THE ROLE OF β-ENDORPHIN AND C-PEPTIDE IN STREPTOZOTOCIN INDUCED DIABETIC RATS WITH EFFECT OF ACU-TENS AT ZUSANLI (ST36) ACUPOINTS

THE ROLE OF β-ENDORPHIN AND C-PEPTIDE IN STREPTOZOTOCIN INDUCED DIABETIC RATS WITH EFFECT OF ACU-TENS AT ZUSANLI (ST36) ACUPOINTS

R. Subbulakshmi ^{* 1} and Girija Sivakumar ²

Department of Anatomy ¹, Vivekanandha Dental College for Women, Tiruchengode, Namakkal - 637205, Tamil Nadu, India.

Department of Anatomy ², Karpaga Vinayaga Institute of Medical Sciences, Maduranthagam, Chennai - 603308, Tamil Nadu, India.

ABSTRACT: Streptozotocin is 2-Deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose drug is commonly used to induce diabetes in experimental rats. This study was designed to evaluate the role of β-endorphin and C-peptide in Acu-TENS therapy at Zusanli (ST36) acupoint. Diabetic was induced by Streptozotocin (45mg/kg/body weight) with a single dose of intraperitoneal injection in male albino Wistar rats. The diabetic rats were treated with Acu-TENS electrical stimulation with a pulsed current of low frequency and high intensity at bilateral Zusanli (ST36) acupoint without anesthesia for 60 days, and the effect was compared with pioglitazone (7.5 mg/kg/body weight) treated diabetic rats on fasting blood glucose, β-endorphin & C-peptide levels. The results showed that a significant reduction (p≥0.01) in fasting blood glucose and increased β-endorphin and C-peptide level in Zusanli (ST36) acupoint treated diabetic rats. The anti-diabetic effect of Zusanli (ST36) acupoint may be due to stimulation of Qi (life energy) and regulating the circulation of blood and an increase in insulin secretion through the mediation of β- endorphin.

Zusanli (ST36), Acupoint, Streptozotocin, Diabetes, Acu-TENS, β-endorphin

INTRODUCTION: ‘Streptozotocin is a glucos-amine-nitrosourea compound, and alkylating agent that damages DNA and is particularly toxic to pancreatic beta cells is widely used to induce experimental diabetic animal models’ ¹. Diabetes mellitus is a metabolic disorder characterized by hyperglycemia (elevated blood glucose) with abnormalities of carbohydrates, protein, and fat metabolism due to absolute or relative deficiency of insulin secretion and varying degrees of insulin resistance.

The long term complications of diabetes lead to diseases of large blood vessels (macrovascular disease including coronary heart disease and peripheral arterial disease) and small blood vessels (microvascular disease, including retinal and renal vascular disease) ². Diabetes is a significant and growing threat to global health ³. The global prevalence of diabetes as in 2016, 422 million people have diabetes worldwide, and the number of people with diabetes will expect to rise up to 592 million by 2035 ⁴. In India, diabetes is gaining the status of a potential epidemic disease ^{5, 6} and becoming the diabetes capital of the World with a projected 109 million people with diabetes by 2035 ⁷. Type 2 diabetes (NIDDM) is a common type of diabetes, adult-onset with the age group of > 40 years, occurs due to the combination of insulin resistance and reduced insulin secretion.

Obesity and physical inactivity are the leading risk factors for type 2 diabetes. Though medical treatment is convenient and effective, certain treatments can lead to serious adverse effects and are not always satisfactory in treating and preventing the long-term complications of diabetes ^{8, 9}. Patients with poorly controlled type 2 diabetes (non-insulin-dependent diabetes) who frequently use oral hypoglycemic agents often present with unstable glucose levels ¹⁰. Increasing evidence suggests that C-peptide may be useful in predicting future levels of glycemic control, response to hypoglycemic agents, and risk of future diabetes complications. C-peptide and insulin are synthesized as a single polypeptide chain called proinsulin in the beta cells of pancreatic islet. Proinsulin cleaved and formed equal amounts of insulin and C-peptide, both entered the portal vein ¹¹. The great interest in C-peptide is due to the limitations of the use of serum insulin as a measure of insulin secretion because insulin, after its secretion into the portal vein, passes through the liver where approximately 50% of the delivered insulin is extracted ¹². Within the physiologic range of insulin concentrations, there is a variable fraction of hormone extracted by the liver. Peripheral insulin concentration therefore is the post hepatic insulin delivery rather than the secreted insulin by the pancreatic beta cells ¹². The measurement of C-peptide, which is co-secreted with insulin from beta cells of the pancreas, thus provides a better index of endogenous insulin production and pancreatic beta-cell function than insulin measurements ¹³.

