Effects of pestle needle on nerve conduction velocity and inflammatory injury in patients with diabetic peripheral neuropathy

Fang Wang,Shun-Qi Liao,Jun Wen,Han Wang,Yao Wang,Xi-Mei Weng,Rong Wu,Ya-Ling Huang

1Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu 610072, China.2Chengdu University of Traditional Chinese Medicine, Chengdu 610075,China.

Abstract Background: Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes mellitus.Impaired neurological function is one of the main characteristics of DPN and is strongly associated with the inflammatory response.Our previous studies have confirmed that pestle needle can improve the nerve function of patients with DPN.But the mechanism of pestle needle treatment of DPN is still unclear.Methods: A total of 70 DPN patients who met the inclusion criteria were randomly divided into two groups.Control group (CG) (n = 35)received DPN conventional treatment and the pestle needle group (PNG) (n = 35) received pestle needle therapy at Zhiyang (DU09) eight array, Mingmen (DU04) eight array and Heche Road (from Mingmen (DU04) to Changqiang (DU01)), Zusanli (ST36), Sanyinjiao (SP06), Taixi(KI03) and Yongquan(KI01).Patients in the PNG group were required to take this treatment for 4 weeks, 5 times a week.Examination indexes were collected before and after treatment,respectively.Nerve function was examined using the Toronto clinical scoring system and nerve conduction velocity detection.Serum inflammatory factors were measured by enzyme linked immunosorbent assay.Results: The Toronto clinical scoring system was significantly reduced in the PNG compared with the CG after treatment.The sensory nerve conduction velocity and motor nerve conduction velocity of the right peroneal and median nerves were significantly faster in the PNG than those in the CG (P ＜0.05).After treatment, serum interleukin-1 beta,interleukin-6 and tumor necrosis factor-alpha levels decreased in both groups, and the improvement of PNG was better than CG (P ＜0.05).Conclusion: The pestle needle can significantly improve the symptoms and nerve conduction velocity of DPN, and its mechanism may be related to the reduction of inflammatory factors.

Keywords: diabetic peripheral neuropathy; pestle needle; nerve conduction velocity;inflammatory factors

Background

Diabetes mellitus(DM) is a serious chronic non communicable disease.The number of people with DM is up to 500 million worldwide, and this number continues to rise.Concomitant with the rise in the number of DM, the complications of DM also increase [1].Diabetic peripheral neuropathy (DPN) is one of the most common complications of DM.Its occurrence is correlated with DM duration,glycemic, and other factors.With the development of the disease, at least 50% of DM patients have DPN [2].DPN usually occult under the condition of poor glucose.Early symptoms are not obvious, so it is easy to be ignored by patients.When the condition progresses to a certain extent, it causes irreversible nerve damage, even foot ulceration or amputation.DPN is a major cause of disability in DM [3].Moreover, it not only adds psychological pressure to patients, but also brings serious economic burden to families [4].At present, the treatment of DPN by Western medicine is still based on the treatment of the pathogenesis of DPN on the basis of lowering lipids and blood sugar.Antioxidants, aldose reductase inhibitors, neurotrophic factors,cell metabolism agonists, and vasodilators are used for pathogenesis drugs.These medications play a role in reducing symptoms.But they do not significantly improve neurological function and have various side effects, including insomnia, decreased appetite, arrhythmias,allergies, and so on [2, 5].Therefore, we seek new and effective DPN treatment strategies from the perspective of complementary medicine.At present, it is generally accepted that the development of DPN is associated with abnormalities in nerve polyols, hexosamine, and protein kinase C pathways [4, 6, 7].These pathways can cause signal transduction changes in cells, which can cause mitochondrial damage,trigger oxidative stress, and promote the secretion of a variety of inflammatory factors [8-10].Ultimately leading to neurological,vascular, and tissue dysfunction.The level of serum inflammatory mediators is an important factor in nerve injury and always involved in the occurrence and development of DPN [11].Multiple studies have confirmed that the inflammatory response occurs early in DM, and long-term chronic inflammation promotes DPN [11, 12].A large number of inflammatory factors can cause nerve damage and affect nerve regeneration [13, 14].

