Sheng-Xing Ma, M.D., Ph.D. University of California Los Angeles David Geffen School of Medicine

1. Biochemical Physiology of Nitric Oxide-cGMP on the Skin Acupuncture Points and Meridians: An Innovative Approach in Humans Sheng-Xing Ma, M.D., Ph.D. University of California Los Angeles David Geffen School of Medicine Harbor-UCLA Medical Center

4. Question • Do meridian systems exist in our body? • What are the chemicals, structures, and functions of acupuncture points/meridians?

5. Background • Acupuncture points (acupoints)/meridians possess characteristics of low electrical resistance. • Higher neural components, hair follicles, and sweat glands exist at acupoints. • Skin electrical resistance depends upon the activity of the sympathetic nervous system. • NO stimulates norepinephrine release from the central and peripheral nervous systems, which increases sympathetic functions.

6. 经穴一氧化氮浓度及其合成酶表达的变化

7. Goals • Determine if NO can be collected and quantified from the skin surfaces of humans. • Examine the influences of hot or electrical stimuli on NO-cGMP levels in acupoints/meridians.

8. 工欲善其事 必先利其器 The essence of science is method

9. Synthesis, Biomedical Activities, Reactions, and Metabolism of NO-cGMP

10. Do NO Concentrations along the Skin Surface of Acupoints Differ from Meridian Lines without Acupoints and Non-Meridian Controls? • The Pericardium meridian (PC) points 4 to 7 were defined as meridian with points. • The distance between PC 3 to 4 were defined as meridian without point (MWOP). • Controls were obtained in non-meridian areas close to the PC Meridian Without Point Non- Meridian control Meridian With Point

11. The Bladder Meridian (BL) points 56 and 57 were defined as meridian with point • Controls were obtained in non-meridian areas close toBL Meridian With Point Non- Meridian control

12. Characteristics of Participants in the Study (n = 125)

13. NOx- Levels along the PC and BL Meridians Were Increased vs. Control following NO Collecting Solution on Skin Surface for 20 Minutes in Humans

14. Illustrates the Targeted Areas for NO-cGMP Collection from the Skin Surface over the Lung Meridian LU-C indicates non-meridian control. LU-WO represents the distance between LU 6 to 7 as meridian line without acupoint. LU-P corresponds to the region from LU 8 to 10 defined as acupoint (3 acupoints). LU-11 denotes acupoint in which hot stimulation is applied.

15. Without Hot Side 14 * Hot Side # * p < 0.05 vs. Without Hot Side # p < 0.05 vs. Without Acupoint 12 10 8 6 4 2 0 Non-meridian Control Without Acupoint Acupoint Hot Stimulation-Induced Nitric Oxide Release over LU Acupoint NOx- Concentrations (µM/cm²)

16. Without Stimulation 1.2 Hot Stimulation * p<0.05 vs. Non-Meridian Control # * #p<0.05 vs. Acupoint without Stimulation 1.0 0.8 cGMP Concentration (nM/cm2) 0.6 0.4 0.2 0.0 Non-Meridian Control Acupoint Lung Meridian Concentrations of cGMP on Acupoints and Non-meridian Regions in Lung Meridian following Hot Stimulation in Humans * * # p<0.05 vs. Non-Meridian Control # p<0.05 vs. Acupoint without Stimulation

17. Illustrates the Targeted Areas for NO-cGMP Collection from the Skin Surface over the Pericardium Meridian • PC-C indicates non-meridian control as non-meridian area adjacent to the meridian. PC-WO represents the distance between PC 3 to 4 as meridian line without acupoint. PC-P corresponds to the region from PC 4 to 7 defined as acupoint. PC-8 denotes acupoint in which electrical stimulation is applied.

