| Part 1 Clinical researchObjective To observe the clinical therapeutic effect and safety of acupuncture and sodium vitrate eye drops in the treatment of aqueous humour deficiency(ATD)dry eyes,and to provide an effective and safe treatment option for the clinic.Methods Sixty patients with dry eyes were randomly divided into control and treatment groups.30 patients in each group were given sodium glassate drops 4 times a day for 14 days.Patients in the treatment group were treated with acupuncture by selecting Jingming,Cuanzhu,Sizhukong,Taiyang,and Tongziliao,once a day,with a break every 6 days,for a total of 12 times for 14 days.The OSDI scale score,tear river height(TMH),non-contact tear film rupture time(NIBUT),sodium fluorescein staining score(FLS),and tear secretion(SIT)were recorded before and after treatment in both groups,and adverse effects were also noted.Results The OSDI scale scores,TMH,NIBUT,FLS,and SIT were not significantly different between the two groups before treatment(p>0.05);compared with before treatment,TMH and NIBUT of dry eyes were significantly higher after treatment in both groups,with a significant difference(p<0.01),FLS was significantly lower in both groups,with a significant difference(p<0.05),SIT was significantly higher after treatment in the treatment group(p<0.01)and SIT increased in the control group(p<0.05),and OSDI scale scores decreased significantly in the treatment group with a significant difference(p<0.01),while OSDI scale scores did not decrease significantly in the control group after treatment(p>0.05);compared between groups after therapy,the treatment group was better than the control group in OSDI scale scores,TMH and SIT,with a significant difference,The difference between the two groups was not statistically significant for NIBUT and FLS(p>0.05).Conclusion(ⅰ)Both acupuncture and sodium vitreous acid eye drop treatment can improve the ocular surface symptoms of ATD,increase the height of tear rivers,prolong tear film rupture time,reduce the corneal staining score,and increase tear secretion.However,acupuncture has a significant advantage in promoting tear secretion and increasing the height of the tear river;(ⅱ)acupuncture treatment can reduce the OSDI scale scores of ATD patients and effectively reduce the ocular self-discomfort symptoms of ATD patients,while sodium vitrate eye drops treatment is not effective in reducing the ocular self discomfort symptoms of ATD patients;(ⅲ)acupuncture treatment ATD eye is more easily accepted by patients,and has no obvious adverse effects.Part 2 Experimental StudyObjective To observe the effects of acupuncture on the Phenol red cotton wool tear production(PRT),tear film break-up time(BUT),and corneal fluorescein staining(FLS),and the expression of vasoactive intestinal peptide(VIP),cyclic adenosine monophosphate(cAMP),phosphorylated protein kinase A(p-PKA),protein kinase A(PKA)and aqueous channel protein 5(AQP5)in the lacrimal gland tissues of guinea pigs with aqueous deficiency dry eye(ATD)model,and to investigate the mechanism of action of acupuncture in the treatment of aqueous deficiency dry eye.Methods Experiment 1:Healthy British tricolor short-haired guinea pigs were randomly divided into blank group,model group,acupuncture group,sham acupuncture group,and medicine group,with eight animals in each group.The animal model of ATD was induced by subcutaneous injection of scopolamine hydrobromide for 10 d.The model was successfully established after 10 d.After model creation,the model group was continuously injected with scopolamine hydrobromide subcutaneously until the end of the experiment.In the acupuncture group,acupuncture points were selected for Jingming,Cuanzhu,Sizhukong,Taiyang,and Tongziliao,all without operation,and the needles were left in place for 15 min once daily for 14 days,while scopolamine hydrobromide was injected subcutaneously until the end of the experiment.The sham acupuncture group was treated with blunt needle on the same acupoints,and the needles were not inserted into subcutaneous area avoiding "deqi",once every 5 minutes for 15 minutes,once daily for 14 days,while scopolamine hydrobromide was injected subcutaneously until the end of the experiment.In the medicine group,0.1%sodium glass drops were administered in both eyes,1 drop three times a day for 14 days,while scopolamine hydrobromide was continuously injected subcutaneously until the end of the experiment.Phenol red cotton wool tear production(PRT),tear film break-up time(BUT),and corneal fluorescein staining(FLS)were measured in each group before modeling,after film creation,and after the intervention.After the intervention,the lacrimal index was calculated,histopathological changes in the lacrimal gland were observed by light