after the treatment of CQQNC for 5 days. Fever-related mediators (IL-1β, TNF-α, cAMP and PGE2) in serum, cerebrospinal fluid (CSF), hypothalamus or lung tissues were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits based on the operating instructions. Simultaneously, LPS-stimulated RAW264.7 cell fever model was constructed, and CQQNC effects on febrile factors were also detected by ELISA kits. Subsequently, the analgesic effects of CQQNC were systematically assessed using classic physical and chemical stimuli pain models of hot plate and acetic acid writhing tests. Furthermore, to validate the analgesic effects and unveil its mechanism, the formalin-induced pain rats model was established. The paw licking/biting time and foot swelling were chosen as evaluation metrics. Meanwhile, pain-related mediators (SP, NO, and cGMP) in serum or spinal cord tissue were quantified by ELISA kits. Protein expression of c-fos in spinal cord tissue was detected by immunohistochemistry. Finally, Western blotting was employed to un-reveal the antipyretic and analgesic mechanisms of CQQNC.

RESULTS: Our results proved that CQQNC has good antipyretic and analgesic functions. Concretely, CQQNC significantly reduced the body temperature of fever rats, and markedly decreased the levels of febrile mediators (IL-1β, IL-6, TNF-α, cAMP and PGE2) in both serum, cerebrospinal fluid, cell supernatant, and lung tissue in a dose-dependent manner. Its antipyretic mechanism involved the inhibition of TLR4/MyD88/PI3K pathway, as evidenced by reducing protein expression of TLR4, MyD88, and phosphorylated PI3K in LPS-induced fever models in vitro and in vivo. Results on the analgesic reevaluation of CQQNC showed that CQQNC notably increased the pain thresholds in hot plate and acetic acid writhing tests, and reduced paw licking/biting time and swelling in formalin-induced rats. Meanwhile, ELISA tests demonstrated that CQQNC significantly diminished the levels of pain mediators including SP, NO, and cGMP in serum and spinal cord tissue. Protein expression results analysis identified that CQQNC could significantly downregulate the protein expressions of c-fos, NMDAR2B, CaMKIIa, NO, and PKG in spinal cord tissue of formalin-induced rats, indicating the analgesic mechanism of CQQNC was via the modulation of the NMDAR2B/CaMKIIa/NO/PKG pathway.

CONCLUSION: CQQNC exhibits significant antipyretic, analgesic, and anti-inflammatory properties. The antipyretic effects and mechanism of CQQNC involve the regulation of TLR4/MyD88/PI3K pathway to inhibit the productions of febrile mediators including IL-1β, IL-6, TNF-α, cAMP and PGE2. CQQNC showed good analgesic effects in both physical and chemical stimuli. Its analgesic mechanism may be associated with the regulation of NMDAR2B/CaMKIIa/NO/PKG pathway. Collectively, this studies provide novel insights into CQQNC's antipyretic and analgesic mechanism of actions, and support its clinical application for treating fever and pain symptoms caused by respiratory tract infections.