Figure 1 Experimental flowchart for the construction of a depression-pain comorbidity model in pregnant mice
Note： CUS, chronic unpredictable stress; CFA, complete Freund's adjuvant; SPT, sucrose preference test; FST, forced swimming test; TST, tail suspension test; OFT, open field test; PTT, pain threshold test; T0, before CUS intervention; T4, after CUS intervention; T8, after pregnancy.

Figure 2 Depressive behavior evaluation and pain threshold detection in the CUS model of mice
Note： A, Results of the sucrose preference test at the end of intervention periods before the start of the experiment (T0), one week after the experiment (T1), two weeks after the experiment (T2), three weeks after the experiment (T3), and four weeks after the experiment (T4) for the control group (n=40) and the chronic unpredictable stress (CUS) group (n=40); B, Forced swimming immobility time at the end of intervention periods T0 and T4 for each group; C, Tail suspension immobility time at the end of intervention periods T0 and T4 for each group; D, Percentage of time spent exploring the center of an open field for each group at the end of intervention periods T0 and T4; E, Right hind foot stabbing pain thresholds for each group at the end of intervention periods T0 and T4; F, Time to tolerate thermal radiation pain in the right hind foot for each group at the end of intervention T0 and T4. ^nsdifferences were not statistically significant, ^*P＜0.05, ^**P＜0.01, ^***P＜0.001, n=40.

Figure 3 Depression affects the ability to perceive pain in pregnant mice
Note： Control-blank group, i.e., without any treatment; Control-A group, i.e., without chronic unpredictable stress (CUS) but injected with formalin; CUS-A group, i.e., stimulated by CUS and injected with formalin; Control-C group, i.e., without CUS but injected with complete Freund's adjuvant (CFA); CUS-C group, i.e., stimulated by CUS and injected with CFA; Control-A+C group, i.e., without CUS but injected with formalin and CFA; CUS-blank group, i.e., stimulated by CUS but not injected with formalin and CFA; and CUS-A+C group, i.e., stimulated by CUS and injected with formalin and CFA. A, Pain threshold of the right hind foot in the control-blank, control-A, and CUS-A groups after delivery (T8); B, Pain threshold of the right hind foot at the end of gestation week 2 (T6) in the control-blank, control-C, and CUS-C groups; C, Pain threshold of the right hind foot at the end of gestation week 2 (T6) and after delivery (T8) in the control-C and CUS-C groups; D, Pain threshold of the right hind foot in control-blank, control-A+C, CUS-blank, and CUS-A+C mice before the CUS intervention (T0), after the CUS intervention (T4), at the end of the 2nd week of gestation (T6), and after delivery (T8); E, Pain threshold of the right hind foot after delivery (T8) of mice in the control-blank, control-A+C, and CUS-A+C groups; F, Plantar thermal radiation tolerance time of the right hind foot after delivery (T8) in control-blank, control-A+C, and CUS-A+C groups. ^nsdifferences were not statistically significant, ^**P＜0.01, ^***P＜0.001, n=10.

Figure 4 Pain during pregnancy impacts depressive behaviors of mice
Note： Control-A+C group, i.e., without chronic unpredictable stress (CUS) but injected with formalin and complete Freund's adjuvant (CFA); CUS-blank group, i.e., stimulated with CUS but not injected with formalin and CFA; CUS-A group, i.e., stimulated with CUS and injected with formalin; CUS-C group, i.e., stimulated with CUS and injected with CFA; CUS-A+C group, i.e., stimulated with CUS and injected with formalin and CFA. A-D, Preference for sugar water, swimming immobility time, tail suspension immobility time, and center exploration time in an open field as a percentage of time in each group after delivery (T8). ^**P＜0.01, ^***P＜0.001, n=10.

Figure 5 Representative open field trajectory maps and heat maps for each group
Note： Control-blank group, i.e., without any treatment; control-A+C group, i.e., without chronic unpredictable stress (CUS) but injected with formalin and complete Freund's adjuvant (CFA); CUS-blank group, i.e., stimulated with CUS but not injected with formalin and CFA; and CUS-A+C group, i.e., stimulated with CUS and injected with formalin and CFA. A-D, Trajectory maps and heat maps of open field test in Control-blank, Control-A+C, CUS-blank, and CUS-A+C mice after delivery (T8); E-F, Ratio of central exploration distance to total distance after delivery (T8) in each group of mice. ^**P＜0.01, ^***P＜0.001, n=10.

Figure 6 Higher hypothalamic-pituitary-adrenal axis （HPA axis） hormone levels and hippocampal inflammatory factor levels in mice with depression-pain comorbidity during pregnancy
Note： Control-blank group, i.e., without any treatment; Control-A+C group, i.e., without chronic unpredictable stress (CUS) but injected with formalin and complete Freund's adjuvant (CFA); CUS-blank group, i.e., stimulated with CUS but not injected with formalin and CFA; and CUS-A+C group, i.e., stimulated with CUS and injected with formalin and CFA. A-D, Serum cortisol and ACTH levels, as well as hippocampal IL-6 and TNF-α levels, in mice from each group. ^**P＜0.01, ^***P＜0.001, n=10.