SNI surgery induced anxiety- and depression-like behaviors after surgery
The anxiety- and depression-like behaviors were measured from POD14 onward, and the results showed that SNI surgery decreased center distance (group F1,14 = 25.7, P = 0.001; time F1,14 = 0.367, P = 0.329; interaction F1,14 = 0.001, P = 0.971; Fig 1A) and center/total distance (group F1,14 =23.3, P = 0.001; time F1,14 =0.492, P = 0.329; interaction F1,14 = 0.037, P = 0.851; Fig 1 C) compared to sham group in the open field test from POD14 to POD 21 after surgery. SNI surgery did not affect total running distance in the open field test after surgery (group F1,14 = 0.274, P = 0.609; time F1,14 =0.430, P = 0.523; interaction F1,14 = 0.059, P = 0.811; Fig 1B). In the sucrose preference test, SNI decreased sucrose intake percentage from POD14 to POD21(group F1,14 = 64.9, P = 0.001; time F1,14 = 0.076, P = 0.787; interaction F1,14 = 0.116, P = 0.739; Fig 1D). In addition, SNI decreased food intake (group F1,14 = 35.6, P = 0.001; time F3,14 = 1.86, P = 0.152; interaction F1,14 = 11.5, P = 0.001; Fig 1E) and weight gain (group F1,14 = 58.3, P = 0.001; time F1,14 = 76.8, P = 0.329; interaction F1,14 = 134, P = 0.001; and Fig 1F respectively) compared to sham after surgery from POD7 to POD21. The paw withdrawal threshold and response to acetone were recorded before and after SNI surgery. The results showed that SNI surgery decreased paw withdrawal threshold (group F1,14 = 335, P = 0.001; time F1,14 = 75.6, P = 0.001; interaction F1,14 = 61.7, P = 0.001; and Fig 1G) and increased response to acetone (group F1,14 = 162, P = 0.001; time F1,14 = 16.5, P = 0.001; interaction F1,14 = 18.8, P = 0.001; and Fig 1H) after surgery from POD7 to POD21.
ICV injection of α7 nAch receptor agonist PHA and microglia inhibitor MIN decreased anxiety- and depression-like behaviors after SNI surgery
The behavior results showed that SNI surgery decreased center distance and center/total distance from POD10 to POD18, and ICV PHA or MIN improved that decrement of center distance (group F4,45 = 29.7, P = 0.001; time F1,45 = 12.0, P = 0.001; interaction F4,45 = 2.95, P = 0.030; and Fig 2A) and center/total distance (group F4,45 = 23.7, P = 0.001; time F1,45 = 14.4, P = 0.001; interaction F4,45 = 4.38, P = 0.005; Fig 2C) on POD18. SNI decreased sucrose intake percentage from POD10 to POD18 and ICV PHA or MIN decreased that effect on POD18 (group F4,45 = 35.6, P = 0.001; time F1,45 = 11.7, P = 0.001; interaction F4,45 = 5.60, P = 0.001; Fig 2D). The total running distance was not different among four groups in the open field test (group F4,45 = 0.642, P = 0.635; time F1,45 = 0.027, P = 0.870; interaction F4,45 = 0.058, P = 0.994; Fig 2B). SNI surgery decreased food intake and weight gain from POD10 to POD18 compared to sham group, and ICV PHA or MIN decreased that effect on food intake (group F4,45 = 7.82, P = 0.001; time F2,45 = 9.94, P = 0.001; interaction F8,45 = 2.09, P = 0.045; Fig 2E) and weight gain (group F4,45 = 70.4, P = 0.001; time F2,45 = 12.9, P = 0.001; interaction F8,45 = 23.2, P = 0.001; Fig 2F) on POD18. Although SNI decreased paw withdrawal threshold and increased response to acetone, ICV PHA or MIN did not affect paw withdrawal threshold (group F4,45 = 407, P = 0.001; time F3,45 = 206, P = 0.001; interaction F12,45 = 25.8, P = 0.001; Fig 2G) and response to acetone (group F4,45 = 125, P = 0.001; time F3,45 = 102, P = 0.001; interaction F12,45 = 16.4, P = 0.001; Fig 2 H). The time course for surgery and experiment was shown in Fig 2I.
