| Purpose:Clinical ResearchTo explore the clinical efficacy of eye-acupuncture combined with electroacupuncture in the treatment of knee osteoarthritis(KOA)and explore new ideas and methods for the treatment of KOA.Mechanism ResearchBased on p38 MAPK signaling pathway,the anti-inflammatory and analgesic mechanism of eye-acupuncture combined with electroacupuncture therapy on KOA was explored to provide theoretical and experimental support for the mechanism study of KOA.Method:Clinical ResearchAccording to the inclusion and exclusion criteria,100 patients diagnosed with KOA in the Second Department of Bone Injury Rehabilitation,Affiliated Hospital of Liaoning University of Traditional Chinese Medicine from January 2019 to July 2021 were selected and divided into a combined group and an electroacupuncture group,with 50 cases in each group.The electroacupuncture group was treated with electroacupuncture alone,and the combined group was treated with eye-acupuncture combined with electroacupuncture.Patients in both groups were treated for 20 minutes once a day for 2 consecutive weeks.The efficacy observation indexes included VAS score,WOMAC pain score,WOMAC stiffness score,WOMAC daily activity score,WOMAC total score,loxolophen sodium tablet taking rate.The comprehensive efficacy of the two groups was evaluated by calculating the effective rate,cure rate and cure rate plus significant rate.IBM SPSS Statistics 22.0 statistical software was used for data analysis.Mechanism ResearchThirty-six SPF sprague-dawley rats aged 8 weeks,weighing 200±20g,with male and female halves,were selected and provided with water daily and poplar wood shavings regarded as padding were changed every other day.The rats were divided into sham operation group,eye-acupuncture combined with electroacupuncture group,electroacupuncture group and model group by random number table method,with 9 rats in each group.After 1 week of adaptive feeding,all rats were modeled by the modified Hulth method.One week after the modeling operation,the rats were driven for 30 minutes a day for 8 weeks.Eye-acupuncture combined with electroacupuncture group and electroacupuncture group alone were intervened20 minutes each time,once a day,continuous intervention for 2 weeks,sham operation group and model group did not receive any intervention.The analgesic effect of different intervention methods was determined by Von Frey fiber pain test after intervention.Then the rats were sampled.After anesthesia,blood was taken from the abdominal aorta of all rats,and supernatant was taken after centrifugation for ELISA test.Six rats were randomly selected from each group,and the knee cartilage of the intervention side was cut for WB test and qRT-PCR test.Knee cartilage of the intervention side belongs to the remaining 3 rats in each group was used for HE staining and IHC test.The expressions of IL-1β,IL-6,TNF-α,PGE2 and IL-10 in serum of rats were tested by ELISA.The m RNA expressions of TAK1,MKK6,p38 and ATF2 in knee cartilage of rats were tested by qRT-PCR.The expression of p-p38 in knee cartilage of rats was detected by IHC.The protein expressions of TAK1,p-TAK1,MKK6,p-MKK6,p38,p-p38,ATF2 and p-ATF2 in the knee cartilage of rats were tested by WB,and the pathological changes of the knee cartilage of rats were observed by HE staining.IBM SPSS Statistics 22.0 statistical software was used for data analysis.Results:Clinical Research1.There was no statistically significant difference in gender composition between combined group and electroacupuncture group(p>0.05),indicating comparability.There was no statistically significant difference in age between combined group and electroacupuncture group(p>0.05),indicating comparability.There was no statistically significant difference in weight between combined group and electroacupuncture group(p>0.05),indicating comparability.There was no statistically significant difference in course of disease between combined group and electroacupuncture group(p>0.05),indicating comparability.There was no statistically significant difference in distribution of affected knee between combined group and electroacupuncture group(p>0.05),indicating comparability.2.Before treatment,there was no significant difference in VAS score between combined group and electroacupuncture group(p>0.05),indicating comparability.Before treatment,there was no significant difference in WOMAC pain score between combined group and electroacupuncture group(p>0.05),indicating comparability.Before treatment,there was no significant difference in WOMAC stiffness score between combined group and electroacupuncture group(p>0.05),indicating comparability.Before treatment,there was no significant difference in WOMAC daily activity score between combined group and electroacupuncture group(p>0.05),indicating comparability.Before treatment,there was no significant difference in WOAMC total score between combined group and electroacupuncture group(p>0.05),indicating comparability.3.There was statistically significant difference in VAS score between the combined groups at 1 week after treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC pain score between the combined groups at 1 week after treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC stiffness score between the combined groups at 1 week after treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC daily activity score between the combined groups at 1 week after treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC total score between the combined groups at 1 week after treatment and before treatment(p<0.01).There was statistically significant difference in VAS score between the electroacupuncture groups at 1 week of treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC pain score between the electroacupuncture groups at 1 week of treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC stiffness score between the electroacupuncture groups at 1 week of treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC daily activity score between the electroacupuncture groups at 1 week of treatment and before treatment(p<0.01).There was statistically significant difference in WOMAC total score between the electroacupuncture groups at 1 week of treatment and before treatment(p<0.01).4.After 1 week of treatment,there was statistically significant difference in VAS score between combined group and electroacupuncture group(p<0.01).After 1 week of treatment,there was statistically significant difference in WOMAC pain score between combined group and electroacupuncture group(p<0.01).After 1 week of treatment,there was statistically significant difference in WOMAC total score between combined group and electroacupuncture group(p<0.01).5.After 1 week of treatment,there was no statistically significant difference in WOMAC stiffness score between combined group and electroacupuncture group(p>0.05).After 1 week of treatment,there was no statistically significant difference in WOMAC daily activity score between combined group and electroacupuncture group(p>0.05).6.There was statistically significant difference in VAS score between 2 weeks and 1week of treatment in combined group(p<0.01).There was statistically significant difference in WOMAC pain score between 2 weeks and 1 week of treatment in combined group(p<0.01).There was statistically significant difference in WOMAC stiffness score between 2weeks and 1 week of treatment in combined group(p<0.01).There was statistically significant difference in WOMAC daily activity score between 2 weeks and 1 week of treatment in combined group(p<0.01).There was statistically significant difference in WOMAC total score between 2 weeks and 1 week of treatment in combined group(p<0.01).There was statistically significant difference in VAS score between 2 weeks and 1 week in the electroacupuncture group(p<0.01).There was statistically significant difference in WOMAC pain score between 2 weeks and 1 week in the electroacupuncture group(p<0.01).There was statistically significant difference in WOMAC stiffness score between 2 weeks and 1 week in the electroacupuncture group(p<0.01).There was statistically significant difference in WOMAC daily activity score between 2 weeks and 1 week in the electroacupuncture group(p<0.01).There was statistically significant difference in WOMAC total score between 2weeks and 1 week in the electroacupuncture group(p<0.01).7.After 2 weeks of treatment,there was statistically significant difference in VAS score between combined group and electroacupuncture group(p<0.01).After 2 weeks of treatment,there was statistically significant difference in WOMAC pain score between combined group and electroacupuncture group(p<0.01).After 2 weeks of treatment,there was statistical difference in WOMAC total score between combined group and electroacupuncture group(p<0.05).8.After 2 weeks of treatment,there was no statistical difference in WOMAC stiffness score between combined group and electroacupuncture group(p>0.05).After 2 weeks of treatment,there was no statistical difference in WOMAC daily activity score between combined group and electroacupuncture group(p>0.05).9.After 2 weeks of treatment,there was statistically significant difference in rate of loxoprofen sodium tablets between combined group and electroacupuncture group(p<0.01).10.There was no statistical difference in clinical effective rate between combined group and electroacupuncture group(p>0.05).There was statistical difference in clinical cure rate between combined group and electroacupuncture group(p<0.05).There was statistically significant difference in clinical cure rate plus significant rate between combined group and electroacupuncture group(p<0.01).11.There were no adverse reactions and abnormal acupuncture conditions in combined group and electroacupuncture group during treatment.Mechanism Research1.Comparison of Von Frey fiber pain test after driving,there was statistically significant difference between sham operation group and eye-acupuncture combined with electroacupuncture group(p<0.01).Comparison of Von Frey fiber pain test after driving,there was statistically significant difference between sham operation group and electroacupuncture group(p<0.01).Comparison of Von Frey fiber pain test after driving,there was statistically significant difference between sham operation group and model group(p<0.01).There was no statistical difference between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p>0.05).There was no statistical difference between eye-acupuncture combined electroacupuncture group and model group(p>0.05).There was no statistical difference between electroacupuncture group and model group(p>0.05).2.Comparison of Von Frey