microscopy,the expression and distribution of AQP5 in the lacrimal gland were detected by immunofluorescence(IF),the levels of VIP and AQP5 in the cornea and lacrimal gland were measured by enzyme-linked immunosorbent assay(ELISA),and VIP,cAMP,PKA,P-PKA and AQP5 were detected in the corneal and lacrimal gland tissues of guinea pigs in each group by western blot(WB).Experiment 2:Healthy British tricolor shorthair guinea pigs were randomly divided into blank group,model group,agonist group,antagonist group,and needle antagonist group,with 8 guinea pigs in each group.The animal model of ATD was induced by subcutaneous injection of scopolamine hydrobromide for 10 d.The model was successfully established after 10 d.After modeling,the model group was continuously injected with scopolamine hydrobromide subcutaneously until the end of the experiment.The agonist group was given an intraperitoneal injection of VIP agonist(Aviptadil)once every other day for 14 days for a total of 7 times,while scopolamine hydrobromide was continuously injected subcutaneously until the end of the experiment.In the antagonist group,VIP antagonist([D-p-Cl-Phe6,Leul7]-VIP TFA)was injected intraperitoneally once every other day for 14 days,7 times in total,while scopolamine hydrobromide was injected subcutaneously until the end of the experiment.In the acupuncture group,on top of the acupuncture treatment(the acupuncture method was the same as in Experiment 1),[D-p-Cl-Phe6,Leu17]-VIP TFA was injected intraperitoneally every other day for 14 days,while scopolamine hydrobromide was injected subcutaneously continuously until the end of the experiment.Observations were the same as in Experiment 1.Results(1)Phenol red cotton thread tear secretion test(PRT):In experiment 1,(ⅰ)on the 1st day of the experiment,there was no statistical difference in PRT between the groups(p>0.05).(ⅱ)On the 10th day of the experiment,compared with the blank group,PRT was significantly lower in the model group,the acupuncture group,the sham acupuncture group,and the medicine group(p<0.01).(ⅲ)On the 24th day of the experiment,compared with the model group,PRT was significantly higher in the acupuncture group and the medicine group(p<0.01);compared with the medicine group,PRT was higher in the acupuncture group(p<0.05).Experiment 2,(ⅰ)On the 1st day of the experiment,there was no statistically difference in PRT between the groups(p>0.05).(ⅱ)On the 10th day of the experiment,PRT was significantly lower in the model group,agonist group,antagonist group,and needle antagonist group compared to the blank group(p<0.01).(ⅲ)On the 24th day of the experiment,compared with the blank group,PRT was significantly lower in the model group,antagonist group,and needle antagonist group(p<0.01);compared with the model group,PRT was significantly higher in the agonist group(p<0.01)and lower in the antagonist group(p<0.05);compared with the antagonist group,PRT was increased to different degrees in the agonist and antagonist groups(p<0.01,p<0.05).(2)Tear film rupture time(BUT):In experiment 1,(ⅰ)on the 1st day of the experiment,there was no statistical difference in BUT in each group(p>0.05).(ⅱ)On the 10th day of the experiment,compared with the blank group,the BUT decreased to different degrees in the model group,the acupuncture group,the sham acupuncture group and the medicine group(p<0.01,p<0.05).(ⅲ)On the 24th day of the experiment,compared with the model group,BUT increased to different degrees in the acupuncture group and the medicine group(p<0.01,p<0.05).Experiment 2,(ⅰ)On the 1st day of the experiment,there was no statistical difference in BUT in each group(p>0.05).(ⅱ)On the 10th day of the experiment,BUT was significantly lower in the model group,agonist group,antagonist group and needle antagonist group compared to the blank group(p<0.01).(ⅲ)On the 24th day of the experiment,compared with the blank group,BUT was reduced to different degrees in the model group,antagonist group and needle antagonist group(p<0.01,p<0.05);compared with the model group,BUT was significantly increased in the agonist group(p<0.01)and reduced in the antagonist group(p<0.05);compared with the antagonist group,BUT was significantly increased in the agonist group and needle antagonist group(p<0.01).(3)Corneal fluorescence staining score(FLS):Experiment 1,(ⅰ)On the 1st day of the experiment,there was no statistical difference in FLS among the groups(p>0.05).(ⅱ)On the 10th day of the experiment,compared with the blank group,FLS was significantly higher in the model group,the acupuncture group,the sham acupuncture group and the medicine group(p<0.01).(ⅲ)On the 24th day of the experiment,FLS was significantly lower in the acupuncture group and the medicine group compared to the model group(p<0.01).Experiment 2,(ⅰ)On the 1st day of the experiment,there was no statistical difference in FLS among the groups(p>0.05).