A study of acupoint specificity and mechanism of electroacupuncture intervention on chronic colitis in rats based on PI3K/AKT/mTOR signaling pathway

TANG Kun-peng, LV Jia-qi, WEN Tan, ZHANG Chun-qing, MA Meng-na, REN Hua-shan,YAN Li-ping

Shanxi University of Chinese Medicine, Jinzhong, Shanxi 030619, China

Keywords:

ABSTRACT Objective: This study aimed to elucidate the differences in effects and mechanisms of action of electric-needle therapy at Tian Shu (ST25), Da Chang Shu (BL25), Zu San Li (ST36)and Shang Ju Xu (ST37) acupoints on chronic experimental colitis in rats through the PI3K/AKT/mTOR signaling pathway.Methods: Sixty pathogen-free SD rats were randomly assigned to six groups: the normal, model, Tian Shu (ST25), Da Chang Shu (BL25), Zu San Li (ST36) and Shang Ju Xu (ST37) groups, each with 10 rats.Chronic colitis was induced in rats by combining immunization and local stimulation.After model establishment, electrical needle intervention combined with dispersing wave of 2 Hz/50 Hz with a current intensity of 2 mA once daily for 20 min was applied on acupoints of each group.Subsequently, the inflammation of colonic mucosa and serum levels of inflammatory factors (IL-23, IL-17, IL-10) were observed; ELISA was used to detect mRNA expressions of PI3K, Akt and mTOR in colitic tissues by RT-PCR as well as protein content of p-PI3k/PI3K, p-Akt/Akt, and p-mTOR/mTOR in colitic tissues by Western blotting.Result: Compared with the normal group, the model rats showed a poor general condition, serious damage to the colonic mucosa with a large number of inflammatory cells infiltration.The serum IL-23 and IL-17 expressions were significantly increased (P ＜ 0.01), while the serum IL-10 expression was significantly decreased (P＜0.01); the mRNA and protein expressions of PI3K, Akt, mTOR and p-PI3K,p-Akt and p-mTOR were significantly increased (P＜0.05, P＜0.01).Compared with the model group, the pathological slices of rats in each acupoints intervention group showed obvious improvement of colitis inflammatory reaction and tissue damage; the serological levels of IL-23 and IL-17 were significantly reduced (P ＜ 0.01), while the serology level of IL-10 was significantly increased (P ＜ 0.01); the expressions of PI3K, Akt, mTOR mRNA and p-PI3K,p-Akt, p-mTOR proteins were significantly decreased (P＜0.05, P＜0.01).Compared with Tian Shu (ST25), Da Chang Shu (BL25), Zu San Li (ST36) groups, the recovery degree of mucosa layers in Shang Ju Xu (ST37) group was closer to that of normal group, and the curative effect was relatively the best; in terms of serological levels of IL-23 and IL-17, the Shang Ju Xu (ST37) group was significantly lower (P＜0.05), while the level of IL-10 was significantly higher (P＜0.01); the expressions of PI3K, Akt, mTOR mRNA and p-PI3K, p-Akt, p-mTOR proteins were significantly decreased (P＜0.05, P＜0.01).Conclusion: Results indicate that electrical acupuncture at Tian Shu (ST25), Da Chang Shu (BL25), Zu San Li (ST36) and Shang Ju Xu(ST37) show similar effects in relieving the colitis-induced damage in the mucosa of chronic colitis rats, as well as inflammatory response.Among them, Shang Ju Xu(ST25) has a superior overall effect in treating chronic colitis compared to Tian Shu (ST25) , Da Chang Shu (BL25) and Zu San Li (ST36).The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.

1.Introduction

Inflammatory Bowel Disease (IBD) is a type of chronic nonspecific inflammatory intestinal disorder[1].Its course is long and its condition hard to cure, usually manifested as periods of acute exacerbations alternating with periods of chronic remission.In recent years, its incidence has been increasing year by year in our country, and is mostly seen in teenagers[2].At present, it is generally believed that its onset is closely related to intestinal inflammation,damage to homeostatic equilibrium, immune response disorder and damage to the epithelial barrier[3,4].Studies have found that the Phosphatidylinositol 3 Kinase (PI3K)/Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Signal Pathway can regulate inflammatory mediators and inflammatory cells, mediate the inflammatory response caused by immune abnormality, and play an important role in relieving colitis.

