A total of 50 rats were randomly divided into five groups (n = 10), including the normal group, model group, and three treatment groups. The experimental protocol was designed according to the previous studies with some modifications,29–31 and the experimental schematic diagram is shown in Figure 1. Briefly, experimental rats in the model group and the three treatment groups were exposed to the CUS for 14 consecutive days. Meanwhile, different doses (50, 100, and 200 mg/kg/day, respectively) of VOZB were administered to rats in the three treatment groups by an intragastric gavage for 14 days, previously suspended in vehicle (1% CMC-Na). Rats in other groups were administered with the vehicle (1% CMC-Na, 10 mL/kg). Since there is no information on the dose of VOZB in the treatment of anxiety either in clinical or preclinical studies, the dose of VOZB in this study was determined by our preliminary experiment. The behavioral tests were conducted 2 h after the final drug administration. On the next day, all rats were anesthetized by 1% pentobarbital sodium (3 mL/kg). The inguinal skin was cut open to expose the femoral vein. Then blood samples were collected from the femoral vein and centrifuged for 10 min at 5000 rpm to prepare the serum samples. After blood sampling, the rats were killed by cervical dislocation and the brain samples were removed quickly from the skull. Some of the brain samples were immersed and fixed in a 4% paraformaldehyde (PFA) solution for immunohistochemical assay, and others were had the hypothalamus isolated for the next process. The serum samples and hypothalamus samples were stored at −80°C. In addition, the abdomen of rats was sterilized by iodophor after barbering on a super clean bench. The whole colon was removed and cut open lengthwise. The stools in colon were collected into sterile tubes and immediately frozen in liquid nitrogen for detection of gut microbiota.

Schematic diagram illustrating the major steps of the experiment. The experimental rats were exposed to the CUS for 14 consecutive days. Meanwhile, different doses (50, 100, and 200 mg/kg/day, respectively) of VOZB were administered for 14 days.

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