A Correlation Study Between Acupoint Signal Transfer And Connexin 43

Acupuncture and moxibustion, which are important ingredients of Chinese traditional medicine, have been used empirically in clinical practice in China for several millennia. Owing to its distinct operation method and marvelous therapeutic efficacy, acupuncture and moxibustion, especially acupuncture analgesia, are approved and accepted gradually by the whole world. Although a large number of previous clinical studies also support the effectiveness of acupuncture, little is known about the intercellular transfer pattern of acupoint signal and the mechanism of the effect created by acupuncture. And the specific material in acupuncture points and meridians still remains mysterious.Gap junctions, which provide low resistance pathways and a possibility for direct coupling of cells to be organized into functional units, are ubiquitous specialized plasma membrane domains composed of collections of the only channels that directly connect neighboring cells. These pathways are responsible for the cell-to-cell transfer of ions and small hydrophilic molecules, including amino acids, nucleotides and second messengers (e.g. Ca2+, cAMP, cGMP, IP3). Gap junctional intercellular communication plays an important role in embryonic development, cell differentiation, proliferation and growth control etc., which is essential for proper development and health in animals and humans. And transfer of electric signal and mechanical stimulation signal have also been attributed to gap junctional communication. Structural studies have demonstrated that each gap junctional channel is composed of a connexon (hemichannel) in the plasma membrane of one cell and a connexon in the apposing cell membrane. Each connexon, which provides a hydrophilic channel, 2 nm in diameter, is an oligomer of 6 protein subunits, termed connexins belonging to the connexins gene family. Although there are more than 20 members in vertebrates, the major components of human keratinocyte gap junctions are Cx43 and Cx26. Immunostaining for Cx43 was observed in epidermal keratinocytes, sebaceous glands, eccrine sweat ducts, and hairs, but Cx26 was only detectable in the two latter epidermal adnexae.On the basis of above facts and our previous research work, we put forward a hypothesis: gap junctions between adjacent cells in skin and subcutaneous layer, which provide low resistance pathways, are the major channels for acupoint signal transfer; gap junctional intercellular communication are the main transfer pattern; intercellular and interstitial fluid are the buffer system. Gap junctions and gap junctional intercellular communication may be activated when the acupoints received stimulation, and signal might be transferred directly through gap junctions along the low resistance area in the co-effect of nerve-humoral factor. The cerebrum might integrate the signal, and there is an intercom among the acupoint, cerebrum and target organ. Therefore, we designed a program to research the correlation between acupoint signal transfer and Cx43. First, to ascertain the structure foundation of connexins in acupoints and meridian, we studied the expression of Cx43 in rat acupoints and meridians, and observed the change after acupuncture (Part I). Secondly, we employed RNA interference technology to construct shRNA plasmid expression vectors to silence the expression of Cx43 (Part II). Third, we injected the vector targeting Cx43 with a better silence effect locally into the acupoint and observed the different acupuncture effect of each group. Part I: Experimental Study of Expression of Connexin 43 in Rat Acupoint and Meridian and the Change after Acupuncture1. Experimental Study of Expression of Connexin 43 in Rat Meridian Objective:To study the expression of connexin 43 in rat Kidney and Bladder meridians and in control lines, and approach the possible relationship between connexin 43 and meridians.Methods:Young adult female Sprague-dawley (SD) rats (210～230g, n=10) were employed. PowerVisionTM two step immunoenzyme technic and ASIAS-2000 automatic image-scan analyzing system was used to detect the distribution and Western Blot was performed to detect the quantity of Cx43 in rat Kidney and Bladder meridians and in control lines.Results:Cx43 was expressed mainly in keratinocytes in spinous layer and some unknown cells in subcutaneous layer and in the dermis. Statistical analysis showed that the expression of Cx43 in Kidney and Bladder meridians was distinctly more than in the controls respectively (P<0.05).Conclusion:Cx43 expression was much abundant in meridians than in the controls, implying there is a relationship between Cx43 and meridians, and gap junctional intercellular communication might be involved in meridian action. 