A large body of research has found that electroacupuncture can effectively control IBD clinical symptoms and intestinal inflammation through multi-stage and multi-target approaches while having low side effects, low recurrence, and long-term good effects,gradually becoming an important auxiliary means for supplementary or alternative drug treatment[5].The four specific points - Tian Shu,Da Chang Shu, Zu San Li, and Shang Ju Xu - are acupuncture points with high frequencies used in the treatment of IBD, all of which have good therapeutic effects[6].Our previous experimental studies found that[7, 8] electroacupuncture of the four points has a different effect on regulating serum IL-6 and myeloperoxidase (MPO) levels of rats with acute/chronic colitis, but the specific mechanism remains unclear.Therefore, this experiment is based on the detection of relevant protein content and inflammatory factors in the PI3K/Akt/mTOR signal pathway, further exploring the differences in the effect of stimulating these four groups of specific acupoints on intestinal chronic inflammation and its mechanism.

2.Materials and methods

2.1 Experimental animals and grouping

Six-week-old SD rats (60, female/male ratio half-half, body weight 200 ± 20 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co.Ltd.[License No.: SCXK(Beijing) 2016-0006], housed in the clean animal room of the Research Experiment Center of Shanxi University of Chinese Medicine, maintained in a temperature of (24 ± 2) ℃ and relative humidity of 50% to 60%, with a light cycle simulating day-night alternation of 12/12 h.They were freely fed and watered for adaptation, and randomly divided into normal group, model group,Tian Shu group, Da Chang Shu group, Zu San Li group, and Shang Ju Xu group with each group containing 10 rats after one week.This experimental scheme was approved by the Ethics Committee of Shanxi University of Chinese Medicine (Ethics Code: 2018DW181).

2.2 Main reagents and instruments

Sigma Complete Freund’s Adjuvant (CFA, F5881), Supersensitive ECL Luminescent Solution (Beijing Solaybio Technology Co.,Ltd., Product No.MA0186-1), Trizol (Ambion, USA, 190906);Reverse transcription Kit (TaKaRa, Japan, RR047A), BCA Protein Concentration Detection Kit, PCR Reagent Kit (Beyotime, Shanghai,P0012S,D7281S); PMSF Proteinase Inhibitor, SDS-PAGE Protein Sampling Buffer 5 × (Bosterbio, Wuhan, batch numbers AR1179,AR1112), Rat IL-17, IL-23, IL-10 ELISA Kit (Boster Engineering Co., Ltd., Wuhan, batch numbers EK0431, EK0866, EK0417), PI3K Antibody, Phosphorylation (p)-PI3K, Akt Antibody; p-Akt Antibody,mTOR Antibody, p-mTOR Antibody (Cell Signaling Technology,USA, batch numbers 4292S, 4228S, 4691S, 4060S, 2983S, 5536S);Goat Anti-Rabbit IgG-HRP, β-Actin Mouse Anti (Beijing Baoao Biological Technology Co., Ltd.), Color PAGE Fast Gel Kit (Abe Sign Bio Technology Co., Ltd., product number abs9367, abs9365).Huatuo Brand SDZ-Ⅱ Electroacupuncture Instrument, Disposable Sterile Acupuncture Needles (Beijing Zhongyan Taihe), High Speed Refrigerated Centrifuge (Xi Ma Centrifuge Co., Ltd.), Enzyme Labeler (BioTek, USA, model ELX808), T100TM Thermal Cycler Real Time Fluorescence Quantitative PCR Instrument, Tanon5200 Chemiluminescence Imaging Instrument (Bio Tek, USA),DYY-6C Electrophoresis Instrument (Beijing Liu Yi).

2.3 Model preparation

Immune Complex Sensitization Method[9] was used to prepare a chronic colitis rat model.Fresh rabbit colonic mucosa was suspended in an appropriate volume of 0.9% NaCl solution and centrifuged, and the supernatant was stored in a -80 ℃ refrigerator.During modeling, the antigen supernatant and CFA were mixed in equal proportions to prepare an antigen emulsion, which was then injected into rat ankle (3rd day), toe (10th day), back subcutaneously(17th day), inguinal grooves (24th day), and peritoneum (31st day).When the serum antibody titer against rabbit colon reached a certain titer, the model group and each acupoint treatment group were fasted for 24 h without water.Then, 2% pentobarbital sodium (3 mL/kg) was administered intraperitoneally for anesthesia, and 2%furamoline solution was used with a soft guide tube to enema for 60 minand then rinsed with saline.After that, antigen solution without CFA was administered by enema for 2 h and subsequently rinsed with saline to complete the modeling.Pathological examination of colonic specimens was performed on one rat per group to verify the successful modeling of the model group[10].For the control group,saline was used instead of the modeling reagents, and the same procedure was performed in parallel.

