Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

ZHANG Chun-qing, TANG Kun-peng, YAN Li-ping, WEN Tan, WANG Hai-jun

Shanxi University of Tradit Chin Med, Jinzhong 030619, China

Keywords:

ABSTRACT Objective: To investigate the mechanism of JAK2 / STAT3 / SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods: 36 SPF SD rats were randomly divided into 6 groups, with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group, with density wave, frequency 2Hz-50 Hz, intensity 2 mA, muscle tremor as the degree 20 min/time, 1 time/day, for 3 consecutive days.Observe the general condition of rats ; the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4 ( IL-4 ) and interleukin-8 (IL-8 ) were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2, STAT3, SOCS1 protein and mRNA in rat colon tissue.Results: In contrast to the normal group, the overall condition of the model group was worse, the colonic mucosa was severely damaged, even necrotic, and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced, and the content of IL-8 was obviously go up ( P ＜ 0.01 ).The protein content of JAK2, STAT3 and the expression of JAK2, STAT3 mRNA in colon tissue of rats were obviously go up, while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced ( P ＜ 0.01 ).In contrast to the model group, the general condition of rats in each acupoint group was significantly improved, the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated, the content of IL-4 in serum was obviously go up, and the content of IL-8 was significantly decreased ( P ＜ 0.01 ).The protein content of JAK2, STAT3 and the expression of JAK2, STAT3 mRNA in colon tissue of rats were obviously reduced, while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up ( P ＜ 0.05, P ＜ 0.01 ).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced, the content of serum IL-4 was significantly increased, and the content of IL-8 was significantly decreased ( P ＜ 0.05,P ＜ 0.01 ).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated, while the protein content and mRNA expression of SOCS1 were significantly go up ( P ＜ 0.05, P ＜ 0.01 ).Conclusion: Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli (ST36) is better than that of Tianshu (ST25), Dachangshu(BL25) and Shangjuxu (ST37).The mechanism may be related to the regulation of JAK2 /STAT3 / SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

1.Introduction

Inflammatory bowel disease ( IBD ) is a debilitating disease characterized by recurrent intestinal inflammation[1].It can be divided into two periods: acute onset and chronic remission, which is difficult to cure[2].Especially in the acute phase, a large number of pro-inflammatory factors will be produced, which will accelerate the transformation to Crohn ‘s disease ( CD ) and ulcerative colitis( UC ) under the stimulation of continuous aseptic inflammatory response[3].In recent years, it has been found that its incidence has increased rapidly in China[4].At present, the treatment of acute colitis in Western medicine is limited, mostly based on hormones,immunosuppressants, etc.[5], but the disease is easy to relapse after withdrawal.Long-term use will cause drug dependence, and there will be different degrees of adverse reactions[6,7].Therefore,effectively inhibiting the infiltration of inflammatory cells and improving the inflammatory microenvironment can help to reduce the occurrence and development of acute colitis.

The janus kinase 2 ( JAK2 ) / signal transducer and activator of transcription 3 ( STAT3 ) / suppressor of cytokine signaling 1 (SOCS1 ) signaling pathway is a classical inflammatory signaling pathway, which can activate downstream pro-inflammatory cytokines.Produce inflammatory cascade reaction and cause colonic mucosal injury[8,9].The previous study of the research group found that electroacupuncture at ‘Tianshu’, ‘Dachangshu’, ‘ Zusanli’ and ‘Shangjuxu’ acupoints had a significant effect on the chronic phase of colitis, and the effect of Shangjuxu acupoint was the best[10].Therefore, on the basis of previous studies, this study intends to take the acute phase of colitis as the research object, by detecting the expression of protein content related to JAK2 / STAT3 / SOCS1 signaling pathway and the content of interleukin in rat serum,aiming to explore the mechanism of electroacupuncture at different acupoints on acute colitis, select the optimal acupoints, and provide the basis for clinical treatment.