The earliest prototype of the pestle needle is the “Chi needle” in theInner Canon of Yellow Emperor(unknown author, written around 2,600 B.C.E.), which is a blunt needle made of bone or wood.Pestle needle originated from Taoism (It is also spelled Daoism, and it is a religion and a philosophy from ancient China which emphasizes living in harmony with the universe.) and is often used for the treatment of poor people.It has a history of more than 300 years.Unlike the regular needle technique, the pestle needles do not penetrate into the skin; rather they are used to rub or press the acupoints and meridians of the body (Figure 1).The pestle needle therapy has not been recorded in the traditional Chinese medical classics.In the process of secret transmission, the method was only dictated without written records.Until the 1970s, Zhong-Yu Li, the 14thgeneration heir,officially promoted pestle acupuncture therapy.Then the theory and operation skills of pestle needle were organized into a document, and a complete school of traditional Chinese medicine was formed.In 1990,Pestle Needle Therapywas the first book on pestle needle to be published [15].In 2020, pestle needle was officially included in China’s national intangible cultural heritage.Nowadays, pestle needle therapy has been widely used in sports trauma diseases,cerebrovascular diseases, osteoarticular diseases, respiratory diseases,and so on[16].

Figure 1 Pestle needle

The pestle needle has special needle tools and manipulations that are different from regular acupuncture.It is achieved to promote the running of human Qi and blood and to patency the meridians, by stimulating special acupoints and body surface acupoints.Our previous study found that the pestle needle treatment of DPN was effective.The pestle needle can effectively reduce the current perception threshold and vibration perception threshold of the patient’s foot [17, 18].Our latest findings showed that the mechanism of DPN treatment by clubbing needle was related to antioxidative stress [19].Compared with the Western medicine method of treating DPN, the patients treated with pestle needle felt more comfortable and had no side effects.So the patient was more willing to accept the pestle needle treatment.But the mechanism of the pestle needle treatment for DPN is still not clear.Therefore, from an inflammatory point of view, we hypothesized that the pestle needle might alleviate DPN nerve damage by inhibiting the inflammatory response of the body.

Methods

General data

From April 2021 to September 2021, 70 inpatients met the inclusion criteria were recruited from Department of Endocrinology of the Hospital of Chengdu University of Traditional Chinese Medicine.

Participants were randomly assigned to the pestle needle group(PNG) and the control group (CG) according to the 1: 1 distribution ratio.Random grouping was conducted by an independent person,and the rest of the researchers did not participate.Random numbers 1 to 70 were generated using Microsoft Excel 2003.Odd numbered subjects were included in the PNG and even numbered subjects were included in the CG.Then, the random number was sealed in opaque envelopes and the patients were allowed to extract the sealed number.

Because of the characteristics of the treatment, it was difficult to allow physicians who implemented the pestle needle treatment or participants to be blinded to its allocation.

The study design was approved by the Ethics Committee of the Hospital of Chengdu University of Traditional Chinese Medicine (No.2021KL-107), and all patients signed informed consent before treatment.This clinical study was registered Chinese Clinical Trial Registry(No.ChiCTR2100053887).

Sample size

Sample size calculation was based on sensory nerve conduction velocity (SNCV) of peroneal nerve.According to the published studies[20],the mean±standard deviation of SNCV of peroneal nerve in CG,and PNG after intervention were (38.78 ± 5.02, 43.23 ± 4.34),respectively.Gpower 3.1.9.2 software was used to calculate the sample size.The sample size of 58 participants was enough to detect a target effect size with a type 1 error of 5% (α =0.05) and 95% power(β = 0.05).Considering a 20% attrition rate, there were a total of 70 participants, 35 in each group.

Diagnostic criteria

World Health Organization diagnostic criteria for DNP are as follows.(1) The patient has a clear history of DM.(2) Neuropathy present at the time of diagnosis or after diagnosis of DM.(3) At least 2 of the following examinations were abnormal, including abnormalities of temperature sensation, nylon wire examination, decreased or absent sensation in the feet, abnormalities of vibration sensation, and ankle reflexes.(4) The presence of median, sural nerve hyperesthesia or hypoesthesia was quantified by sensory nerve[21].

