[Blood-letting plus cupping relieves endotoxin-induced fever by improving hypothalamic mitochondrial metabolism in rabbits].

Objectives: To observe the effect of blood-letting plus cupping on endotoxin-induced fever in rabbits and to explore its central antipyretic mechanism based on mitochondrial energy metabolism.

Methods: Seventy-two male Japanese white rabbits were randomly divided into six groups:blank control, model, medication, blood-letting, cupping, and blood-letting cupping groups, with 12 rabbits in each group. The fever model was established by injection of endotoxin (200 ng/kg) into the ear vein. One hour after modeling, the rabbits in the medication group received gavage of indomethacin, while the other groups were given the same volume of physiological saline via gavage. The rabbits in the blood-letting group received blood-letting at the "Dazhui" (GV14) acupoint, those of the cupping group received cupping at the GV14 area with -0.02 MPa pressure for 15 min, and those of the blood-letting cupping group first underwent blood-letting at GV14 followed by cupping with the same parameters as those of the simple blood-letting and simple cupping groups. Temperature changes in each group were recorded at 1, 2, 3, 4, 5 and 6 h after modeling. A transmission electron microscopy was used to observe the ultrastructure of hypothalamic mitochondria, the ELISA was employed to measure the contents of serum IL-1β and IL-6. An Oxygraph-2k was used to assess the mitochondrial respiratory function, the ATP content was determined with an ATP assay kit, and the expression levels of uncoupling protein 2 (UCP2) and mitofusin 2 (Mfn2) in the hypothalamic tissue were measured using the WES (automatic Western blot quantitative analysis system).

Results: Compared with the blank control group, the model group showed a significant increase in the body temperature from 1 h on after modeling (P<0.01), contents of serum IL-1β and IL-6 (P<0.01), and the expression of hypothalamic UCP2 protein (P<0.01), and a significant decrease in the mitochondrial oxygen consumption (P<0.05) and ATP content (P<0.01), and the expression of Mfn2 protein in the hypothalamic tissue (P<0.01). Compared to the corresponding time points of the model group, the body temperature at 2, 3, 4, 5 and 6 h in both medication and blood-letting cupping groups, at 2, 3, 4 and 6 h in both cupping and blood-letting groups were significantly decreased (P<0.01, P<0.05). No significant differences were found between the medication and blood-letting cupping groups, and between the cupping and blood-letting groups in lowering the body temperature at the five time points. The effects of simple cupping at 2, 3, 5 and 6 h and simple blood-letting at 2, 3, 4, 5 and 6 h were respectively obviously weaker than those of the medication in lowering body temperature at the corresponding time points (P<0.01, P<0.05). The effects of simple cupping at 5 and 6 h and simple blood-letting at 2, 3, 5 and 6 h were obviously weaker in lowering body temperature than those of the blood-letting cupping at the corresponding time points (P<0.01, P<0.05). Compared with the model group, the serum IL-1β and IL-6 contents and UCP2 expression level in hypothalamic tissue were strikingly down-regulated in the 4 intervention groups (P<0.05, P<0.01), except IL-6 in the blood-letting group, while the mitochondrial oxygen consumption, serum ATP content and Mfn2 expression level in hypothalamic tissue were considerably up-regulated in the 4 intervention groups (P<0.05, P<0.01), except ATP content in the blood-letting group. No significant differences were found among the 4 intervention groups in the levels of serum IL-1β and IL-6, UCP2 and Mfn2 expressions in hypothalamic tissue, oxygen consumption and ATP content. Results of the hypothalamic mitochondrial ultrastructure showed severe damage, including blurred and broken cristae or even disappearance of the cristae in the model group, which was milder in the injury severity in the 4 intervention groups, including incomplete structure of the bilayer membrane, but without obvious fracture.

Conclusions: Blood-letting cupping intervention has a significant antipyretic effect in rabbits with endotoxin-induced fever, which may be related to its functions in improving mitochondrial respiratory function and regulating the expression of key proteins (UCP2 and Mfn2) involving ATP synthesis in the hypothalamus.