2.4 Intervention methods

Acupoint localization was guided by “Experimental Acupuncture”[11].The bilateral acupoints of “Tianshu (ST25)”, “Dachangshu(BL25)”, “Zusanli (ST36)” and “Shangjuxu (ST37)” were selected.A 0.18 X 25 mm needle was inserted perpendicularly 5 mm deep into each acupoint of the treatment group, connecting the same electrode with the same side acupoint.The frequency was 2 HZ /50 HZ and the intensity was 2 mA.The rat muscle or needle handle was vibrated accordingly.Each session was 20 min long and was conducted once a day.The treatment started after the modeling was successful and lasted for 10 days.Blank control group and Model group were fixed in the same way but no treatment was given.

2.5 Take samples and index tests

After all the rats were deeply anesthetized with 2% pentobarbital sodium (3 mL/kg), their abdomen was fully exposed and abdominal aortic blood was collected and centrifuged.The samples were stored in a -20 ℃ refrigerator for ELISA detection.After decapitation of the rats, the colon tissues were washed with physiological saline,part of them were stored in the tissue fixative at room temperature for pathological observation and the other part was rapidly frozen and stored in a -80 ℃ refrigerator for PCR and Western blotting.

2.5.1 Colonic mucosal damage and histopathology of rats by HE staining

The colonic tissues were fixed with 4% tissue fixative, dehydrated,transparentized, and then infiltrated with paraffin wax.The wax blocks were cut into 4 μm thick paraffin slices, and treated in a 60 ℃oven for dewaxing, staining, rinsing and dehydration, before finally being embedded in neutral resin.The morphological structures of the colonic tissues were observed under 40× microscopy.

2.5.2 The serum levels of IL-23, IL-17, and IL-10 were measured by ELISA in rats

The supernatant of centrifuged rat blood was thawed and tested for contents of relevant inflammatory factors in serum of rats with chronic colitis according to the instructions of ELISA kit strictly.After completion of the experiment, the enzyme-linked immunosorbent assay (ELISA) was set to 450 nm wavelength to measure the absorbance value of each hole, draw a standard curve,and calculate the concentration of each sample.

2.5.3 Real time PCR was used to determine the mRNA expression of PI3K, Akt and mTOR in colon tissues

TRIzol reagent was used to extract total RNA from the colon, which was subsequently reverse-transcribed into cDNA and amplified by PCR.The reaction procedure included 95 ℃30 s, 95 ℃ 5 s, 60 ℃ 34 s, 72 ℃ 30 s, with a total of 40 cycles.After the reaction, the melting curve was collected and the Ct value was counted.The primers were designed and synthesized by Comtech Biotechnology Co., Ltd., and the primer sequence is listed in Table 1.

Tab 1 Primer sequences

2.5.4 PI3K / p-PI3k, p-Akt / Akt, and p-mTOR / mTOR protein expression in colon tissues was determined by Western blot

100 mg of pre-frozen colonic tissue was ground in liquid nitrogen,and RIPA lysis buffer, a protease inhibitor and a phosphatase inhibitor were added according to weight and placed in an ice cold homogenate until the proteins were completely digested.The proteins were then centrifuged to collect the protein supernatant and BCA was used to quantify the samples.After determining the amount of sample, the proteins were boiled to denature them,cooled and stored at -20 ℃.According to the measured protein quantity, gelation and sample loading were performed, and then electrophoresis and transfer membranes were performed in electrophoresis solution and electrolyte solution.After washing the membrane, sealing and re-washing the membrane, anti-PI3K,p-PI3k, p-Akt, Akt, p-mTOR, mTOR, and β-actin (1:1000, 1:500,1:2000, 1:1000, 1:1000, 1:1000, 1:1000) were added and incubated at 4 °C overnight.The next day, secondary antibody solution(1:5000) was incubated at room temperature for 2 h, the membranes were washed and ECL luminescent reagents were dropped on the lanes for color exposure, and ImageJ software was used for analysis.The relative value was calculated by the target protein gray value/internal control protein gray value.

2.6 Statistical processing

The experiment data was analyzed using SPSS 26.0 statistical software, expressed as mean ± standard deviation (±s ).All data was subjected to normality test and variance homogeneity test.One-way ANOVA was used for intergroup comparison followed by LSD-t test for pairwise comparison.P＜0.05 was considered different significantly and P＜0.01 was considered extremely significantly different.