Inclusion criteria

(1) All patients met the World Health Organization diagnostic criteria for type 2 DM in 1999 [22].(2) All patients aged between 40 and 80 years.(3) All patients were conscious and coordinated with examinations.(4) All patients volunteered to participate in this study and signed informed consent were included in this study.

Exclusion criteria

Patients with severe diseases (severe infection, severe heart, kidney,brain and other organ damage), acute complications of DM (diabetic ketoacidosis and hyperglycemic hyperosmolar state), mental disorders,and uncooperativeness were excluded.Pregnant or lactating women were also excluded.

Therapeutic method

All subjects received basic treatment for DM, including DM health education, oral hypoglycemic medication, or insulin for glucose control.The CG received oral mecobalamin tablets (manufactured by China Pharmaceutical Co., Ltd.), 0.5 mg each time, 3 times daily for 4 weeks.The PNG chose the pestle needle treatment for 30 minutes a day for 4 weeks.

Selection of pestle needle.Tai Chi brand pestle needles(manufactured in Hospital of Chengdu University of Traditional Chinese Medicine) were used in this study.It consists of “Kui Xing pen”, “Jin Gang pestle”, “Qiyao Hunyuan pestle” and “Wuxing Santai pestle”.Kui Xing pen is 8 cm long and the needle tip is a blunt cone.The length of the Jin Gang pestle is 10.5 cm, which is similar to the shape of the Kui Xing pen.Both are often used for small acupoints(Such as Shaoshang (LU11) and Neiguan (PC6) acupoints).Qiyao Hunyuan pestle is 10.5 cm long with parallel 7 blunt claws at the tip of the needle; Wuxing Santai pestle is 11.5 cm long and has claws at both ends.One end is three claws side by side, and the other end is five claws in the shape of plum blossom(Figure 1).

Acupoints selection of pestle needle therapy.Acupoints were selected according toScience of Acupuncture[23] andScience of Pestle Needle Therapy[24] in this study.Eight array acupoint is a circle drawn with one acupoint as the midpoint and a certain distance outward from the midpoint as the radius.According to the eight trigrams, the circle is divided into eight equal parts, which is named outer eight array.The distance from the midpoint to the outer eight array is further divided into three equal parts and drawn into two circles,which are named middle eight array and inner eight array.The main acupoints are Zhiyang (DU09) eight array, Mingmen (DU04)eight array, Heche Road (from Mingmen (DU04) to Changqiang(DU01)), Zusanli (ST36), Sanyinjiao (SP06), Taixi (KI03) and Yongquan (KI01).Zhiyang eighth array is a circle drawn with Zhiyang(DU09) as its midpoint and the distance from Zhiyang (DU09) to Geguan (BL46) as its radius.Mingmen eighth array is a circle drawn with Mingmen (DU04) as its midpoint and the distance from Mingmen(DU04) to Zhishi (BL52) as its radius.There are seven lines in Heche Road (from Mingmen (DU04) to Changqiang (DU01)) in total.The midline is drawn from Mingmen (DU04) to Changqiang (DU01), and then lines are drawn at distances of 0.5 Cun (the width of the knuckle of the thumb as 1 Cun), 1.5 Cun, and 3 Cun from the midline (Figure 2).