18. 7 Without Stimulation Stimulation Side * # 6 * p< 0.05 vs. Without Stimulation # p< 0.05 vs. Without Acupoint 5 4 NOx -Concentrations (μM/cm2) 3 2 1 0 Without Acupoint Non-meridian Control Acupoint Electrical Stimulation-Induced Nitric Oxide Release over PC Acupoint

19. Without Stimulation Electrical Stimulation *p<0.05 vs. Non-Meridian Control # p<0.05 vs. Acupoint without Stimulation 1 # * 0.8 0.6 cGMP Concentration (nM/cm2) 0.4 0.2 0 Non-Meridian Control Acupoint Pericardium Meridian Concentrations of cGMP on Acupoints and Non-meridian Regions in PC meridian following Electrical Stimulation in Humans * p<0.05 vs. Non-Meridian # p<0.05 vs. Acupoint without # *

20. Summary • NO contents are significantly increased over PC with 4 acupoints and tend to increase over BL with 2 acupoints in humans. • NO and cGMP levels of the LU acupoint receiving hot stimulation were significantly higher than those of the non-meridian control receiving hot stimulation. • NO and cGMP levels were significantly increased in the PC acupoint by electric stimulation but not altered in non-meridian control area.

21. Goals • Determine responses of NO release in dermis and subcutaneous tissue of acupoints/meridians to electroacupuncture in humans. • Define whether NO release is acupoint/meridian specific.

23. Characteristics of Participants in the Study (n = 24)

24. EA Stimulation-Response Curves of Dialysate NOx- from PC4 and Non-meridian Dialysis in Healthy Volunteers The dialysate was collected in 20-minute intervals for 2 hours. The PC dialysis control group did not receive EA stimulation (n=8). The electro-acupuncture stimulation (EA) was applied to PC3 in the acupoint and dialysate was collected from PC 4 (n=13). Eight subjects were received EA, but the dialysate was collected from the non-meridian point.

25. Effect of the Electroacupuncture Stimulation on Dialysate NO Concentration The percentage change of NO concentration was calculated based on the differences of the NO concentrations during the EA, 20 min, 40 min, and 60 min after EA and the basal value of the second collection at 20 minutes after the dialysis. The NO concentration of PC 4 dialysis was increased at 20 min after the EA stimulation vs. non-meridian dialysis (*P < 0.05) and the PC 4 dialysis control group (#P = 0.077).

26. Effect of Electroacupuncture (EA) Stimulation on Dialysate cGMP Concentration The concentration of cGMP was measured from the dialysate collected from the non-meridian dialysis (n = 8) and PC4 acupoint dialysis (n = 13) after EA stimulation. The mean basal level of cGMP in the absence of EA stimulation was used to calculate the percentage change in cGMP. The cGMP concentration of the acupoint dialysate was significantly increased after the EA stimulation compared to this of the non-meridian dialysate (t test; P < 0.05).

27. Summary • There is no difference of the baseline NO and cGMP levels between the acupoints and non-meridian points. • Nitric oxide and cGMP concentrations in acupoints of the pericardium meridian after EA stimulation were significantly higher than those in the no EA and non-meridian controls .

28. Conclusion • These results show that NO-cGMP can be successfully collected from skin acupoints/ meridians. • NO-cGMP is predominantly released/ generated from the skin acupoints by heating, EA and electrical stimulation. NO-cGMP is physiologically released over and in the skin with a high level at acupoints. Stimulus-evoked NO-cGMP release is acupoint/meridian specific, which plays an important role in in signal transduction of meridian stimulations and communication.

29. Represents the Relationshipbetween NO and the Meridian System with Respect to the Environment and Universe

31. 研 究 意 义 • 经穴一氧化氮及其信使传递化合物可以定量测定，并具有可重复性，与经络理论在逻辑上相吻合 • 对古典经络学和现代医学都是一种创新 • 揭示了中医药学的核心理论之一——经络学与现代生物化学、生理学是相互兼容，并相互联系的 由此全面展开该领域的研究 将为中医药科学化、现代化领航

32. This work was supported by grants AT002478 and AT00450 from National Center for Complementary and Alternative Medicine National Institutes of Health U.S. Department of Health and Human Services www.nccam.nih.gov

33. ACKNOWLEDGMENTS Funded Research Grants Principal Investigator: Sheng-Xing Ma R01 AT002478 -- NIH/National Center for CAM R01 AT00450 – NIH/ National Center for CAM KO2 HL04447 – NIH/ Nat'l Heart, Lung & Blood Inst R03 HD39169 – NIH/ National Inst. of Child & Human Dev. R21 AT00209 -- NIH/National Center for CAM Research Award RA0049 -- American Diabetes Association Grant-in-Aid (9950708Y)-- American Heart Association-WS Grant-in-Aid (96011320) -- American Heart Association Grant-in-Aid (94-GS-52) -- American Heart Association-IA

35. Thank you!