To explore the relationship between sensitization points of the body surface and the expression of pituitary adenylate cyclase activating polypeptides (PACAP) in myocardial ischemia (MI) mice, so as to reveal the underlying mechanisms of acupoint sensitization from the perspective of molecular biology.

Male C57BL/6J mice were randomly divided into control and model groups (28 mice/group). The MI-induced visceral pain model was established by intraperitoneal injection of isoprenaline (ISO, 160 mg/kg). The mice of the control group received intraperitoneal injection of the same dose of normal saline. Six days after modeling, the Evans blue (EB) dye was injected into the tail vein of mice to observe the distribution and quantity of the plasma extravasated EB points at the body surface. Meanwhile, the mechanical pain threshold (MPT) was measured to evaluate the level of pain sensitivity in the activated area on their body surface and left forelimb and hindlimb, respectively. Hematoxylin-eosin (H. E.) staining was used to evaluate the morphologic and pathological changes of the heart tissue in the two groups. Then, the expressions of PACAP in the thoracic (T)1—T5 dorsal root ganglia (DRGs), spinal cord and skin in the dominant area of body surface were detected by Western blot and immunofluorescence staining, respectively.

Compared with the control group, the heart tissue of the model group was hypertrophic and the myocardial tissue showed obvious inflammatory cell infiltration and fibrosis. In addition to these pathologic changes, the number of EB exudation points on the body surface was significantly increased (P<0.01), and was mainly distributed in the innervated region of T1—T5 segments of the spinal cord, and the MPT of these EB exudation points was lower than that of non-exudation points (P<0.01). At the same time, the MPTs of left forelimb and hindlimb were significantly decreased in the model group (P<0.001). More importantly, the level of protein and positive expression of PACAP were significantly higher in the model group than those in the control group, which was observed in the innervated body surface, spinal cord and its DRG neurons of T1—T5 segments (P<0.01, P<0.001, P<0.05).

ISO injection resulted in histological lesions and cardiogenic referred pain on the body surface after the formation of MI in mice. The expression of PACAP in the body surface of the sensitization points, the corresponding T1—T5 segments of spinal cord and DRG neurons were significantly increased, which may partly explain the reason for acupoint sensitization in the animal model of visceral pain.

To elucidate the relationship between the auricular point stimulation and the activity of glutamatergic neurons in the anterior cingulate cortex (ACC) from the perspective of intrinsic excitability plasticity of neurons in mice with posttraumatic stress disorder (PTSD), so as to explore the underlying mechanisms of acupuncture at auricular points in improving emotional diseases induced by PTSD.

C57BL/6J male mice were randomly divided into control, PTSD model, sham electroacupuncture (EA), and EA groups, with 5 mice in each group. The glutamatergic neurons were labelled by injection of AAV2/9-CaMK Ⅱ α-EGFP viral fluid into the bilateral ACC. Fourteen days after the injection, the PTSD model was established by single prolonged stress (restraint stress, forced swimming, ether exposure) and plantar electrical shock. EA (2 Hz/15 Hz, 1 mA) was applied to bilateral "Xin" points and the center of auriculae of auricular concha area for 30 min, once daily for 7 days. Mice of the sham EA group were anesthetized for 30 min per day for 7 days, but no EA treatments were given. The anxiety-like behavior of mice was evaluated by open field (OF) and elevated plus maze (EPM) tests after 2 weeks of modeling. The whole-cell patchclamp method was used to record the intrinsic excitability level of ACC glutamatergic neurons.

Compared with the control group, the dwell time and locomotor distance in the central area of the OF, the dwell time and entry times percentage in the open arms of the EPM in the model group were significantly reduced (P < 0.001, P < 0.01). At the same time, the intrinsic excitability of ACC glutamatergic neurons in model mice was suppressed, presenting as enhanced rheobase currents (P < 0.01) and decreased spike number (P < 0.05, P < 0.01, P < 0.001). Compared with the model group, the dwell time and locomotor distance in the central area of the OF, the dwell time and entry times percentage in the open arms of the elevated plus maze were significantly increased (P < 0.001, P < 0.01), and the intrinsic excitability of ACC glutamatergic neurons was significantly improved, presenting as reduced rheobase currents (P < 0.05) and an increased spike number (P < 0.05) in the EA group. Compared with the sham EA group, the dwell time and locomotor distance in the central area of the OF, the dwell time and entry times percentage in the open arms of the EPM were significantly increased (P < 0.001, P < 0.01) in the EA group, and the intrinsic excitability of ACC glutamatergic neurons was significantly improved, presenting as reduced rheobase currents (P < 0.01) and an increased spike number (P < 0.05).

EA of auricular points can effectively alleviate the anxiety-like behavior of PTSD mice and increase the intrinsic excitability level of glutamatergic neurons in ACC, which may be one of the neural mechanisms of auricular point stimulation in the treatment of emotional diseases.