2.Materials and methods

2.1 Experimental animals and grouping

Thirty-six SD rats of SPF grade, 18 males and 18 females, weighing( 180 ± 20 ) g, were purchased from Beijing Spyford Biotechnology Co., Ltd., with license number [SCXK ( Beijing ) 2019-0010 ].All rats were free to eat, natural light, animal room temperature ( 23± 2 ) ℃, humidity ( 50 ± 5 ) %, adaptive feeding for a week.Rats were randomly divided into six groups, normal group, model group,Tianshu group, Dachangshu group, Zusanli group, Shangjuxu group,6 rats in each group.The experiment has been reviewed by the Animal Ethics Committee of Shanxi University of Tradit Chin Med (ethical approval number: 2021DW211 ).

2.2 Reagents and instruments

Main reagents: BCA protein concentration assay kit ( AB0146,Wuhan BOSTER Engineering Co., Ltd.) ; protease inhibitor cocktail( HY-K0010, MCE Biotechnology, USA ) ; IL-4, IL-8 kit ( MM-0191R1, MM-0175R1, Shanxi Ren ‘an Biotechnology Co., Ltd.) ;jAK2 antibody, STAT3 antibody, SOCS1 antibody, goat anti-rabbit IgG-HRP ( AF1489, AF1492, AF8022, AS014, Wuhan BOSTER Engineering Co., Ltd.) ; feikete ultrasensitive ECL luminescent solution ( MA0186, Dalian Meilun ).

Main instruments: electrophoresis instrument ( DYY-6D, Beijing Liuyi ) ; high speed refrigerated centrifuge ( 3K15, Xima Centrifuge Co., Ltd.); microplate reader ( 2108251C, BioTek, USA );tanon5200 chemiluminescence imager ( Tanon-5200 Multi, BioTek,USA ) ; decolorizing shaker ( DS-2H200, Wuhan Servicebio Biotechnology Co., Ltd.) ; paraffin embedding machine ( YB-6LF,Hubei Yaguang Medical Electronic Technology Co., Ltd.).

2.3 Model preparation

Referring to the previous modeling method of the research group[11], the rat model of acute colitis was prepared by enema with 6 % glacial acetic acid solution.During the modeling, except for the normal group, the polyvinyl chloride tubes coated with paraffin oil were slowly inserted from the anus of the rats to 8 cm in the anus,and 1ml of 6 % glacial acetic acid solution was slowly pushed in.After 15 s, 5 mL of normal saline was taken for washing.In this process, rats in each group did not need anesthesia.The normal group was replaced with 6 % glacial acetic acid solution with normal saline, and the remaining treatment was unchanged.

2.4 Acupoint selection and intervention therapy

The specific positioning of acupoints refers to ‘experimental acupuncture’ [12] Select ‘Tianshu’, ‘Dachangshu’, ‘Zusanli’, ‘Shangjuxu’ as electroacupuncture bilateral acupoints.

After modeling, sterile filiform needle and Huatuo brand SDZ-II electronic acupuncture instrument were used to give electroacupuncture stimulation to each acupoint ( bilateral ), with frequency of 2Hz-50Hz, intensity of 2 mA 20 min / time, 1 time / d,for 3 days.The normal group and the model group were only fixed without acupuncture at the same time.

After the end of the last treatment, the rats in each group were fasted and could not help but water.After 12 h, the rats were anesthetized and blood was taken.Centrifugation was performed at 4 ℃.The supernatant was collected and stored in a refrigerator at-20 ℃ for examination.The intercepted rat colon tissue ( anal and above 12 cm ) was placed in a sterile cryopreservation tube, and then immediately placed in a refrigerator at 80 ℃ for preservation.

2.5 Observation indicators and detection methods

(1) Observe the general situation of rats: each group of rats hair color light how, whether the reaction sensitivity, whether active,whether the spirit of depression and weight loss were closely observed.

(2) HE staining was used to observe the mucosal changes of colon tissue: colon tissue ( about 2 mm × 2 mm × 2 mm ) was used for HE staining, and then the colon tissue was fixed, dehydrated and transparent to make wax blocks.Wax block slice thickness of about 5 μm, dewaxing, dehydration, soaking, differentiation, washing, etc.Then add the eosin dye solution, followed by cleaning and sealing.The images were collected under an optical microscope ( 200 × ).

