Intestinal inflammation causes initial axonal degeneration and neuronal death but subsequent axon outgrowth from surviving neurons restores innervation density to the target smooth muscle cells. Elsewhere, the pro-inflammatory cytokines TNF␣ and IL-1␤ cause neurotoxicity, leading us to test their role in promoting enteric neuron death. In a rat coculture model, TNF␣ or IL-1␤ did not affect neuron number but did promote significant neurite outgrowth to twofold that of control by 48 h, while other cytokines (e.g., IL-4, TGF␤) were without effect. TNF␣ or IL-1␤ activated the NFB signaling pathway, and inhibition of NFB signaling blocked the stimulation of neurite growth. However, nuclear translocation of NFB in smooth muscle cells but not in adjacent neurons suggested a dominant role for smooth muscle cells. TNF␣ or IL-1␤ sharply increased both mRNA and protein for GDNF, while the neurotrophic effects of TNF␣ or IL-1␤ were blocked by the RET-receptor blocker vandetanib. Conditioned medium from cytokine-treated smooth muscle cells mimicked the neurotrophic effect, inferring that TNF␣ and IL-1␤ promote neurite growth through NFB-dependent induction of glial cell linederived neurotrophic factor (GDNF) expression in intestinal smooth muscle cells. In vivo, TNBS-colitis caused early nuclear translocation of NFB in smooth muscle cells. Conditioned medium from the intact smooth muscle of the inflamed colon caused a 2.5-fold increase in neurite number in cocultures, while Western blotting showed a substantial increase in GDNF protein. Pro-inflammatory cytokines promote neurite growth through upregulation of GDNF, a novel process that may facilitate re-innervation of smooth muscle cells and a return to homeostasis following initial damage.

Intestinal inflammation causes an increased intestinal wall thickness, in part, due to the proliferation of smooth muscle cells, which impairs the contractile phenotype elsewhere. To study this, cells from the circular muscle layer of the rat colon (CSMC) were isolated and studied, both in primary culture and after extended passage, using quantitative PCR, Western blot analysis, and immunocytochemistry. By 4 days in vitro, both mRNA and protein for the smooth muscle marker proteins α-smooth muscle actin, desmin, and SM22-α were reduced by >50%, and mRNA for cyclin D1 was increased threefold, evidence for modulation to a proliferative phenotype. Continued growth caused significant further decrease in expression, evidence that phenotypic loss in CSMC was proportional to the extent of proliferation. In CSMC isolated at day 2 of trinitrobenzene sulfonic acid-induced colitis, flow cytometry and Western blotting showed that these differentiated markers were reduced in mitotic CSMC, while similar to control in nonmitotic CSMC. By day 35 post-trinitrobenzene sulfonic acid, when inflammation has resolved, CSMC were hypertrophic, but, nonetheless, showed markedly decreased expression of smooth muscle protein markers per cell. In vitro, day 35 CSMC displayed an accelerated loss of phenotype and increased thymidine uptake in response to serum or PDGF-BB. Furthermore, carbachol-induced expression of phospho-AKT (a marker of cholinergic response) was lost from day 35 CSMC in vitro, while retained in control cells. Therefore, proliferation reduces the expression of smooth-muscle-specific markers in CSMC, possibly leading to altered contractility. However, inflammation-induced proliferation in vivo also causes lasting changes that include unexpected priming for an exaggerated response to proliferative stimuli. Identification of the molecular mechanisms of intestinal smooth muscle cell phenotypic modulation will be helpful in reducing the detrimental effects of inflammation.

The salidroside pretreatment alleviated the focal cerebralischemia-reperfusion injury in the rat model, possibly by decreasing the permeability of blood brain barrier, attenuating brain edema and reducing TNF-alpha expression.

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