(ⅱ)On the 10th day of the experiment,FLS was significantly higher in the model group,agonist group,antagonist group and needle antagonist group compared to the blank group(p<0.01).(ⅲ)On the 24th day of the experiment,compared with the blank group,FLS was significantly higher in the model group,antagonist group and needle antagonist group(p<0.01);compared with the model group,FLS was significantly lower in the agonist group(p<0.01);compared with the antagonist group,FLS was significantly lower in the agonist group and needle antagonist group(p<0.01).(4)Lacrimal gland index:In experiment 1,(ⅰ)compared with the blank group,the lacrimal gland index was significantly lower in the model group(p<0.01);(ⅱ)compared with the model group,the lacrimal gland index was increased in the acupuncture group and the medicine group(p<0.05).In experiment 2,(ⅰ)compared with the blank group,the lacrimal gland index was reduced to different degrees in the model group,the agonist group and the needle antagonist group(p<0.01,p<0.05);(ⅱ)compared with the model group,the lacrimal gland index was significantly increased in the agonist group(p<0.01);the lacrimal gland index was reduced in the antagonist group(p<0.05);(ⅲ)compared with the antagonist group,the lacrimal gland index was increased to different degrees in the agonist group and the needle antagonist group(p<0.01,p<0.05)(5)Light microscopic observation:In experiment 1,the overall structure of the lacrimal gland tissue in the blank group was not obviously abnormal,the epithelial cells were uniform in size,rich in cytoplasm,closely arranged and relatively regular,no obvious atrophy of the glandular vesicles or inflammatory reactions were seen;in the model group and the sham acupuncture group,the epithelial cells in the lacrimal gland tissue were obviously atrophied,irregularly arranged,the lumen of the gland was dilated and focal lymphocyte infiltration was seen;in the acupuncture group,the epithelial cell structure of the lacrimal gland was basically normal and a small amount of lymphocyte infiltration was seen.In the medicine group,some epithelial cells in the lacrimal gland were atrophied,the gland’s lumen was dilated,and some lymphocytes were seen to infiltrate.In Experiment 2,the overall structure of the lacrimal gland tissue in the blank group was not significantly abnormal,the epithelial cells were uniform in size,rich in cytoplasm,closely arranged and regular,no obvious atrophy of the glandular vesicles or inflammatory reactions were seen;the epithelial cells in the lacrimal gland tissue of the model group and the needle antagonist group were obviously atrophied,irregularly arranged,with dilated lumen and focal lymphocyte infiltration;the epithelial cells in the lacrimal gland of the agonist group were basically normal in structure,with a small amount of dilated lumen and rich in cytoplasm.The lacrimal gland epithelial cells in the antagonist group were obviously atrophied,arranged in a disorganized manner,with a dilated lumen and a large number of lymphocytes infiltrating the lacrimal gland.(6)Immunofluorescence technique(IF)detection:In experiment 1,(ⅰ)the expression of the AQP5 was significantly reduced in the model group compared with the blank group(p<0.01);(ⅱ)the expression of AQP5 was increased to different degrees in the acupuncture group and the medicine group compared with the model group(p<0.05,p<0.01).The fluorescence staining of AQP5 was more intense in the blank and acupuncture groups,and was mainly expressed in the ducts of the lacrimal gland and the parietal plasma membrane of the vesicular cells;the fluorescence staining of AQP5 was weaker in the western medicine group;the fluorescence staining of AQP5 was extremely weak in the model and sham acupuncture groups,and was faintly seen in the cytoplasm or nucleus of the vesicles.In Experiment 2,(ⅰ)the expression of AQP5 in the model and antagonist groups decreased to different degrees compared with the blank group(p<0.01,p<0.05):(ⅱ)the expression of AQP5 in the agonist group increased compared with the model group(p<0.05);(ⅲ)the expression of AQP5 in the agonist and needle antagonist groups increased to different degrees compared with the antagonist group(p<0.01,p<0.05).The fluorescence staining of AQP5 was stronger in the blank and agonist groups,and was mainly expressed in the ducts of the lacrimal gland and the parietal plasma membrane of the vesicular cells;the fluorescence staining of AQP5 was weaker in the model and needle antagonist groups;the fluorescence staining of AQP5 was extremely weak in the antagonist group,and was faintly seen in the cytoplasm or nucleus of the vesicles.