(3) Detection of serum IL-4 and IL-8 levels by ELISA: The collected supernatant was taken out and thawed on ice.The contents of IL-4 and IL-8 in rat serum were detected under the guidance of the explanation of ELISA kit.

(4) Detection of protein expression in colon tissue by Western blot:100 mg of colon tissue was ground and the supernatant was taken.Then the protein concentration was measured by BCA method, and the protein was stored after denaturation.After the end of the leak detection, the gel was prepared and the sample was loaded.After the end of the sample loading, electrophoresis and membrane transfer were started.After the end of the membrane transfer, the skim milk was blocked.After the blocking was completed, the primary anti-JAK2 antibody, STAT3 antibody, and SOCS1 antibody were incubated ( the ratio was 1: 2 000, 1: 1 000, and 1: 2 000 ).After the incubation was completed, the refrigerator was closed overnight at 4 ℃.On the second day, the goat anti-rabbit IgG ( 1: 5 000 ) was incubated after the T-BST was washed three times.After 2 h, the T-BST was washed, and then the hypersensitive ECL luminescent liquid was used for luminescence development, and the protein band map was saved.After that, the gray value of protein bands was analyzed by ImageJ software.

(5) Real-time PCR was used to detect the expression of JAK2,STAT3 and SOCS1 mRNA in colon tissue: About 50 mg of rat colon tissue was weighed, total RNA was extracted, and cDNA was synthesized.GAPDH was used as the internal reference gene, and the total mRNA was used as a template for reverse transcription into cDNA for amplification.Amplification conditions: pre-denaturation 95 ℃ -10min, 95 ℃ -15 s, 60 ℃ -60 s, 40 cycles.The relative expression of the target gene was calculated by 2-ΔΔCtmethod.The primer design and synthesis were completed by Shanxi Ren‘an Biotechnology Co., Ltd.The primer sequence is shown in Table 1.

Tab 1 Primer sequence

2.6 Statistical analysis

Statistical analysis was performed using SPSS26.0 statistical software, expressed as mean ± standard deviation (±s).When all sample data were in accordance with normal distribution and homogeneity of variance, one-way analysis of variance and LSD test were used for comparison between groups.P ＜ 0.05 indicated significant difference, P ＜ 0.01 indicated extremely significant difference, and P ＞ 0.05 indicated no statistical significance.

3.Results

3.1 Comparison of general conditions of rats in each group

Before modeling, the rats in each group were full of spirit,responsive, shiny hair, and normal stool ; after modeling, the hair of rats in each group was dim and dull, the spirit was apathetic and lazy, and there was dilute water stool.In severe cases, there was blood stool and weight loss.After intervention, the hair color of each acupoint group was significantly improved compared with the model group, the body weight and activity increased, the blood stool decreased, the stool shape gradually returned to normal shape, and the overall situation was significantly improved compared with the model group.Among them, the improvement of Zusanli acupoint group was the most significant compared with other acupoint groups.

3.2 Comparison of pathomorphology of colon tissue in rats of each group

In the normal group, the glands were arranged in an orderly manner, the surface of the crypt was regular, the mucosal layer and the muscular layer had no congestion, edema, and the structure was complete ; in the model group, the gland arrangement was seriously disordered, a large number of inflammatory cells infiltrated,congestion and edema, mucosal layer and muscular layer were seriously damaged, and the structure was obviously abnormal.Compared with the model group, the lesions in each acupoint group were significantly improved, the inflammatory response was significantly reduced, but the mucosal layer and muscular layer were still defective, and the gland arrangement was partially abnormal.Among them, the mucosal layer, muscular layer and glandular structure of Zusanli acupoint group were significantly improved compared with other acupoint groups.See Figure 1.

Fig 1 Comparison of pathological changes of colon tissue ( HE,×200 )

3.3 Comparison of the contents of IL-4 and IL-8 in serum of rats in each group

Compared with the normal group and the model group, it was found that the level of IL-4 in the serum of the model group was significantly decreased and the level of IL-8 was significantly increased ( P ＜ 0.01 ).Compared with the model group, the levels of IL-4 in the serum of rats after electroacupuncture intervention were significantly increased, and the levels of IL-8 were significantly decreased ( P ＜ 0.01 ).Among them, compared with the Tianshu,Dachangshu and Shangjuxu groups, the level of IL-4 in the serum of rats was significantly increased, and the level of IL-8 was significantly decreased ( P ＜ 0.05, P ＜ 0.01 ).There was no significant difference in the content of IL-4 and IL-8 in the serum of rats among the three groups of the Tianshu group, the Dachangshu group and the Shangjuxu group ( P ＞ 0.05 ).See Table 2.