In recent years, with the advancement of medicine and treatments, alternative therapies like acupuncture therapy, herbal medicine, dietary supplementation, yoga therapy, and hot tub therapy have become increasingly popular due to less adverse effects for treating diabetes. Acupuncture therapy, one of the oldest and most commonly used forms of alternative medicine, has existed for more than 2500 years. Various methods like exercise, diet control, and medication are also included for controlling blood glucose levels in patients with diabetes mellitus. Researchers have, therefore, searched for alternative or complementary treatment methods with no side effects to increase insulin sensitivity ^14-17. The effectiveness of acupuncture in diabetes had observed both in reducing the symptoms and preventing the long-term complications of diabetes. The effects of acupuncture on diabetes have been observed both experimentally and clinically ¹⁸. Acupuncture can act on the pancreas to enhance insulin synthesis, increase the number of receptors on target cells, and accelerate the utilization of glucose, resulting in the lowering of blood sugar ¹⁹. Various studies have shown the beneficial effect of acupuncture on obesity, which is the most modifiable risk factor for type 2 diabetes. The therapeutic effect of acupuncture on diabetes is mainly based on the action on multiple systems ²⁰. Nowadays, electro-acupuncture has been a growing interest in the glucose-lowering effect.

In our study, Zusanli (ST36) acupoint electric stimulation was selected for the treatment of diabetes based on traditional Chinese medicine²¹. The release of β-endorphin lowers the plasma glucose levels has been demonstrated by electro-acupuncture at the Zusanli acupoints in rats ²². Endorphins (endogenous morphine) are endogenous opioid neuropeptides and peptide hormones. They are produced by CNS, pituitary gland, and endocrine pancreas ²³. Endorphins, a multi-functional chemical, are emitted to counteract and deal with sensations by transmitting electrical impulses through the body to the nervous system. Acupuncture therapy and meditation stimulates the release of endorphin. Beta-endorphin acts as a stimulant for an appetite that helps in the release of insulin from the beta cells of pancreas. Reid RL, in his study, explained the effect of beta-endorphin (2.5 mg IV bolus) to type 2 DM patients, showed a progressive decrease in plasma glucose ²⁴. The lowering in plasma glucose action in diabetes is believed to involve the promotion of β- endorphin secretion of insulin ¹⁵. Electro-acupuncture (EA) is commonly used to stimulate the acupoints, an invasive technique with a needle than manually.

In this study, Transcutaneous electric nerve stimulation at acupoints (Acu-TENS) therapy, a non-invasive method, was selected to stimulate the acupoints, which combines the advantages of both electrical stimulation and acupuncture therapy. The application of acupuncture stimulation facilitates the local circulation system. It enhances glucose levels in serum, and these effects may be comparable to those produced as a result of physical exercise ¹⁵.

In this study, we aimed to investigate the effects of Zusanli acupoints stimulation with Acu-TENS (15Hz) on levels of fasting blood glucose, C-peptide, and β-endorphin in diabetic rats.

FIG. 1: POSSIBLE MECHANISM WITH ACU-TENS STIMULATION AT ACUPOINTS

FIG. 2: STREPTOZOTOCIN - MODE OF ACTION

MATERIALS AND METHODS:

Experimental Animals: Male Wistar rats with a weight of 200-250gms were selected for this study. The experimental animals were kept well ventilated and maintained at controlled temperatures with 12 hours of light and dark cycles. Animals are given with standard laboratory diet and water ad libitum. The animal experiment was carried out as per CPCSEA guideline, and this study was approved by the Institutional Animal Ethical Committee (Reg. no. 889/PO/Re/S/05/CPCSEA/2018) of Swamy Vivekanandha College of Pharmacy, Elayampalayam, Tiruchengode.

Experimental Groups: Four groups with six animals in each were divided.

Group 1:  Rats were kept in control.

Group 2: Rats received a single dose of Streptozoto-cin (STZ) (45 mg/kg/body weight) intraperitonally, the nitrosourea alkylating agent with selective uptake into pancreatic β cells produce diabetes in the rats. The induction of diabetes mellitus was confirmed after 72 h of Streptozotocin treatment by estimation of elevated fasting blood glucose level above ≥250 mg/dl included in this study.

Group 3: STZ induced diabetic rats given pioglitazone drug orally via intragastric (7.5 mg/kg/body weight) for 60 days.

Group 4: STZ induced diabetic rats treated with Zusanli (ST36) acupoint with Acu-TENS stimulator with 15HZ for 60 days.