fiber pain test after intervention showed that there was no statistical difference between sham operation group and electroacupuncture group(p>0.05).Comparison of Von Frey fiber pain test after intervention showed that there was statistically significant difference between sham operation group and electroacupuncture group(p<0.01).Comparison of Von Frey fiber pain test after intervention showed that there was statistically significant difference between sham operation group and model group(p<0.01).Comparison of Von Frey fiber pain test after intervention showed that there was statistically significant difference between eye-acupuncture combined electroacupuncture group and model group(p<0.01).There was statistical difference between eye-acupuncture combined with electroacupuncture group and electroacupuncture group(p<0.05).3.As to Von Frey fiber pain test in eye-acupuncture combined with electroacupuncture group,there was statistical difference between the timing after the end of intervention and the timing after driving(p<0.05).As to Von Frey fiber pain test in model group,there was no statistical difference between the timing after the end of intervention and the timing after driving(p>0.05).4.There was statistical difference in IL-1β between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.05).There was statistical difference in IL-1β between electroacupuncture group and eye-acupuncture combined electroacupuncture group(p<0.05).There was statistically significant difference in IL-1βbetween sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in IL-1β between sham operation group and model group(p<0.01).There was statistically significant difference in IL-1β between eye-acupuncture combined electroacupuncture group and model group(p<0.01).5.There was statistical difference in IL-6 between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.05).There was statistical difference in IL-6 between electroacupuncture group and eye-acupuncture combined electroacupuncture group(p<0.05).There was statistically significant difference in IL-6between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in IL-6 between sham operation group and model group(p<0.01).There was statistically significant difference in IL-6 between eye-acupuncture combined electroacupuncture group and model group(p<0.01).6.There was no statistical difference in TNF-α between sham operation group and eye-acupuncture combined electroacupuncture group(p>0.05).There was statistically significant difference in TNF-α between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in TNF-α between sham operation group and model group(p<0.01).There was statistically significant difference in TNF-αbetween eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in TNF-α between eye-acupuncture combined electroacupuncture group and model group(p<0.01).7.There was statistically significant difference in PGE2 between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).There was statistically significant difference in PGE2 between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in PGE2 between sham operation group and model group(p<0.01).There was statistically significant difference in PGE2 between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in PGE2 between eye-acupuncture combined electroacupuncture group and model group(p<0.01).8.There was statistically significant difference in IL-10 between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).There was statistically significant difference in IL-10 between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in IL-10 between sham operation group and model group(p<0.01).There was statistically significant difference in IL-10 between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in IL-10 between eye-acupuncture combined electroacupuncture group and model group(p<0.01).9.There was statistically significant difference in HE staining Mankin’s score between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).There was statistically significant difference in HE staining Mankin’s score between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in HE staining Mankin’s score between sham operation group and model group(p<0.01).There was statistically significant difference in HE staining Mankin’s score between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in HE staining Mankin’s score between eye-acupuncture combined electroacupuncture group and model group(p<0.01).10.The comparison of the average optical density of p-p38 detected by IHC showed statistically significant difference between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).The comparison of the average optical density of p-p38 detected by IHC showed statistically significant difference between sham operation group and electroacupuncture group(p<0.01).The comparison of the average optical density of p-p38 detected by IHC showed statistically significant difference between sham operation group and model group(p<0.01).The comparison of the average optical density of p-p38 detected by IHC showed statistically significant difference between combined group and electroacupuncture group(p<0.01).The comparison of the average optical density of p-p38 detected by IHC showed statistically significant difference between eye-acupuncture combined electroacupuncture group and model group(p<0.01).11.There