Tab 2 Comparison of serum IL-4 and IL-8 levels in each group of rats (pg/ml, n=6,±s )

Tab 2 Comparison of serum IL-4 and IL-8 levels in each group of rats (pg/ml, n=6,±s )

Note: Compared with the normal group, ＊＊P ＜ 0.01 ; compared with the model group, ##P ＜ 0.01 ; compared with Zusanli group, △P ＜ 0.05, △△P ＜0.01.

Group IL-4 IL-8 Normal group 96.00±4.68 53.18±7.03 Model group 43.54±6.39＊＊ 140.39±7.90＊＊Tianshu group 70.88±7.46##△ 95.55±7.61##△Dachangshu group 66.86±7.45##△△ 98.62±6.91##△△Zusanli group 78.78±5.01## 83.65±8.62##Shangjuxu group 65.78±5.66##△△ 102.16±8.83##△△F 46.164 77.861 P＜0.0001 ＜0.0001

3.4 Comparison of JAK2, STAT3 and SOCS1 protein expression in colon tissue of rats in each group

Compared with the normal group and the model group, it was found that the content of JAK2 and STAT3 protein in the colon tissue of the model group was significantly increased, and the content of SOCS1 protein was significantly decreased ( P ＜ 0.01 ).Compared with the model group, the content of JAK2 and STAT3 protein in the colon tissue was significantly decreased and the content of SOCS1 protein was significantly increased after electroacupuncture intervention( P ＜ 0.05, P ＜ 0.01 ).Among them, compared with Tianshu,Dachangshu and Shangjuxu groups, the content of JAK2 and STAT3 protein in colon tissue of rats decreased significantly, and the content of SOCS1 protein increased significantly ( P ＜ 0.05 ).There was no significant difference in the content of JAK2, STAT3 and SOCS1 protein in colon tissue between Tianshu group, Dachangshu group and Shangjuxu group ( P ＞ 0.05 ).See figure 2, table 3.

3.5 Comparison of JAK2, STAT3 and SOCS1 mRNA expression in colon tissue of rats in each group

Compared with the model group, the expression of JAK2 and STAT3 mRNA in the colon tissue of the model group was significantly increased, and the expression of SOCS1 mRNA was significantly decreased ( P ＜ 0.01 ).Compared with the model group,the expression of JAK2 and STAT3 mRNA in the colon tissue of rats after electroacupuncture intervention was significantly decreased( P ＜ 0.05, P ＜ 0.01 ), and the expression of SOCS1 mRNA was significantly increased ( P ＜ 0.01 ).Among them, compared with the Tianshu, Dachangshu and Shangjuxu groups, the expression of JAK2 and STAT3 mRNA in the colon tissue of rats was significantly decreased, and the expression of SOCS1 mRNA was significantly increased ( P ＜ 0.05, P ＜ 0.01 ).There was no significant difference in the expression of JAK2, STAT3 and SOCS1 mRNA in colon tissue among the three groups ( P ＞ 0.05 ).See Table 4.

Fig 2 Expression of JAK2, STAT3 and SOCS1 protein in colon tissue of rats in each group

Tab 3 Comparison of JAK2, STAT3 and SOCS1 protein expression in colon tissue of rats in each group (n=6, ±s)

Tab 3 Comparison of JAK2, STAT3 and SOCS1 protein expression in colon tissue of rats in each group (n=6, ±s)

Note: Compared with the normal group, ＊＊P ＜ 0.01 ; compared with the model group, #P ＜ 0.05, ##P ＜ 0.01 ; compared with Zusanli group, △P ＜0.05.

