Abstract:Objective To investigate whether loperamide hydrochloride（LOP）- induced constipated mice exhibit comorbid depressive-like behaviors，and to elucidate the potential mechanism of the colonic macrophage SOCS1/JAK2-STAT3/IL - 6 signaling pathway. Methods Twelve adult male C57BL/6J mice were randomly divided into a control group and an LOP group（n=6 per group）. Mice in the LOP group were intragastrically administered LOP twice daily for10 consecutive days to establish a slow transit constipation model；the control group received an equal volume of 0.9%sodium chloride solution. The constipation phenotype was verified by measuring whole gut transit time （WGTT），colonic transit time（CTT），fecal water content, and 2 hour defecation frequency. Depressive-and anxiety-like behaviors were assessed using the tail suspension test（TST），forced swim test（FST），open field test（OFT），and elevated plusmazetest（EPM）. Additionally，bone marrow - derived macrophages（BMDMs）were extracted and treated with fecal supernatants from control group and LOP group，respectively，to simulate the intestinal microenvironment. Quantitative reverse transcription polymerase chain reaction （qRT-PCR），enzyme -linked immunosorbent assay （ELISA），immunofluorescence，and Western blot were applied to detect the levels of proinflammatory factors［interleukin-6（IL）-6，tumor necrosis factor - α（TNF - α），IL - 1β，etc.］in the colon，serum，and brain tissues（prefrontal cortex and hippocampus）. Results Behavioral tests revealed a significant increase in the immobility time of LOP group in both the TST and FST（TST：61.95%±9.76% vs 41.92%±8.74%，t=3.754，P=0.004；FST：57.59%±9.16% vs 35.07%±7.80%，t=4.590，P=0.001）. Molecular analyses indicated that compared with control group，SOCS1 mRNA and protein expressions were significantly downregulated，whereas p-JAK2 and p-STAT3 levels were elevated in the colonic tissues of LOP mice，accompanied by elevated colonic IL-6 mRNA levels（3.71±1.13 vs 1.06±0.24，t=5.644，P<0.01）and an increased number of F4/80 +IL -6 + macrophages［（7.17±2.32）cells/high power field vs（3.67±1.21）cells/high power field，t=3.280，P=0.008］. Furthermore，serum IL - 6 levels were significantly elevated in the LOP group［（86.15 ±16.38）pg/mL vs（66.09±8.49）pg/mL，t=2.663，P=0.024］. Central，pro -inflammatory such as in IL -6 mRNA levels were upregulated（prefrontal cortex：2.53±1.43 vs 1.10± 0.31，t=2.411，P=0.037；hippocampus：3.17±1.55 vs 1.05±0.29，t=3.308，P=0.008），and Iba1+ microglia were significantly activated（prefrontal cortex：1.63±0.32 vs 1.03±0.15，t=4.171，P=0.002；hippocampus：1.63±0.37 vs 1.02±0.13，t=3.955，P=0.003）. In vitro experiments further confirmed that intervention with fecal supernatant from LOP group significantly downregulated SOCS1 in BMDMs，activated theJAK2-STAT3 pathway，and promoted the release of IL-6（6.43±0.29 vs 2.29±0.45，q=13.220，P<0.01）.Conclusion LOP-induced constipated mice exhibit obvious depressive-like behaviors，and the mechanism may be related to the alteration of the intestinal microenvironment caused by constipation，which inhibits SOCS1 expression in colonic macrophages，thereby activating the JAK2 - STAT3 signaling pathway，promoting IL - 6 release，and inducing central neuroinflammation via systemic circulation.