3.Results

3.1 General condition of rats after modeling

Before modeling, the body indicators of each group of rats were normal, with no obvious differences, bright and tidy coat color, quick response, and good mental state.During the modeling process, the body weight of rats decreased slowly, gradually appeared sluggish hair standing, loose stool and other symptoms, sleepiness and piled up phenomenon were more serious, after the modeling, anal area dirt and some rats appeared gross hematuria and black stool.After electroacupuncture intervention, the symptoms of each acupoints group were improved compared with the model group, such as reducing or disappearing black stool and hematuria, significantly increasing food intake and weight.

3.2 Comparison of colonic histopathology between rats in each group

Histopathological sections showed that the colon structure of rats in the blank group was intact, without any inflammation, necrosis or tissue damage; after the successful modeling, the colorectal mucosa of the model group had a large number of inflammatory cells infiltrated into the submucosal and muscular layers, the boundaries between the layers of the mucosa are not clear, and the mucous glands are necrotic and atrophic; after electroacupuncture therapy,each acupoints treatment group’s colorectal mucosal injury was reduced than that of the model group, and the mucosal epithelial injury was partially repaired, especially the Shang ju xu group had less inflammation and damage to the colon mucosa than other acupoints treatment groups.See Figure 1.

Fig 1 Colonoscopic observation of rats in each group (HE, × 40)

3.3 Serum levels of IL-23, IL-17 and IL-10 were compared between rats in each group

Comparing with the normal group, the serum IL-23 and IL-17 levels of rats in the model group were significantly increased and the level of IL-10 was significantly decreased (P＜0.01); compared with the model group, the serum IL-23 and IL-17 levels of rats in the Tian Shu, Da Chang Shu, Zu San Li, Shang Ju Xu groups were significantly decreased, and the level of IL-10 was significantly increased (P＜0.01); among them, compared with Tian Shu, Da Chang Shu, Zu San Li groups, the serum IL-23, IL-17 levels of rats in Shang Ju Xu group were significantly decreased (P＜0.05), and the level of IL-10 was significantly increased (P＜0.01); there was no statistically significant difference in the serum IL-23, IL-17, IL-10 levels between Tian Shu group, Da Chang Shu group, Zu San Li group (P＞0.05).See Table 2.

Tab 2 Comparison of serum levels of IL-23, IL-17 and IL-10 in rats from each group (pg/mL,±s ,n=9)

Tab 2 Comparison of serum levels of IL-23, IL-17 and IL-10 in rats from each group (pg/mL,±s ,n=9)

Note: Compared with the normal group, **P＜0.01; compared with the model group,##P＜ 0.01; compared with Shang Ju Xu group, ΔP＜ 0.05,ΔΔP＜0.01.

group IL-23 IL-17 IL-10 Normal 34.14±4.16 10.50±0.63 99.34±2.57 Model 102.40±8.88** 39.57±1.22** 65.38±2.83**ST25 69.48±6.14##△ 17.48±1.69##△ 77.72±7.43##△△BL25 70.61±15.31##△ 24.42±1.45##△ 79.43±3.23##△△ST36 71.01±9.70##△ 23.04±1.46##△ 78.80±3.76##△△ST37 55.68±8.82## 17.17±0.85## 89.88±3.13##F 49.85 60.776 69.78 P＜0.0001 ＜00.0001 ＜00.0001

3.4 Comparison of mRNA expression of PI3K, Akt and mTOR in colonic tissue of rats from each group

Compared with the normal group, the mRNA expression of PI3K,Akt and mTOR in the model group colon tissues was significantly increased (P＜0.01); compared with the model group, the mRNA expression of PI3K, Akt and mTOR in the colon tissues of each acupoint intervention group was significantly decreased (P＜0.05, P＜0.01); among them, compared with Tian shu, Da chang shu and Zu san li, the mRNA expression of PI3K, Akt and mTOR in the colon tissues of Shang Ju xu group was significantly decreased (P＜0.05, P＜ 0.01); there was no significant difference between Tian shu group,Da chang shu group and Zu san li group (P＞0.05).See table 3.