Group JAK2 STAT3 SOCS1 Normal group 0.24±0.11 0.29±0.05 1.69±0.26 Model group 1.40±0.46＊＊ 1.69±0.29＊＊ 0.23±0.09＊＊Tianshu group 0.78±0.10#△ 0.91±0.12##△ 0.89±0.09#△Dachangshu group 0.78±0.09#△ 0.92±0.20##△ 0.89±0.17#△Zusanli group 0.22±0.09## 0.36±0.07## 1.45±0.34##Shangjuxu group 0.83±0.05#△ 0.92±0.29##△ 0.86±0.06#△F 14.04 19.72 20.38 P＜0.0001 ＜0.0001 ＜0.0001

Tab 4 Comparison of relative expression of JAK2, STAT3 and SOCS1 mRNA in colon tissue of rats in each group(n=6, ±s)

Tab 4 Comparison of relative expression of JAK2, STAT3 and SOCS1 mRNA in colon tissue of rats in each group(n=6, ±s)

Note: Compared with the normal group, ＊＊P ＜ 0.01 ; compared with the model group, #P ＜ 0.05, ##P ＜ 0.01 ; compared with Zusanli group, △P ＜0.05, △△P ＜ 0.01.

Group JAK2mRNA STAT3mRNA SOCS1mRNA Normal group 1.00±0.07 1.00±0.02 1.00±0.02 Model group 2.06±0.12＊＊ 1.96±0.07＊＊ 0.25±0.04＊＊Tianshu group 1.72±0.18#△△ 1.62±0.06#△ 0.62±0.10##△Dachangshu group 1.66±0.08##△ 1.65±0.16##△△ 0.60±0.10##△△Zusanli group 1.29±0.02## 1.29±0.05## 0.87±0.08##Shangjuxu group 1.63±0.11##△ 1.62±0.09##△△ 0.60±0.05##△△F 33.58 42.74 36.92 P＜0.000 1 ＜0.000 1 ＜0.000 1

3.Discussions

In Tradit Chin Med, colitis belongs to the category of ‘intestinal abscess’, ‘diarrhea’, ‘ abdominal pain’, ‘dysentery’ and so on.The disease is located in the intestine and is mainly responsible for the spleen and stomach[13].As ‘Suwen · Yin and Yang should be like the big theory’ mentioned: ‘the stomach, the water valley of the road, the five flavors into the stomach...Losing to the kidney,don’ t be clear.’ In recent years, a large number of literature studies have shown that acupuncture has unique advantages in clinical treatment, and also has obvious therapeutic effect on colitis, which can effectively inhibit intestinal inflammatory response[14,15,16,17].The preliminary study of the research group also found that electroacupuncture ‘Tianshu, Dachangshu, Zusanli, Shangjuxu’four groups of acupoints have a significant therapeutic effect on chronic colitis, and the specificity of acupoints was further studied.It was found that electroacupuncture at Shangjuxu acupoint had a significant effect on chronic colitis[10].Based on this, this experiment continued to take colitis as the research object, and carried out basic research in the acute phase.Four groups of specific acupoints of‘Tianshu, Dachangshu, Zusanli and Shangjuxu’ were used to observe their therapeutic effects and further analyze their specificity.The Tianshu point belongs to the Yangming Stomach Meridian, which is the Mu point of the large intestine.It has the effect of regulating Qi and regulating the intestines.Dachangshu belongs to the bladder meridian of foot sun, which is the back-shu point of the large intestine meridian, and has the effect of regulating the intestines and regulating qi stagnation.Zusanli is the combined acupoint of the stomach meridian of foot yangming.According to the theory of “ combined treatment of internal organs, “ Zusanli can not only treat the related diseases of spleen and stomach, but also treat the related diseases of six fu organs.Shangjuxu belongs to the lower He-sea point of the large intestine meridian, which has the function of regulating qi movement, dredging meridians and activating collaterals.Studies have found that electroacupuncture ‘ Tianshu’and ‘Zusanli’ play a role in protecting colonic mucosa by regulating the content of related inflammatory factors in serum and reducing inflammatory response[11].The results of this study showed that after electroacupuncture treatment, the general condition of rats in each acupoint group was significantly improved, bloody stools were reduced, body weight and activity were increased, and the improvement of Zusanli acupoint group was the most significant compared with other acupoint groups.