2. The Effect of Acupuncture on the Expression of Cx43 at Rat Acupoint ST36Objective:To probe into the expression of connexin 43 in the effect of acupuncture at rat ST36 acupoint, and discuss the possible relationship among Cx43, acupoint and acupuncture.Methods:Young adult female Sprague-dawley (SD) rats (210～230g, n=20) were divided randomly into two groups: non-acupuncture group (n=10) and acupuncture group (n=10). PowerVisionTM two step immunoenzyme technic and ASIAS-2000 automatic image-scan analyzing system was used to detect the distribution in ST36, RT-PCR and Western Blot was performed to detect Cx43 mRNA and protein level in the acupoints of each group.Results:Cx43 was expressed mainly in keratinocytes in spinous layer and some unknown cells in subcutaneous layer and in the dermis. There were aggregated positive cells for Cx43 immunostaining in rat ST36, especially in the subcutaneous layer, and even more so after acupuncture. In non-acupuncture group, the expression of Cx43 mRNA and protein level in acupoints was significantly higher than in the non-acupoints respectively (P<0.01). After acupuncture, the expression of Cx43 in acupoint was obviously increased, which formed a striking contrast to the acupoint in the non-acupuncture group (P<0.05).Conclusion:Cx43 expression was much abundant in acupoints than in the controls, and acupuncture could significantly increase the Cx43 mRNA and protein level at acupoint. Part II:Construct an shRNA Expression Vector Targeting Cx43 and Identify the Efficiency of RNA Interference in vitroObjective:To construct an shRNA expression vector targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines.Methods:Aiming directly at the two target of Cx43 mRNA sequence of rat and mouse homology region, we synthetize two pair complementary oligonucleotide strands in vitro. Double strands were formed after annealing, which were then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombined plasmids named P-Cx43-shRNA (1), P-Cx43-shRNA (2) and P-con-shRNA were transfected into NIH/3T3 cells. RT-PCR and Western Blot were used to detect the effect of the plasmid on the mRNA and protein level of Cx43 after screened by G418.Results: The results of enzyme cutting and sequencing showed that we successfully construct two shRNA expression vectors targeting Cx43 and a control expression vector for rat and mouse. The Cx43 mRNA level was decreased 68.6%(P<0.01)and 16.3% by P-Cx43-shRNA (1) and P-Cx43-shRNA (2) respectively in NIH/3T3 cells, and the Cx43 protein level was decreased 73.5%(P<0.01)and 10.8% accordingly. The Cx43 mRNA and protein level were not influenced by transfection of P-con-shRNA.Conclusion : The plasmid P-Cx43-shRNA (1) can specificly silence better the expression of Cx43 in NIH/3T3 cells. Part III: The Influence on Acupuncture Effect of Silencing the Expression of Cx43 in AcupointObjective:To investigate the influence on acupuncture effect of silencing the expression of Cx43 in acupoint.Methods:We used mifepristone to result in the model of nidation disorder in rat, and RNA interference technology to silence the expression of Cx43 in acupoint. The mated female rats were randomly divided into five groups (n=8 of each group): Normal (control), Model (mifepristone), Acupuncture (Mife+Acupunc.), Interference (Mife+P-Cx43-shRNA (1)+Acupunc.) and Interference-con (Mife+P-con-shRNA+Acupunc.). Acupuncture was performed in the acupoints"Housanli","Sanyinjiao"and"Taichong"in Acupuncture, Interference and Interference-con group from the first day of pregnancy for seven consecutive days. P-Cx43-shRNA (1) was injected locally into the acupoints in Interference group, and P-con-shRNA was used in Interference-con group. On the d8 of pregnancy, the quality of blastocyst, pregnancy rate, average amount of blastocyst and hormone level (E2, P4, PRL) in serum of each group were observed.Results:①The mRNA and protein level of Cx43 in acupoints in Interference group were significantly lower than in the Acupuncture Group (P<0.05,P<0.01), there was no significant difference between Interference-con and Acupuncture group.②Compared with Normal group, the quality of blastocyst in Model group was bad,the size was comparatively small and inhomogeneous with maldistribution and poor blood supply. The results in acupuncture group and Interference-con group were similar to the Normal. The results in Interference group were similar to the Model group.③The pregnancy rate in Normal, Model, Acupuncture, Interference and Interference-con group was 100%, 50%, 75%, 50%, 75% respectively, but the difference was not statistically significant.④The average amount of blastocyst in Model and Interference group were significantly lesser than in Normal group(P<0.01). Compared with Acupuncture group, the average amount of blastocyst in Interference group was lower (P<0.05).⑤The serum PRL level in Model and Interference group was significantly lower than in Normal group (P<0.05) ; Compared with Interference group, the serum PRL level in Interference-con group was higher (P<0.05). The difference of the serum E2 and P4 level (E2, P4) in each group was not statistically significant.Conclusion:Acupuncture could improve the blastocyst quality in the model of nidation disorder of rat, and have the tendency of raising the average amount of blastocyst. Local injection of Cx43 shRNA expression vetor could silence the expression of Cx43 in acupoint and markedly influence acupuncture effect, demonstrating Cx43 is involved in acupuncture effect.