(7)The contents of VIP and AQP5 in corneal tissues:In experiment 1,(ⅰ)compared with the blank group,the contents of VIP and AQP5 were reduced to different degrees in the model group(p<0.05,p<0.01);(ⅱ)compared with the model group,the contents of VIP were increased in the acupuncture group and the medicine group(p<0.05),while the contents of AQP5 were increased to different degrees(p<0.01,p<0.05).In experiment 2,(ⅰ)compared with the blank group,the expression of both VIP and AQP5 was decreased in the model group(p<0.05),and the expression of both VIP and AQP5 was significantly decreased in the antagonist group(p<0.01);(ⅱ)compared with the model group,the expression of AQP5 was increased in the agonist group(p<0.05),and the expression of both VIP and AQP5 was decreased in the antagonist group(p<0.05);(ⅲ)compared with the antagonist group,the expression of VIP and AQP5 was significantly higher in the agonist group(p<0.01),and the expression of VIP and AQP5 was increased to different degrees in the needle antagonist group(p<0.01,p<0.05).(8)Content of VIP and AQP5 in lacrimal gland tissues:In experiment 1,(ⅰ)compared with the blank group,the content of both VIP and AQP5 was significantly reduced in the model group(p<0.01).(ⅱ)Compared with the model group,the contents of VIP and AQP5 were significantly higher in both the acupuncture and medicine groups(p<0.01).In experiment 2,(ⅰ)compared with the blank group,the expression of VIP and AQP5 was reduced to different degrees in the model group(p<0.01,p<0.05),and the expression of both VIP and AQP5 was significantly reduced in the antagonist group(p<0.01);(ⅱ)compared with the model group,the expression of VIP and AQP5 was increased to different degrees in the agonist group(p<0.01,p<0.05);(ⅲ)compared with the antagonist group,the expression of VIP and AQP5 was significantly higher in the agonist group(p<0.01),and the expression of VIP and AQP5 was differentially higher in the needle antagonist group(p<0.01,p<0.05).(9)Protein expression levels of VIP,cAMP,p-PKA,PKA and AQP5 in corneal tissue:Experiment 1,(ⅰ)compared with the blank group,VIP,cAMP,P-PKA/total PKA and AQP5 were reduced to different degrees in the model group(p<0.01,p<0.05);(ⅱ)compared with the model group,VIP,cAMP,P-PKA/total PKA and AQP5 were increased in the acupuncture group(p<0.05);(ⅲ)compared with the medicine group,cAMP was increased in the acupuncture group(p<0.05).In experiment 2,(ⅰ)compared with the blank group,VIP5 cAMP,P-PKA/total PKA,and AQP5 were reduced to different degrees in the model group(p<0.01,p<0.05),and VIP,cAMP,P-PKA/total PKA and AQP5 were significantly reduced in the antagonist group(p<0.01);(ⅱ)compared with the model group,VIP,cAMP,P-PKA/total PKA and AQP5 were increased to different degrees in the agonist group compared to the model group(p<0.01,p<0.05);(ⅲ)VIP,cAMP,P-PKA/total PKA and AQP5 were significantly increased in the agonist group and VIP,cAMP,P-PKA/total PKA and AQP5 were increased to different degrees in the needle antagonist group compared to the antagonist group(p<0.01,p<0.05).(10)Protein expression levels of VIP,cAMP,p-PKA,PKA and AQP5 in lacrimal gland tissues:In experiment 1,(ⅰ)compared with the blank group,VIP,cAMP,P-PKA/total PKA,and AQP5 were decreased to different degrees in the model group(p<0.01,p<0.05);(ⅱ)compared with the model group,VIP,cAMP,P-PKA/total PKA,and AQP5 were increased to different degrees in the acupuncture group(p<0.01,p<0.05),and VIP and AQP5 in the medicine group protein expression were increased(p<0.05).In experiment 2,(ⅰ)compared with the blank group,VIP,cAMP,P-PKA/total PKA,and AQP5 were significantly decreased in the model and antagonist groups(p<0.01);(ⅱ)compared with the model group,the protein expression of VIP,AQP5,and cAMP were increased to different degrees in the agonist group(p<0.01,p<0.05),and the protein expression of cAMP was decreased in the antagonist group(p<0.05));(ⅲ)compared with the antagonist group,VIP,cAMP,P-PKA/total PKA and AQP5 were differentially increased in the agonist group(p<0.01,p<0.05)and VIP,cAMP,P-PKA/total PKA and AQP5 were increased in the needle antagonist group(p<0.05).Conclusion(ⅰ)Acupuncture treatment can promote lacrimal gland secretion,increase tear secretion,prolong tear film rupture time,improve tear film stability,and reduce ocular surface damage in ATD guinea pigs.(ⅱ)Acupuncture treatment could upregulate the expression of VIP,cAMP,PKA,p-PKA and AQP 5 in the cornea and lacrimal glands of ATD guinea pigs,and improved the morphology of the lacrimal gland.(ⅲ)The VIP/cAMP-PKA/AQP5 signaling pathway exists in corneal and lacrimal gland tissues of guinea pigs,and acupuncture treatment of ATD may be associated with the activation of this signaling pathway. |
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