The imbalance of pro-inflammatory and anti-inflammatory cytokines is closely related to the occurrence and development of acute colitis[18].As two important inflammatory factors, IL-4 and IL-8 play an important role in the occurrence and development of inflammatory bowel disease.IL-4 is an anti-inflammatory cytokine produced by Th2, which mainly acts on immune-active cells and induces the proliferation of activated mature T cells, and can inhibit inflammatory response and immune cell infiltration[19,20].IL-8 is a pro-inflammatory factor that can directly mediate when inflammation occurs.It is also a strong neutrophil chemokine and activator[21].When the acute phase of colitis occurs, the insufficient and excessive secretion of IL-4 and IL-8 will lead to the destruction of the intestinal mucosal barrier and the intensification of the inflammatory response.It can be seen that the mechanism of IL-4 and IL-8 in acute colitis is mainly through the promotion and inhibition of inflammatory response and immune cell infiltration,leading to the destruction of intestinal mucosal barrier and the aggravation of inflammatory response.Therefore, effective control of the secretion of inflammatory factors IL-4 and IL-8 is helpful to alleviate the symptoms and inflammatory response of acute colitis.The results of this study showed that the secretion of inflammatory cytokines IL-4 in acute colon tissue decreased and the secretion of IL-8 increased.After electroacupuncture treatment, it can increase the secretion of inflammatory cytokine IL-4 and reduce the secretion of IL-8, indicating that electroacupuncture can improve the symptoms of colonic mucosa and protect colonic mucosa by increasing IL-4 and reducing IL-8, avoiding the infiltration of inflammatory cells, thus maintaining the stability of inflammatory microenvironment.The content of IL-4 in Zusanli group was higher than that in other acupoint groups, and the content of IL-8 was lower than that in other acupoint groups.

JAK2 / STAT3 / SOCS1 signaling pathway, as a classic proinflammatory signaling pathway, can mediate the signal transduction of cytokines, transmit a variety of stimulus signals, and regulate its corresponding cell response through target genes[22,23].Among them,JAK2 is a tyrosine kinase.When the activity of JAK2 increases, it can promote the occurrence of inflammatory response[24].STAT3 is a transcription factor whose main role is to regulate cell growth and differentiation[25].In the inflammatory microenvironment, the activity of STAT3 is usually elevated, thereby exacerbating the inflammatory response[26].SOCS1 is a negative immunomodulatory factor that provides negative feedback to the JAK / STAT pathway that regulates cytokine signaling[27].When the expression level of SOCS1 increases, it will inhibit the activity of JAK / STAT signaling pathway and reduce the infiltration of inflammatory cells.The results of this study showed that after intraperitoneal injection of 6 % glacial acetic acid solution in rats, the colon tissue showed obvious injury symptoms, and the expression of JAK2 and STAT3 was significantly increased.The expression of SOCS1 was significantly decreased.Electroacupuncture at each acupoint can reduce the expression of JAK2 and STAT3 and increase the expression of SOCS1.Therefore,by inhibiting the activity of JAK2 / STAT3 / SOCS1 signaling pathway, the inflammatory response can be alleviated and the symptoms of acute colitis can be improved.Therefore, this signaling pathway may become a new target for the treatment of acute colitis.In summary, electroacupuncture at each acupoint can improve the damage of colonic mucosa in rats with acute colitis and reduce the inflammatory response.The therapeutic effect of Zusanli is better than that of Tianshu, Dachangshu and Shangjuxu.The mechanism may be related to the inhibition of JAK2 / STAT3 / SOCS1 signaling pathway related proteins, the increase of inflammatory cytokine IL-4 and the decrease of IL-8 secretion.

Description of the author’s contribution:

Zhang Chunqing: Complete the experiment, carry out index detection, analyze and sort out data, and write papers ; tang Kunpeng,literary world: to assist in the completion of the experiment, in the index detection, data analysis to give corresponding guidance ; yan Liping: Communication author, experimental design, guidance and article revision ; wang Haijun: Support and help the successful completion of the experiment.

All authors declare that there is no conflict of interest.