Figure 2 Acupuncture points of pestle needle

Pestle needle operation method.The method of applying pestle needle follows the operation specifications described inScience of Pestle Needle Therapy[24].The pestle needle operation of this study was completed by 5 graduate students majoring in integrated traditional Chinese and Western medicine nursing.They have received the pestle needle operation training of the Pestle Needle Committee and passed the examination.The suitable manipulation technique is selected according to the characteristics of acupoints.and each operation method takes 7 times as a cycle.(1) The patient was placed prone on the bed.The Zhiyang (DU09) eight array was repeatedly tapped with the tip of the Wuxing Santai pestle.The tapping method was like a bird pecking.It took about 3-5 minutes to reach the flushing of the skin.Then the doctor used the needle handle of the Qiyao Hunyuan pestle to press against the skin of the Zhiyang (DU09)eight array to perform repeated Tai Chi movements.The direction was from the inside to the outside, and then from the outside to the inside,about 3-5 minutes.(2) The operation method of the Mingmen (DU04)eight array was the same as Zhiyang (DU09) eight array.(3) The doctor used the tip of the Qiyao Hunyuan pestle to travel on the 7 lines of the Heche Road.The operation method was to push left and right, then push up and down, about 5 minutes.Then use the Wuxing Santai pestle to tap 7 lines repeatedly for about 2 minutes.(4) Patients were taken in the supine position.The doctor hold a Jin Gang pestle in Zusanli (ST36).First, the needle tip was pressed down to the extent that the patient can tolerate it.Second, the needle tip was slowly lifted up, but the needle tip did not leave the skin.The above operation was continued for 2-3 minutes.Then, the doctor used the Kui Xing pen to tap the Zusanli (ST36), about 2-3 minutes; Finally the doctor used the needle handle of the Wuxing Santai pestle to press against the skin of the Zhiyang (DU09) eight array to perform repeated Tai Chi movements, about 2-3 minutes.(5) The manipulation of Sanyinjiao(SP06), Taixi (KI03) and Yongquan (KI01) was the same as Zusanli(ST36).

Observed indices

Nerve conduction velocity (NCV).NCV is the gold standard for the diagnosis of DPN [25].The NCV test is an electromyographic test used to evaluate and diagnose nerve injury.NCV is generally slower in patients with neurological disorders.The peroneal and median nerves are commonly used to assess for DPN.SNCV and motor nerve conduction velocity (MNCV) of the right peroneal and median nerves in both groups were detected using electromyographic detection instruments before and after treatment.

Toronto clinical scoring system (TCSS).TCSS was developed by Perkins in 2001.It is used to assess the severity of DPN[26].The TCSS includes symptom scores (foot pain, numbness, tingling, soreness and weakness, ataxia, and symptoms in the upper limbs), reflex scores(knee reflexes and ankle reflexes), and sensory detection (pain sensation, temperature sensation, light touch sensation, vibration sensation, position sensation).The patient was symptomatic with a score of 1, hyporeflexia with a score of 1, areflexia with a score of 2 and paresthesia with a score of 1.The total score ranged from 0 to 19 points.The higher the score, the more severe the impairment of neurological function.This scale was used in this study to assess whether the patient’s clinical symptoms were relieved before and after treatment.

Serum inflammatory factor level.Before and after treatment, 5 mL of peripheral venous blood was collected in the morning.Serum was separated by centrifugation at 3,000 revolutions/minute for 20 minutes.Enzyme linked immunosorbent assay was used to determine the levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α).The kits for enzyme linked immunosorbent assay were all purchased from Shanghai Enzyme Linked Biotechnology Co., Ltd.And the operation procedures were performed according to the instructions.Wells of standards were added with 50 μL each of standard samples at different concentrations.100 μL enzyme reagent is added to each well,except for the blank well.Incubation at 37 °C for 60 minutes.Wash well with wash solution,discard after standing for 30 seconds, and repeat this 5 times.First,add 100 μL of color developer A and B to each well, shake gently to mix, and develop color at 37 °C for 15 minutes in the dark.Then add 50 μL stop solution to each well to stop the reaction.Measure the absorbance of each hole in sequence at 450 nm wavelength.

Statistical analysis

SPSS 25.0 statistical software was used to analyze the data.Mean and standard deviation described continuous variables.Categorical variables were described as absolute numbers and percentages.Continuous variables were evaluated using normality and variance homogeneity tests.Within-group differences were compared using pairedt-test.Two independent samplest-test was used to analyze intergroup differences.Differences between the groups after the study were analyzed with the covariance model.For categorical data, the Chi-square test analyzed the difference.Two-sided test criteria were used in this study.AP-value ＜0.05 was statistically significant.

Results

Comparison of general data

A total of 70 DPN patients were enrolled.6 patients withdrew from the study for family or work reasons.64 (91%) patients with DPN completed the treatment plan and were included in the analysis.There was no significant difference between the groups at baseline, and the two groups were well comparable (P＞0.05) (Table 1).In addition,based on the doctor’s observations and participant feedback, no adverse reactions were found during treatment.

