Five-month-old C57 BL/6J female mice (Slaccas Laboratory Animal Co. Ltd. Shanghai, China), were randomly divided into six groups of ten each. After one week adaptation period, the animals were given daily one of the following preparations subcutaneously for eight weeks: (I) vehicle control; (II) D-galactose at 100 mg/kg; (III) D-galactose at 100 mg/kg plus pine pollen at 500 mg/kg by intragastric injection; (IV) D-galactose at 100 mg/kg plus pine pollen at 1000 mg/kg by intragastric injection; (V) D-galactose at 100 mg/kg plus pine pollen at 1500 mg/kg by intragastric injection; (VI) D-galactose at 100 mg/kg plus AG at 100 mg/kg by intragastric injection. Mice were sacrificed at the end of treatment, and sera, organs and tissues were immediately collected for experiments or stored at −70°C for later experiments. Sera could be used for detection directly while tissue samples should be performed according to the following treatment.
Tissue Homogenization: tissue samples were weighed and homogenized in normal saline (NS) for studies, and homogenates of 5% were obtained. Tissue homogenates were sonicated two times at 30 sec, intervals. Homogenization and sonication were performed at 4°C. After sonication, homogenates for biochemical studies were centrifuged at 3 000 rpm for 10 min and at 12 000 rpm for 15 min, respectively. Aliquots of the supernatants were used for the studies. The assayed parameters were expressed per mg protein, and protein content of the aliquots was determined by a BCA protein assay kit (Pierce Chemical Co.). All experimental procedures used in this study had been approved by the ethics committee within our hospital and all animal experiments had been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The authors who performed experiments had given their informed consent prior to the study and had followed “principles of laboratory animal care” published by the National Institutes of Health (NIH publication no. 86-23, revised 1985).