PHA decreased anxiety- and depression-like behaviors after SNI surgery by the activation of α7 nAch receptors and suppression of brain inflammation
The results showed that ICV co-administration of α7 nAch receptor antagonist MLA decreased anti- anxiety- and anti-depressive effect of PHA in center distance (F = 19.8, P = 0.001; Fig 3A) and center/total distance (F = 14, P = 0.001; Fig 3C) in the open field test, and the total distance running (F = 0.427, P = 0.789; Fig 3B) was not different between groups after SNI surgery on POD18. PHA increased sucrose intake percentage and food intake and weight gain after SNI, while co-administration MLA decreased that effect of PHA on sucrose intake percentage (F = 15.5, P = 0.001; Fig 3D), food intake (F = 8.63, P = 0.001; Fig 3E) and weight gain (F = 35.5, P = 0.001; Fig 3F). MLA did not affect the effect of PHA on paw withdrawal threshold (group F3,36 = 0.548, P = 0.653; time F2,36 = 680, P = 0.001; interaction F8,45 = 1.05, P = 0.401; Fig 3G) and response to acetone (group F3,36 = 1.22, P = 0.316; time F2,36 = 196, P = 0.001; interaction F8,45 = 0.625, P = 0.710; Fig 3 H).
In addition, ICV PHA increased IL-10 in the mPFC (F = 4.79, P = 0.003; Fig 4A), BA (F = 13.4, P = 0.001; Fig 4E) and VH (F = 11.9, P = 0.001; Fig 4I) compared to the Vehicle group, and MLA decreased those effect. In contrast, SNI increased IL-1β in the mPFC, BA and VH, compared to Naive group, and ICV PHA decreased IL-1β in the mPFC (F = 26.7, P = 0.001; Fig 4B), BA (F = 39.2, P = 0.001; Fig 4F) and VH (F = 27.4, P = 0.001; Fig 4J) compared to the Vehicle group, whereas the effect was compromised by MLA. Similarly, SNI increased TNF-α in the mPFC, BA and VH compared to Naive group, and ICV PHA decreased their content in the mPFC (F = 23.0, P = 0.001; Fig 4C), BA (F = 23.4, P = 0.001; Fig 4G) and VH (F = 22.1, P = 0.001; Fig 4K) compared to the Vehicle group, while MLA decreased the effects of PHA. Further, SNI increased IL-18 in the mPFC, BA and VH compared to Naive group, and ICV PHA decreased their content in the mPFC (F = 28.9, P = 0.001; Fig 4 D), BA (F = 15.7, P = 0.001; Fig 4 H) and VH (F = 21.0, P = 0.001; Fig 4 L) compared to the Vehicle group, whereas those effect was compromised by MLA.
PHA decreased anxiety- and depressive-like behaviors after CFA injection by the activation of α7 nAch receptors and suppression of brain inflammation
The time course for surgery and experiment was similar as Fig 2I, except for CFA injection replaced the SNI surgery. The results showed that CFA induced anxiety-like behaviors in the open field testand PHA improved those behaviors, whereas those effect of PHA on center distance (F = 16.9, P = 0.001; Fig 5A) and center/total distance (F = 14.3, P = 0.001; Fig 5C) was compromised by MLA. The total running distance moved were not different among those groups (F = 0.145, P = 0.964; Fig 5B). CFA decreased sucrose intake percentage, food intake and weight gain, and ICV PHA improved the behavior results, whereas, the effect of PHA on sucrose intake percentage (F = 21.6, P = 0.001; Fig 5D), food intake (F = 7.8, P = 0.001; Fig 5E) and weight gain (group F3,36 = 7.68, P = 0.001; time F2,36 = 14.5, P = 0.316; interaction F6,36 = 9.74, P = 0.001; Fig 5F) was compromised by MLA. CFA decreased mechanical anodynia and increased response to cold, whereas ICV PHA and MLA did not affect those response to mechanical stimulation (group F3,36 = 0.051, P = 0.985; time F2,36 = 86.4, P = 0.001; interaction F6,36 = 0.187, P = 0.980; Fig 5G) and acetone (group F3,36 = 0.423, P = 0.738; time F2,36 = 52.4, P = 0.001; interaction F6,36 = 0.531, P = 0.783; Fig 5H). the ELISA results showed that CFA did not affect IL-10 content in the mPFC BA and VH, and ICV PHA increased IL-10 content in the mPFC (F = 7.29, P = 0.001) and BA (F = 8.68, P = 0.001), and those effect was decreased by MLA (Fig 6A). CFA increased IL-1β content in the mPFC BA and VH, and ICV PHA decreased IL-1β increment in mPFC (F = 29.0, P = 0.001), BA (F = 7.29, P = 0.001), and VH (F = 24.7, P = 0.001), and the effect was compromised by MLA (Fig 6B). CFA increased TNF-α content in the mPFC BA and VH, and ICV PHA decreased TNF-α content in the mPFC (F = 19.5, P = 0.001), and BA (F = 18.4, P = 0.001) , the effect was abolished by MLA (Fig 6C). CFA increased IL-18 content in the mPFC BA and VH, and ICV PHA decreased IL-18 content in the mPFC (F = 14.1, P = 0.001) and VH (F = 16.7, P = 0.001), while the effect of PHA was compromised by MLA (Fig 6D).