Table 1 Baseline characteristics of participants

Comparison of TCSS

Before treatment, there was no significant difference in TCSS between the two groups (P＞0.05).After treatment, the TCSS of both groups significantly reduced (P＜0.05).The comparison between groups the scores of TCSS in the PNG were lower than those in the CG, and the difference was statistically significant (P＜0.05) (Table 2).

Table 2 Comparison of TCSS before and after treatment

Comparison of NCV

Before the treatment, there was no significant difference in the comparison of MNCV and SNCV of the right peroneal and median nerves between the two groups (P＞0.05).SNCV and MNCV of the right peroneal and median nerves in the two groups were faster after treatment than before.The comparison between groups of SNCV and MNCV of the right peroneal and median nerves in the PNG were faster than those in the CG (P＜0.05) (Table 3).

Table 3 Comparison of NCV before and after treatment

Comparison of serum inflammatory factors

There were no significant difference in the levels of IL-6, IL-1β and TNF-α between the two groups before treatment (P＞0.05).After treatment, serum IL-1β, IL-6 and TNF-α levels decreased in both groups, and the improvement of PNG was better than CG (P＜0.05)(Table 4).

Table 4 Comparison of serum inflammatory factors

Discussion

DPN is one of the most common chronic complications of DM [27].It is irreversible.Hyperglycemia is a initiating factor in DPN.Numerous studies have proved that the inflammatory response is closely related to DPN[28,29].Under a chronic hyperglycemic environment, glucose and lipoproteins bind to various transport receptors on neurons and microvascular endothelial cells to initiate inflammatory signaling mechanisms.A large amount of inflammatory mediators infiltration makes neurons damaged or die.At the same time, it also causes edema of unmyelinated nerve fiber axons, demyelination of myelinated nerve fibers, and inhibition of nerve fiber regeneration.Eventually cause pain, numbness, hyperalgesia as well as muscle weakness and other symptoms [11, 30, 31].The level of serum inflammatory factors(TNF-α, IL-1 and IL-6) in DPN patients were found to be significantly higher than those in healthy patients, and there was a linear association between the concentration of TNF-α and peripheral nerve velocity [28].Kajal’s study found that TNF-α inhibitors can alleviate the symptoms of DPN rats [32].TNF-α, IL-1β, IL-6 and monocyte chemoattractant protein-1 were significantly increased in the spinal dorsal horn of db/db mice [33].The results of these studies have confirmed that inflammatory factors are involved in the development of DPN.Therefore, a moderate anti-inflammatory treatment may be effective for DPN.However, due to toxic side effects, non-steroidal anti-inflammatory drugs are not incorporated into the conventional treatment of DPN.With the booming development of traditional Chinese medicine, Chinese and Western rehabilitation techniques provide new ideas for DPN treatment.

Pestle needle therapy is simple to perform.It not only has the advantages of traditional acupuncture and Tuina, but also the needle do not pierce the skin.So, the patient feels very comfortable during the treatment.Meanwhile, it is considered a traditional physiotherapy widely applicable to the population.Our previous studies found that pestle needle acting on its special acupoints could exert the effects of relaxing muscles, relieving pain and promoting microcirculation [34,35].And the pestle needle has a good effect on DPN, cervico shoulder low back and leg pain, constipation.In addition, studies have found that the pestle needle can stimulate the nerve, relax the lumbar musculature, reduce the pressure around the nerve root, and eliminate edema [36, 37].Also, serum levels of inflammatory mediators were significantly improved in patients with lumbar disc herniation by pestle needle therapy[38].