Electrical stimulation was carried out for 5 min daily at the frequency of 15Hz, pulse width 5 sec, intensity 15mA, using Acu-TENS unit without anesthesia. The positive and negative charges were introduced through electrodes to the right and left Zusanli acupoints, respectively. The experiment was carried out for 60 days, and the blood sample was collected from the tail vein and by retro-orbital venous plexus puncture. Fasting Blood glucose was measured and compared periodically between the groups. β-endorphin and C-peptide levels in the blood serum were measured and compared between the groups.

FIG. 3: Acu-TENS THERAPY AT ZUSANLI ACUPOINTS WITH LOW- FREQUENCY(15HZ), HIGH INTENSITY AND THE PULSE WIDTH OF 5 sec, FOR 5 min DAILY WITHOUT ANESTHESIA FOR 60 DAYS

Statistical Analysis: The results were expressed as Mean± SD. The data of blood glucose level, β-endorphin and C-peptide were analyzed by one-way ANOVA followed by Tukey post hoc test for pairwise comparison between the means. The value of p≤ 0.05 was considered as statistically significant.

RESULTS: A significant decrease (p≤0.01) in fasting blood glucose in the toxicity rats was observed at the end of 60^th day treatment and was compared to the control and pioglitazone (7.5 mg/kg/bwt) groups showed in Table 1. Graph 1 & 2 showed the serum β-endorphin and C- peptide levels were significantly (p≤0.01) increased in the bilateral Zusanli acupoint treated rats and compared with pioglitazone group. Increasing β-endorphin activates the specific receptors in the pancreatic β cells and secrete insulin that results in a decrease in blood glucose levels, considered the mechanism of Acu-TENS therapy at Zusanli acupoints.

TABLE 1: EFFECTS OF ST36 ON FASTING BLOOD GLUCOSE LEVEL (mg/dl)

The results were expressed as Mean ± SD. A comparison was done between control and diabetic groups with one way ANOVA and Tukey test with (p≤0.01).

GRAPH 1: EFFECTS OF ZUSANLI (ST36) ACUPOINT ON β-ENDORPIN (pg/dl). The results were expressed as mean ± SD. A comparison was done between the control and diabetic group with one-way ANOVA and Tukey test with (p≤0.01).

GRAPH 2: EFFECTS OF ZUSANLI (ST36) ACUPOINT ON C-PEPTIDE (ng/dl). The results were expressed as mean ± SD. Comparison was done between control and diabetic groups with one way ANOVA and Tukey test with (p≤0.01).

DISCUSSION: Streptozotocin, a compound of glucosamine-nitrosourea, is toxic to pancreatic beta cells and induces diabetes in experimental animals. Diabetes is one of the major public health problems worldwide. For patient everyday medication and insulin was economically high compared to acupuncture treatment. Now a day peoples showing interest in alternative therapies, mainly acupuncture, due to low therapeutic costs. Recently acupuncture therapy has spread throughout the World. Increasing evidence from scientific research is demonstrating the effectiveness of electro-acupuncture for treating various diseases and revealing the mechanism of action. From a biochemical analysis, acupuncture is believed to stimulate the nervous system, influencing the productions of the body’s communication substances like hormones and neurotransmitters. The process of homeostasis activating the body’s self regulating systems, thus stimulating its natural healing abilities and promoting physical and emotional well being ²⁵.

In this study, we found that Acu-TENS therapy at bilateral Zusanli acupoints resulted in a significant reduction in fasting blood glucose levels with an increase in β-endorphin and C-peptide levels in the toxicity induced diabetic rats. Hui - Ching Pai et al., study shows EA (15HZ) at bilateral Zusanli (ST36) acupoint stimulation with rosiglitazone proves hypoglycemic effect due to an increase in insulin secretion and through the mediation of β- endorphin in normal and STZ-induced type 2 diabetic rats ²². In this study, we stimulated Zusanli acupoint with Acu-TENS unit and found a significant reduction of fasting blood glucose with an increase in serum β- endorphin and compared with the standard drug (pioglitazone) group. Shih-Liang Chang et al., explained EA(2HZ) stimulation at both Zusanli acupoints (ST36) resulted in a hypoglycemic effect with an increase in plasma β- endorphin and insulin in STZ induced diabetic rats²⁶.In this study, we selected Acu-TENS (15HZ) to stimulate Zusanli acupoint and found the hypoglycemic effect with an increase in serum β-endorphin and C-peptide levels. Dr. Mehmet and Cho Sha et al., explained electro-acupuncture has a significant influence in lower blood glucose levels of type 2 DM, lowering cholesterol level, decreasing free-fatty acid, eliminating diabetic neuropathy, and lowering body mass index ^{27, 28}.

