WO2016011800A1 - Uses of recombination ganoderma lucidum immunomodulatory protein in senescence delay medicines - Google Patents

Abstract

The present invention provides uses of a recombination ganoderma lucidum immunomodulatory protein (rLZ-8) in senescence delay medicines. According to the measurement of the content of the total protein, the growth hormone, the sex hormone, the thyroid hormone and the thyroid stimulating hormone in experimental animal serum, it is found that the rLZ-8 can adjust the hormone level in the senescence process, enable the hormone level to recover to normal level, and delay the senescence process of an organism.

Description

Application of recombinant Ganoderma lucidum immunomodulatory protein in delaying aging drugs Technical field

The invention belongs to the field of biopharmaceutical and relates to the application of the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) obtained by genetic recombination in the preparation of anti-aging drugs.

Background technique

Delaying human aging is a human desire, and developing anti-aging drugs is the object of the present invention.

Ageing (senescence), also known as aging, usually refers to the decline of self-function, the stability of internal environment and the ability of stress, and the gradual degeneration of structures and components. Death, irreversible phenomenon. The aging process is reflected in the whole, organization, cells, and even the molecular level. With the increase of age, the number of parenchymal cells, response sensitivity and function of organs and tissues gradually decreased. However, the aging rate and aging method of different organs are different. When aging, the cell proliferation ability decreases, the number of functional cells gradually decreases, the hormone secretion is imbalanced, the protease activity decreases, collagen, elastin, and connective tissue are filled and cross-linked, causing the organ to shrink and function.

With the development of medical science and technology, there are various aging drugs with different therapeutic mechanisms. Among them, chemical drugs: antioxidants (such as vitamin E, vitamin C, antioxidant enzyme SOD, etc.), anti-aging hormones (such as blackened Hormone, thymosin, etc., nutrients (such as nucleic acids, etc.), monoamine oxidation Enzyme inhibitors (such as procaine), immunomodulators (such as transfer factors, etc.), biochemical agents (saliva gland, etc.), brain function promoting drugs (such as brain rehabilitation). The main characteristics of chemical drugs are quick effect, poor stability, easy rebound, and relatively large side reactions, which cannot increase the maximum life of the species.

The invention studies the hormone levels of aging rats by rLZ-8, and proves that rLZ-8 delays the progression of aging rats.

Summary of the invention

The invention relates to the application of rLZ-8 in the preparation of anti-aging drugs. Through a series of experimental means and the results, it is shown that rLZ-8 has a significant regulation effect on D-galactose-induced hormone levels in aging rats, and the specific invention contents are as follows:

In the present invention, Wistar rats are used as research objects, and D-galactose is used to induce a sub-acute aging rat model of aging, and the design includes a normal control group, a model group, a positive control drug vitamin E (100 mg/kg), and a low rLZ-8. Six experimental groups including the dose group (5 μg/kg), the rLZ-8 medium dose group (10 μg/kg), and the rLZ-8 high dose (50 μg/kg). The mode of administration and procedure were designed to be administered intraperitoneally, once a day for 8 weeks, and each group of rats was weighed once a week for 8 weeks after treatment. Body weight of aging model rats, serum total protein content, serum growth hormone (GH), sex hormones [testosterone (T) progesterone (P) estradiol (E ₂ ) follicle stimulating hormone (FSH) luteinizing body The levels of pheromone (LH), thyroid hormone (FT ₄ ) and thyrotropin (TSH) were measured using iodine [ ¹²⁵ I] free thyroxine radioimmunoassay kit, iodine [ ¹²⁵ I] free Triiodothyronine radioimmunoassay kit, iodine [ ¹²⁵ I] human thyrotropin radioimmunoassay kit, iodine [ ¹²⁵ I] progesterone radioimmunoassay kit, iodine [ ¹²⁵ I] human luteinizing hormone Radioimmunoassay kit, iodine [ ¹²⁵ I] human growth hormone radioimmunoassay kit, iodine [ ¹²⁵ I] human follicle stimulating hormone radioimmunoassay kit, iodine [ ¹²⁵ I] testosterone radioimmunoassay kit, etc. The above aspects were measured. The results showed that rLZ-8 can significantly improve the general state of D-galactose-induced subacute aging rats. The serum GH content in rats is significantly increased, and the contents of E ₂ , P and T in blood are significantly increased. Elevated, FSH, LH content decreased, and FT ₃ , FT ₄ and TSH levels increased.