Zusanli(ST36),Sanyinjiao(SP06),Taixi(KI03)and Yongquan(KI01)acupoints are located in the lower limbs and are often used to treat DPN [39].Zhiyang (DU09) eight array, Mingmen (DU04) eight array and Heche Road (from Mingmen (DU04) to Changqiang (DU01)) are all located on the back of the body, and the positions of these acupoints correspond to the stomach, spleen and kidney.A previous study confirmed that stimulation of Zhiyang (DU09) eight array,Mingmen(DU04) eight array and Heche Road (from Mingmen (DU04)to Changqiang (DU01)) acupoints could increase the SNCV of the peroneal and median nerves [40].This may be related to the fact that stimulation of acupoints on the back and feet with pestle needle can initiate the mechanism of reflex regulation of the body’s nerves,improve spleen and stomach function, enhance tissue metabolism, and promote blood circulation [35].A recent study [19] used the same acupoints as this study.Its result [19] have shown that pestle needle can regulate the Keap1/Nrf2/ARE signaling pathway to increase the production of the antioxidant enzymes superoxide dismutase and glutathione peroxidase.The body’s antioxidant capacity is enhanced to protect and repair damaged peripheral nerves in patients with DPN.Oxidative stress and the pathogenic mechanisms of inflammation are inextricably linked [41].Inflammation and oxidative stress affect each other [11, 12].Reducing the production of inflammatory factors can activate the Nrf2 signaling pathway [42].Thereby, it can improve the body’s antioxidant capacity and promote the body to restore the dynamic balance of oxidation and antioxidant [43].The interaction of inflammation and antioxidant stress pathways plays an important role in the development of DPN.Therefore, based on our previous findings this study explored the mechanism of pestle needle treatment of DPN from the perspective of inflammatory response.

NCV testing is the gold standard for the diagnosis of DPN.In this study, we found that the SNCV and MNCV of the right peroneal and median nerves were significantly faster in the PNG than those in the CG (P＜0.05).This indicates that the pestle needle treatment can protect and repair the damaged peripheral nerve.This is consistent with our previous study which found that the pestle needle improved the current threshold and shock threshold results in the foot region of patients with DPN [34, 35].And the TCSS was significantly reduced in the PNG compared with the CG after treatment.This indicates that the needle pestle may improve symptoms such as pain, numbness, and reduced sensation in patients with DPN.Consistent with the findings of Liu who applied pestle needle therapy to patients with low back and leg pain.Therefore, pestle needle therapy can prevent or delay the development of DPN, and resist oxidative stress and inflammatory response to nerve invasion.Eventually peripheral nerve conduction function was improved.

The inflammatory response cannot be ignored in the pathogenesis of DPN.Among the pathological mechanisms of DPN, sustained low-grade chronic inflammatory response is closely related to endothelial cell dysfunction and nerve injury.And, the inflammatory effect is amplified through the chain reaction of inflammatory mediators.Thereby the repair and regeneration of nerves are severely hampered [12].Cytokines such as TNF-α and interleukin are important immune cell activation products and are involved in the development and progression of DPN [44].The chain reaction between various inflammatory cytokines aggravates the inflammatory response in DPN, causing demyelinating lesions, pain, and slowing of NCV.TNF-α is a multifunctional inflammatory cytokine that can induce microangiopathy and neuronal apoptosis [45].High concentrations of IL-6 can lead to endothelial cell dysfunction and eventually cause nerve ischemia and nerve fiber damage.IL-1 has two biological effects, pro-inflammatory and anti-inflammatory.While long-term inflammatory responses can lead to imbalanced regulation of inflammation [46].At the same time, IL-1 can also aggravate nerve damage by inducing a variety of cytokines to produce such as TNF-α and IL-6.Then if the inflammatory response is suppressed, the symptoms of DPN can be relieved.The results of this study showed that after treatment, serum IL-1, IL-6 and TNF-α levels decreased in both groups, and the improvement of PNG was better than CG (P＜0.05).Therefore, the mechanism of the pestle needle treatment of DPN may be related to the anti-inflammatory effect [47, 48].Previous studies found that resveratrol and electroacupuncture could reduce inflammation and improve neurological function by inhibiting the nuclear factor-kappaB pathway.This is consistent with the results of this study.

Although we have reached preliminary conclusions, our research is still insufficient.First,our sample size is small,which may lead to bias.Second, it is generally accepted that oxidative stress is one of the pathogenesis of DPN.Meanwhile,oxidative stress in turn promotes the occurrence of inflammatory responses.In the current study, we only focus on the anti-inflammatory effect of pestle needle.Therefore, we can explore the mechanism of pestle needle for the treatment of DPN from the perspective of oxidative stress.Moreover, in the future,animal experiments can be performed to further explore the mechanism of pestle needle treatment of DPN from the molecular level and morphology and so on.

Conclusion

Our results indicate that the needle pestle can improve clinical symptoms as well as nerve conduction velocities in patients with DPN.The mechanism may be related to the inflammatory response.