In our previous study, we noted that Acu-TENS therapy at Zusanli acupoint restored the body weight, food, and water intake in induced diabetic rats. Fengxia Liang et al., explained the hypoglycemic effect with low frequency (3HZ) electro-acupuncture stimulation at ST36/Zusanli acupoint in type 2 diabetic mice ²⁹. In this study, we found that the hypoglycemic effect of Acu-TENS Zusanli acupoint in diabetic rats. Yu-Chen Lee et al. explained electro-acupuncture at the ST36 acupoint produces a hypoglycemic effect due to stimulation of a cholinergic nerve in STZ- induced diabetic rats ¹⁰.

In our study, the Acu-TENS stimulation at Zusanli acupoint reduces the blood glucose level may be due to selective stimulation of muscle contraction in STZ induced diabetic rats. According to Chung-Yuh Tzeng et al., explained intracellular signaling pathways associated with glucose- lowering effect of ST36 electro-acupuncture in STZ-induced diabetic rats³⁰. P. V. Ingle et al., stated the decrease in fasting blood glucose and lower level of TC, TG, LDL, and increase HDL with stimulation of ST36 acupoint with Accu-stimulator apparatus in type 2 diabetes ³¹. In our study, using the Accu-Stimulator apparatus shows the reduction of fasting blood glucose with an increase in β-endorphin and C-peptide levels.

According to traditional Chinese medicine, acupuncture therapy can be practiced either manual or electrical with Acu needles. Zhi-Qi-Zhao et al., mentioned that in electro-acupuncture, the stimulating current is delivered to acupoints via the acu needles connected to an electrical stimulator. Instead of the insertion of acupuncture needles, a surface electrode on the skin over the acupoints is also described as electro-acupuncture ³².

Hence, in our study, we selected the Acu-TENS apparatus to stimulate the Zusanli acupoint with pen-type electrodes in rats, which results in a hypoglycemic effect. Wu Zhiyuan et al., explained the effect of TENS at specific acupoints (LI11, LI4, ST36, SP6) on type 2 diabetes patients for 30 minutes for each session and 5 times a week for 2 months. In our study, stimulation of a single Zusanli acupoint for 5 minutes daily for 60-day treatment in diabetic rats shows a significant decrease in fasting blood glucose (FBG) level.

Acu-TENS, non-invasive technique, has the beneficial effect of preventing the risk of infection, especially to diabetic patients ³³. Some previous research shows that the transcutaneous electrical muscle stimulation with a low stimulation frequency could be an effective method to enhance glucose metabolism ^{34, 35}. We selected Acu-TENS stimulator due to its combined effect of both electrical and acupoints stimulation. Wu Zhiyuan et al., explained treatment with Acu-TENS (10 HZ for 30 minutes) at several acupoints in type 2 diabetes patients produced a beneficial effect on blood glucose level ³³. Catalogna et al., explained utilization of glucose and improved hepatic insulin sensitivity in rats with peripheral electrical stimulation (PES) treatment with 2-3 min ³⁷.

In this study, Acu-TENS with the frequency of 15 HZ (low frequency) produced a positive effect in reducing blood glucose level in type 2 diabetes with increase β-endorphin. Hence, the result of the present study suggests that non-invasive acupuncture therapy is a beneficial and cost-effective treatment for elderly type 2 diabetes mellitus patient.

CONCLUSION: We concluded that Acu-TENS therapy at Zusanli (ST36) acupoints produced its hypoglycemic effect might due to the role of β-endorphin. The other advantages of Acu-TENS therapy with low frequency and high-intensity electrical stimulation at bilateral ST36 acupoint causes muscle contraction and mild muscular twitching, which was similar to that of the physiological process of physical exercise. Hence, this therapy can be useful for elderly diabetes patients who were not able to perform physical exercise. We suggest Acu-TENS therapy a cost-effective non-invasive alternative therapy for diabetic patients not only to control diabetes but also to prevent its complications.

ACKNOWLEDGEMENT: Nil

FINANCIAL SUPPORT AND SPONSORSHIP: Nil

CONFLICTS OF INTEREST: There are no conflicts of interest.

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    How to cite this article:

    Subbulakshmi R and Sivakumar G: The role of β- endorphin and c-peptide in streptozotocin induced diabetic rats with effect of Acu-TENS at Zusanli (ST36) acupoints. Int J Pharm Sci & Res 2020; 11(12): 6532-38. doi: 10.13040/IJPSR.0975-8232.11(12).6532-38.

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