Acupuncture promotes tear secretion by up-regulating VIP/cAMP/PKA/AQP5 signaling in guinea pigs with aqueous tear deficiency dry eye. / 基于VIP/cAMP/PKA/AQP5信号通路探讨针刺治疗水液缺乏型干眼的作用机制.

OBJECTIVES: To observe the effect of acupuncture on the ocular surface symptoms and the protein expression of vasoactive intestinal peptide (VIP) / cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) / aquaporin 5(AQP5) signaling pathway in lacrimal gland tissue of aqueous tear deficiency (ATD) type dry eye model, so as to investigate its mechanism underlying improvement of ATD. METHODS: British shorthair guinea pigs were randomly divided into blank control, model, acupuncture, sham-acupuncture and medication group, with 8 guinea pigs in each group. The ATD model was established by subcutaneous injection of scopolamine hydrobromide (0.6 mg/dose, 4 times/d for 10 days). For guinea pigs of the acupuncture group, filiform needles were inserted into bilateral "Jingming"(BL1), "Cuanzhu"(BL2), "Sizhukong"(TE23), "Taiyang"(EX-HN5), and "Tongziliao"(GB1) for 15 min. For guinea pigs of the sham-acupuncture group, a blunt filiform needle was used to repeatedly prick (not pierce) the skin of the same acupoints mentioned above. The treatment in the above two groups was conducted once daily for 14 days. The guinea pigs in the medication group received administration of sodium hyaluronate eye drops in both eyes, three times a day for 14 days. The objective tests of tear film break-up time (BUT), corneal fluorescein staining score (FLS) and phenol red thread (PRT) test were conducted before and after modeling and after the intervention. After the intervention, the lacrimal index (weight of lacrimal gland/body weight) was calculated. Histopathological changes of the lacrimal gland were observed after H.E. staining. The expression of AQP5 in the lacrimal gland were detected by immunofluorescence, and the contents of VIP and AQP5 in the lacrimal gland were measured by ELISA, the protein expression levels of VIP, cAMP, PKA, p-PKA and AQP5 in the lacrimal gland were detected by Western blot. RESULTS: In comparison with the blank control group, the PRT, BUT, lacrimal index, AQP5 immunoactivity, contents of VIP and AQP5, and protein expression levels of VIP, cAMP, PKA, p-PKA and AQP5 were significantly decreased(P<0.01, P<0.05), and FLS was obviously increased (P<0.01) in the model group . Compared to the model group, the PRT, BUT, lacrimal index, AQP5 immunoactivity, contents of VIP and AQP5, and expression levels of VIP and AQP5 in both acupuncture and medication groups, and the expression levels of cAMP, PKA, p-PKA in the acupuncture group were considerably increased (P<0.01, P<0.05), while the FLS was markedly decreased in both acupuncture and medication groups (P<0.01, P<0.05). Compared with the medication group, the acupuncture group had increased PRT (P<0.05). CONCLUSIONS: Acupuncture intervention is effective in reducing ocular surface damage and promoting tear secretion in guinea pigs with ATD, which may be related to its function in activating VIP/cAMP/PKA signaling, and promoting the expression of AQP5 in the lacrimal gland.

[Acupuncture mitigates ocular surface inflammatory response via α7nAChR/NF-κB p65 signaling in dry eye guinea pigs].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the ocular surface inflammation and α7 nicotinic acetylcholine receptor (α7nAChR) / nuclear factor kappa-B (NF-κB) p65 signal pathway in guinea pigs with dry eye, so as to explore its underlying mechanism. METHODS: A total of 32 male British tricolor short haired guinea pigs were randomized into blank control, model, EA and sham acupuncture groups, with 8 guinea pigs in each group. The dry eye model was established by subcutaneous injection of scopolamine hydrobromide solution (0.6 mg/0.2 mL each time, 4 times a day for 10 days). Guinea pigs of the EA group was treated with EA at bilateral "Cuanzhu" (BL2) and "Taiyang" (HN5), and manual acupuncture at bilateral "Jingming" (BL1), "Sizhukong" (SJ23), "Tongziliao" (GB1) for 15 min, once daily for 14 days. For animals of the sham acupuncture group, a blunt needle was used to prick the skin surface of the acupoints, the acupoint selection and stimulation time were the same as those in the EA group. Before and after modeling and after the intervention, the breakup time (BUT) of lacrimal film, sodium fluorescein coloring (Fl) state of corneal epithelium and phenol red thread (PRT) moist length were recorded for assessing the severity of dry eye. The density of activated immune cells around the corneal epithelial stromal cells was determined by corneal confocal microscopy. The contents of interleukin-4 (IL-4), IL-6, IL-10, tumor necrosis factor α (TNF-α) in the cornea and lacri-mal gland tissues were determined by ELISA, and the expression levels of α7nAChR and NF-κB p65 in the cornea and lacrimal gland were detected by immunohistochemistry and Western blot, separately. RESULTS: Compared with the blank control group, the corneal Fl, density of activated immune cells of corneal epithelium, contents of IL-6, IL-10 and TNF-α in both corneal and lacrimal gland tissues, NF-κB p65 cell positive rate and protein expression of lacrimal gland and corneal tissues were significantly increased (P<0.01, P<0.05), while the BUT, PRT and lacrimal gland α7nAChR cell positive rate considerably decreased (P<0.01) in the model group. In comparison with the model group, the level of corneal Fl, density of the activated immune cells of corneal epithelium, contents of corneal and lacrimal IL-6 and TNF-α, and corneal and lacrimal NF-κB p65 cell positive rates and protein expressions were remarkably down-regulated in the EA group (P<0.01, P<0.05), rather than in the sham acupuncture group (P>0.05) except content of corneal IL-10, lacrimal NF-κB p65 cell positive rate and lacrimal α7nAChR protein expression, whereas the levels of BUT, PRT, corneal and lacrimal IL-10 and corneal and lacrimal α7nAChR cell positive rates and protein expressions significantly up-regulated in the EA group (P<0.01, P<0.05), rather than in the sham acupuncture group (P>0.05) except corneal TNF-α and corneal NF-κB p65 protein expression. CONCLUSION: EA can improve corneal and lacrimal function in dry eye guinea pigs, which may be associated with its actions in increasing the expression of α7nAChR, inhibiting the nuclear translocation of NF-κB, and reducing the activated immune cells and inflammatory reaction.