Tab 3 Comparison of relative expression of PI3K, Akt and mTOR mRNA in colon tissues of rats in each group(±s ,n=9)

Tab 3 Comparison of relative expression of PI3K, Akt and mTOR mRNA in colon tissues of rats in each group(±s ,n=9)

Note: Compared with the normal group, **P＜0.01; compared with the model group, #P＜ 0.05, ##P＜0.01; compared with Shang Ju Xu group, ΔP＜0.05,ΔΔP ＜0.01.

group PI3K mRNA Akt mRNA mTOR mRNA Normal 1.02±0.26 1.01±0.17 0.86±0.33 Model 5.41±0.53** 2.54±0.04** 2.49±0.14**ST25 3.39±0.51##△ 1.55±0.10##△ 1.58±0.26##△BL25 3.94±0.36#△△ 1.69±0.18##△△ 1.67±0.10##△ST36 4.10±0.67#△△ 1.71±0.07##△△ 1.64±0.18##△ST37 2.13±0.12## 1.07±0.32## 1.00±0.14##F 36.14 31.21 23.73 P＜0.0001 ＜0.0001 ＜0.0001

3.5 Comparison of the relative expression of p-PI3k / PI3K,p-Akt / Akt, and p-mTOR / mTOR proteins in colonic tissues of rats from each group

Compared with the normal group, the relative expression of p-PI3K, p-Akt and p-mTOR proteins in the colon tissues of the model group was significantly increased (P＜0.01); compared with the model group, the relative expression of p-PI3K, p-Akt and p-mTOR proteins in the colon tissues of each acupoint intervention group was significantly decreased (P＜0.01).Among them, compared with Tian shu, Da chang shu and Zu san li, the relative expression of p-PI3K, p-Akt and p-mTOR proteins in the colon tissues of Shang Ju xu group was significantly decreased (P＜0.05, P＜0.01); there was no significant difference between Tian shu group, Da chang shu group and Zu san li group (P＞0.05).See figure 2 and table 4.

Fig 2 protein expression of p-PI3k, PI3K, p-Akt, Akt, p-mTOR and mTOR in colonic tissue of rats from each group

Tab 4 Comparison of the relative expression of p-PI3k / PI3K, p-Akt / Akt, and p-mTOR / mTOR proteins in colonic tissues of rats from each group (±s ,n=9)

Tab 4 Comparison of the relative expression of p-PI3k / PI3K, p-Akt / Akt, and p-mTOR / mTOR proteins in colonic tissues of rats from each group (±s ,n=9)

Note: Compared with the normal group, **P＜0.01; compared with the model group,##P＜0.01; compared with Shang Ju Xu group, ΔP＜0.05,ΔΔP＜0.01.

group p-PI3K/PI3K p-Akt/Akt p-mTOR/mTOR Normal 0.39±0.05 0.11±0.03 0.27±0.03 Model 1.66±0.14** 1.95±0.46** 1.31±0.23**ST25 0.81±0.07##△ 0.89±0.10##△ 0.70±0.09##△BL25 0.96±0.24##△△ 1.11±0.24##△△ 0.78±0.10##△ST36 0.92±0.11##△△ 0.80±0.09##△ 0.82±0.05##△△ST37 0.46±0.05## 0.18±0.05## 0.38±0.06##F 37.77 28.44 54.836 P＜0.0001 ＜0.0001 0.000

4.Discussion

In Chinese ancient literature, there is no specific name for inflammatory bowel diseases.According to its related symptoms,it belongs to the disease categories of “intestinal obstruction”,“diarrhea”, “dysentery” and “bloody stool” in traditional Chinese medicine[12].Traditional Chinese medicine believes that this disease belongs to the syndrome of internal and external etiology, the virtual standard real syndrome[13].The causes of this disease are body spleen and stomach deficiency, disruption of transportation and transformation function, invasion of wet heat evil qi in the intestine, stagnation of Qi and blood, and damage of lipid membrane blood vessels[14].Its pathological position is located in the large intestine, but it is closely related to the spleen and stomach.If the disease is chronic, it will involve the liver and kidney.Therefore,in the treatment, it is mainly based on supplementing spleen and strengthening stomach, coordinating intestine qi and dispersing qi and blood.In this experiment, Tianshu acupoint was selected as the large intestine recruiting point, which is the closest to the large intestine in the abdomen, and Dachangshu, located at the Du meridian on the back, is also the convergence of large intestine qi.It has the effect of loosening Yangming qi and blood and stimulating large intestine qi; Zhusanli, Shangjuxu acupoints are located in the lower limbs, which belong to Yangming stomach meridian respectively, and they have the effects of adjusting spleen and stomach, passing through intestine and descending qi.They can improve various gastrointestinal diseases such as diarrhea,abdominal distension, dysentery and bloody mucus.The results of this experiment showed that after modeling, the body weight of rats decreased, the stool became thin and soft with mucopurulent bobbies, and the perianal region was generally polluted; pathological sections showed that hemorrhage, ulceration and a large number of inflammatory cells infiltrated, glands disappeared, mucosa disappeared, suggesting successful model preparation.After acupuncture stimulation, the general condition of each acupoint group was improved; the pathological section showed that the number of inflammatory cells decreased and the mucosal layers were restored to varying degrees.Among them, in the Shangjuxu group,the inflammation was significantly relieved than other acupoint groups, and the gland arrangement was close to the normal group.