was statistically significant difference in TAK1 m RNA expression between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).There was statistically significant difference in TAK1 m RNA expression between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in TAK1 m RNA expression between sham operation group and model group(p<0.01).There was statistically significant difference in TAK1 m RNA expression between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in TAK1 m RNA expression between eye-acupuncture combined electroacupuncture group and model group(p<0.01).12.There was statistically significant difference in MKK6 m RNA expression between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).There was statistically significant difference in MKK6 m RNA expression between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in MKK6 m RNA expression between sham operation group and model group(p<0.01).There was statistically significant difference in MKK6 m RNA expression between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in MKK6 m RNA expression between eye-acupuncture combined electroacupuncture group and model group(p<0.01).13.There was statistical difference in p38 m RNA expression between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.05).There was statistically significant difference in p38 m RNA expression between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in p38 m RNA expression between sham operation group and model group(p<0.01).There was statistically significant difference in p38 m RNA expression between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in p38 m RNA expression between eye-acupuncture combined electroacupuncture group and model group(p<0.01).14.There was statistical difference in ATF2 m RNA expression between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.05).There was statistically significant difference in ATF2 m RNA expression between sham operation group and electroacupuncture group(p<0.01).There was statistically significant difference in ATF2 m RNA expression between sham operation group and model group(p<0.01).There was statistically significant difference in ATF2 m RNA expression between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There was statistically significant difference in ATF2 m RNA expression between eye-acupuncture combined electroacupuncture group and model group(p<0.01).15.There were no statistical differences in TAK1 protein expression,MKK6 protein expression,p38 protein expression and ATF2 protein expression between sham operation group and eye-acupuncture combined electroacupuncture group(p>0.05).There were no statistical differences in TAK1 protein expression,MKK6 protein expression,p38 protein expression and ATF2 protein expression between sham operation group and electroacupuncture group(p>0.05).There were no statistical differences in TAK1 protein expression,MKK6 protein expression,p38 protein expression and ATF2 protein expression between sham operation group and model group(p>0.05).There were no statistical differences in TAK1 protein expression,MKK6 protein expression,p38 protein expression and ATF2 protein expression between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p>0.05).There were no statistical differences in TAK1 protein expression,MKK6 protein expression,p38 protein expression and ATF2 protein expression between eye-acupuncture combined electroacupuncture group and model group(p>0.05).16.There were statistically significant differences in p-TAK1 protein expression,p-MKK6 protein expression,p-p38 protein expression and p-ATF2 protein expression between sham operation group and eye-acupuncture combined electroacupuncture group(p<0.01).There were statistically significant differences in p-TAK1 protein expression,p-MKK6 protein expression,p-p38 protein expression and p-ATF2 protein expression between sham operation group and electroacupuncture group(p<0.01).There were statistically significant differences in p-TAK1 protein expression,p-MKK6 protein expression,p-p38 protein expression and p-ATF2 protein expression between sham operation group and model group(p<0.01).There were statistically significant differences in p-TAK1 protein expression,p-MKK6 protein expression,p-p38 protein expression and p-ATF2 protein expression between eye-acupuncture combined electroacupuncture group and electroacupuncture group(p<0.01).There were statistically significant differences in p-TAK1 protein expression,p-MKK6 protein expression,p-p38 protein expression and p-ATF2 protein expression between eye-acupuncture combined electroacupuncture group and model group(p<0.01).Conclusion:The effect of eye-acupuncture combined with electroacupuncture was similar to that of electroacupuncture alone in improving the symptoms of knee stiffness and daily activities in KOA patients,but the analgesic effect of eye-acupuncture combined with electroacupuncture was superior to that of electroacupuncture alone in KOA patients.The anti-inflammatory and analgesic mechanism of eye-acupuncture combined with electroacupuncture therapy on KOA may be realized by affecting the expression of p-TAK1,p-MKK6,p-P38 and p-ATF2 in p38 MAPK signaling pathway,inhibiting the expression of IL-1β,IL-6,TNF-α and PGE2,and promoting the expression of IL-10. |
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