PHA decreased anxiety- and depressive-like behaviors via WNT/β-catenin signal pathway
PHA increased center distance, center/total distance ratio, sucrose intake percentage, food intake and weight gain after SNI surgery and signal pathway inhibitors of TGFβ, mTOR, WNT/β, notch, JAK were co-administered to explore the potential the anti-anxiety anti-depressive mechanism of PHA. The results found WNT/β-catenin inhibitor IWR reversed the anti-anxiety and anti-depressive effect of PHA on center distance (F = 4.05, P = 0.002; t = 3.35, P = 0.004; Fig 7A), center/total distance ratio (F = 4.11, P = 0.002; t = 3.44, P = 0.003; Fig 7C), sucrose intake percentage (F = 4.55, P = 0.001; t = 3.22, P = 0.006; Fig 7D), food intake percentage (F = 6.17, P = 0.001; t = 3.24, P = 0.004; Fig 7E) and weight gain (F = 6.10, P = 0.001; t = 3.16, P = 0.006; Fig 7F). The total running distance were not different among six groups (F = 0.458, P = 0.837; Fig 7B) Surprisingly, mTOR inhibitor AZD8055 also reversed the effect of PHA on food intake percentage (t = 3.53, P = 0.002; Fig 7E respectively) and weight gain (t = 3.21, P = 0.005; Fig 7 F). And TGFβ, notch and JAK pathway inhibitors did not affect the anti-anxiety anti-depressive behaviors of PHA.
PHA decreased brain inflammation via WNT/β-catenin and mTOR pathway
The cytokines were measured after behavior tests. The results showed that PHA increased IL-10 and decreased IL-18 after SNI surgery, and those effect on IL-10 (Fig 8A) in mPFC (t = 4.78, P = 0.001), BA (t = 4.34, P = 0.001) and VH (t = 3.53, P = 0.003) and on IL-18 (Fig 8 D) in mPFC (t = 3.49, P = 0.003), BA (t = 4.53, P = 0.001) and VH (t = 3.62, P = 0.002) were abolished by WNT/β-catenin inhibitor IWR. In addition, PHA decreased IL-1β content in mPFC and VH after SNI surgery, and the change was compromised by mTOR inhibitor AZD8055 in the mPFC (t = 3.72, P = 0.002) and VH (t = 4.04, P = 0.001) and WNT/β-catenin inhibitor IWR in the mPFC (t = 3.32, P = 0.004) and VH (t = 4.64, P = 0.001) (Fig 8B). Similarly, PHA decreased the content of TNF in mPFC, BA and VH after SNI surgery, and the content change of TNFα in mPFC (t = 3.21, P = 0.005) and BA (t = 4.83, P = 0.001) was reversed by mTOR inhibitor AZD8055 and in mPFC (t = 3.55, P = 0.002), BA (t = 4.24, P = 0.001) and VH (t = 4.44, P = 0.002) was reversed by WNT/β-catenin inhibitor IWR (Fig 8C).