Modern medicine research has revealed that the onset of IBD is influenced by multiple factors, involving the involvement of numerous cells and their secreted cytokines.This leads to the impairment of the intestinal mucosal immune barrier through the participation of multiple inflammatory signaling pathways, mainly manifested as intestinal mucosal inflammation and ulceration[15,16].Studies suggest that blocking of the PI3K/Akt/mTOR pathway can inhibit the activation of nuclear factor kappa B (NF-κB),reducing the secretion and expression of relevant inflammatory cells, thus ameliorating the damages of the inflammatory response in the intestinal mucosa[17, 18, 19].PI3K is an intracellular enzyme which can catalyze the phosphorylation of membrane lipids, and its second messenger PIP3 can serve as an anchoring point for the AKT protein with PH structural domain, promoting the phosphorylation of threonine (Thr308) and serine (Ser473) on AKT under the action of lipid-dependent protein kinases 1,2 (PDK1, 2)[20], and the activated AKT can exhibit anti-inflammatory effects by regulating downstream molecules such as mTOR[21], and the activated mTOR plays a vital role in intestinal inflammation by regulating autophagy[22].At the same time, PI3K can also promote the expression and secretion of IL-6, IL-23 and other cell factors by driving the activation of NFκB[23].Additionally, studies have also found that PI3K signaling can boost the production of anti-inflammatory factor IL-10 that is mediated by Toll-like receptors (TLRs) during the inflammatory response[24].Moreover, the PI3K/Akt/mTOR pathway may also lead to the occurrence of IBD by regulating CD4+T cells and activating Th17 cells to secrete IL-17[25].

The results of this experimental study showed that compared with the normal group, the mRNA of PI3K, Akt, mTOR and p-PI3K,p-Akt, p-mTOR proteins in the colonic tissue of model group rats were increased, and at the same time, the levels of IL-23, IL-17 in serum were increased while the level of IL-10 decreased.Compared with the model group, after electroacupuncture intervention,the levels of IL-23, IL-17 in the serum of rats in each acupoint treatment group were reduced, the level of IL-10 increased, and the mRNA and protein expression of PI3K, Akt, mTOR and p-PI3K,p-Akt and p-mTOR in the colonic tissue were reduced to varying degrees, suggesting that electroacupuncture may effectively treat chronic colitis rats by inhibiting the activation of PI3K/Akt/mTOR signal pathway and regulating the levels of proinflammatory and anti-inflammatory factors.Further comparison showed that Shujuxu group had a better regulation of inflammatory factors and proteins related to PI3K/Akt/mTOR signal pathway than Tianshu, Dachangshu and Zusanli groups, suggesting that Shujuxu has relatively specific treatment effect for chronic colitis, and also verifying the classical theory of “treating internal organs”.In addition, this study showed that the efficacy of Tianshu group was better than Dachangshu and Zusanli groups, further suggesting that “Hebu Peixue” can be used as the basis of acupoint selection for inflammatory bowel disease, which is consistent with previous studies[26, 27].

In summary, electroacupuncture of Tian shu, Da chang shu, Zu san li and Shang ju xu can improve the general condition of chronic colitis rats to varying degrees, and has different therapeutic effects on intestinal mucosal injury and intestinal inflammatory response.The mechanism may be realized by inhibiting the activation of PI3K/Akt/mTOR signal pathway, regulating the pro-inflammatory cytokines IL-23, IL-17 and anti-inflammatory cytokines IL-10, relieving intestinal inflammation, and modulating the body’s immunity.Among them, Shang ju xu has the best effect.

Authors’ contributions:

Tang Kunpeng was responsible for the writing of the article, the construction of animal models, electric acupuncture intervention,index detection and collection, arrangement and statistical analysis of data; Lv Jiaqi, Ma Mengna and Ren Huashan assisted in the execution of experiments and gave guidance; Wen Tan and Zhang Chunqing participated in index detection and statistical audit and verificaion of experimental data; Yan Liping was in charge of experimental design, guidance and recheck.No conflicts of interest exist for all authors.