To investigate whether electroacupuncture (EA) at “Yanglingquan” (GV34) and “Xuanzhong” (GB34) can alleviate anxio-depression-like behaviors induced by neuropathic pain and whether the effect of EA is in connection with the activity of glutamatergic neurons in the ventrolateral orbital cortex (VLO).

The neuropathic pain model was established by common peroneal nerve ligation (CPNL). GV34 and GB34 were selected in EA treatment (2 Hz/15 Hz, 1 mA), 15 min each time, once a day for 7 d. Chemogenetic mixture viruses rAAV-VGLUT1-CRE-WPREs and rAAV-Ef1a-DIO-hM3Dq-mCherry-WPREs or rAAV-Ef1a-DIO-hM4Di-mCherry-WPREs were injected into the VLO, respectively. Clozapine-N-oxide (CNO) was injected intraperitoneally to regulate the activity of glutamatergic neurons in the VLO precisely. Mechanical withdrawal threshold (MWT) and paw withdrawal latency (PWL) were measured by dynamic plantar tactile instruments and plantar thermal nociceptor instruments. Anxiodepression-like behaviors were measured by behavioral tests, including open field test, elevated plus maze test, forced swimming test, and tail suspension test. The activation and inhibition levels of neurons were observed by immunofluorescence staining.

Compared with the sham group, the MWT and PWL of CPNL mice were significantly decreased (P<0.001) on the 3^rd day after surgery and lasted until the 28^th day. After CPNL, the activity time spent in the central area of open field test reduced (P<0.001), the percentage of entries and activity time in the open arm of elevated plus maze test were significantly reduced (P<0.001, P<0.05) ；the immobile duration of forced swimming test and tail suspension test were remarkably prolonged (P<0.01, P<0.05) of mice in the CPNL group. However, after EA treatment, compared with the CPNL group, the anxio-depression-like behaviors of the mice were alleviated (P<0.05, P<0.01, P<0.001). Activating glutamatergic neurons of VLO produced an anti-anxio-depression effect (P<0.05, P<0.01) in neuropathic pain mice. Compared with the inhibited+EA+saline group, inhibiting glutamatergic neurons of VLO reversed (P<0.001, P<0.01, P<0.05) the effect of EA.

EA at GV34 and GB34 can alleviate anxio-depression-like behaviors induced by neuropathic pain through the modulation of glutamatergic neuron activity in the VLO.

To analyze the rules of acupoint selection and compatibility of acupuncture and moxibustion in treatment of inflammatory bowel disease(IBD) based on complex network technology and provide the reliable evidences for acupoint selection in treatment of this disease with acupuncture and moxibustion.

The clinical studies on acupuncture-moxibustion treatment of IBD were searched from the databases including CNKI, Wanfang, VIP, PubMed and Embase. The studies were screened and the acupoint prescriptions were extracted to set up the database of acupuncture-moxibustion treatment for IBD. Using Microsoft Excel 2021 software, the use times of acupoint, the use frequency(%) of acupoint, meridian tropism and the use of special point were imported. With SPSS Modeler 18.0 software adopted, the association rules were analyzed on the acupoint prescriptions. The acupoint co-occurrence network diagram, k-core network diagram, and community analysis diagram were drawn by Gephi 0.9.5 software.

A total of 156 studies were included, composed of 175 acupoint prescriptions, 75 acupoints, with 1378 use times in total and around 8 acupoints in one prescription. Regarding the top use frequency, Tianshu(ST25), Zusanli(ST36), Guanyuan(CV4), Zhongwan(CV12) and Pishu(BL20) were listed. The top meridians involved were the foot-yangming stomach meridian, the foot-taiyang bladder meridian and the Conception Vessel. The front-mu point had been used with the highest frequency among the special points. ST36 and ST25 were a pair of points with the highest frequency in treatment. The k-core hierarchical analysis was adopted to optimize acupoint prescriptions, and 22 core acupoints were obtained, i.e. ST25, ST36, CV4, CV12, BL20, Dachangshu(BL25), Shangjuxu(ST37), Shenshu(BL23), Qihai(CV6), Sanyinjiao(SP6), Mingmen(GV4), Xingjian(LR2), Yinlingquan(SP9), Neiting(ST44), Taichong(LR3), Xiajuxu(ST39), Shuifen(CV9), Shenque(CV8), Ganshu(BL18), Weishu(BL21), Hegu(LI4) and Quchi(LI11), which were classified into three core acupoint groups by community analysis.

Through complex network analysis, it is found that the local acupoints on the chest and abdomen are generally selected in treatment with acupuncture-moxibustion for IBD, the combination of the nearby and distal points is considered simultaneously, and the acupoint prescription is modified according to syndrome/pattern differentiation; and among special points, the front-mu point is widely used in treatment. All of these rules provide the ideas for the acupoint selection of acupuncture-moxibustion in treatment of IBD.