A series of results of the above experiments showed that rLZ-8 can delay the aging of rats by regulating the levels of GH, sex hormones and thyroid hormones in subacute aging rats induced by D-galactose.

The objectives, features, and advantages of the invention are disclosed in the Detailed Description of the Detailed Description of the Drawings.

detailed description

Example 1: Establishment of a aging animal model

Experimental material

Healthy Wistar rats, male and female, provided by the Experimental Animal Center of Jilin University; balance, 1ml disposable syringe, rLZ-8 (provided by Jida Diamond Center), D-galactose (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) , vitamin E.

2. Experimental methods

In this experiment, D-galactose was used to induce a subacute aging model in rats. First, D-galactose was dissolved in physiological saline to prepare a 5% D-galactose physiological saline solution, and the rats were intraperitoneally injected at a dose of 500 mg/kg per day. The normal control rats were intraperitoneally injected with an equal volume. The saline was continuously molded for 8 weeks, and the rats in each group were weighed once a week, and the total protein content in the serum of the rats was measured.

3. Experimental results

Table 1 Changes in body index of rat subacute aging model

Modeling days	Weight (g)	Total protein content (g/L)
			Week 4	259.38±44.71	60.03±7.66
Week 8	232.23±62.96	50.63±5.83

The experimental results show that in terms of body weight, with the prolongation of modeling time, the weight of rat model has a significant downward trend, and the total protein content in serum also decreases with the prolongation of modeling time, indicating that the modeling method is suitable for this experiment. , indicating that the modeling experiment was successful.

Example 2 Relief effect of rLZ-8 on rat aging model

Experimental material

Healthy Wistar rats, male and female, provided by the Experimental Animal Center of Jilin University; balance, 1ml disposable syringe, rLZ-8 (provided by Jida Diamond Center), D-galactose (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) , vitamin E. Iodine [ ¹²⁵ I] free thyroxine radioimmunoassay kit, iodine [ ¹²⁵ I] free triiodothyronine radioimmunoassay kit, iodine [ ¹²⁵ I] human thyrotropin radioimmunoassay kit, iodine [ ¹²⁵ I] Progesterone radioimmunoassay kit, iodine [ ¹²⁵ I] human luteinizing hormone radioimmunoassay kit, iodine [ ¹²⁵ I] human growth hormone radioimmunoassay kit, iodine [ ¹²⁵ I] human follicle stimulating hormone Radioimmunoassay kit, iodine [ ¹²⁵ I] testosterone radioimmunoassay kit, the above analysis kits were purchased from Beijing North Institute of Biotechnology, batch number: 130902. The radioimmunoassay kit for iodine [ ¹²⁵ I]estradiol was purchased from Shenzhen Lalwin Bioengineering Technology Co., Ltd., batch number: 130820, Coomassie Brilliant Blue Kit, purchased from Nanjing.

2. Experimental methods

2.1 Experimental grouping and drug administration procedures:

Seventy-two healthy Wistar rats weighing between 200-250 g were randomly divided into 6 groups according to body weight, 12 rats in each group, administered intraperitoneally and once a day. Normal control group: equal volume of normal saline; model group: equal volume of normal saline; vitamin E group: vitamin E 100 mg/kg; rLZ-8 high dose rLZ-850 μg/kg; rLZ-8 medium dose group for rLZ -8 protein 10 μg / kg; rLZ-8 low dose group was given rLZ-8 protein 5 μg / kg.

2.2 Experimental testing items and methods:

In this experiment, D-galactose was used to induce a subacute aging model in rats. First, D-galactose was dissolved in physiological saline to prepare a 5% D-galactose physiological saline solution, and the rats were intraperitoneally injected at a dose of 500 mg/kg per day. The rats in the normal control group were intraperitoneally injected with the same volume of physiology. Saline was continuously molded for 8 weeks, and rats of each group were weighed once a week. After the successful modeling, the rats were continuously administered for 8 weeks, and the rats in each group were weighed once a week, and were taken after 8 weeks of treatment. The changes in general state such as appetite, body weight, behavior, coat color and gloss were observed. Rats were harvested from the eyeballs and centrifuged at 3500 r/min for 10 min. The supernatant was taken and the total protein content in the serum was determined by Coomassie Brilliant Blue method. Radioimmunoassay (RIA) measures changes in growth hormone (GH) levels in rat serum. The levels of testosterone (T), progesterone (P), estradiol (E ₂ ), follicle stimulating hormone (FSH) and luteinizing hormone (LH) in serum of each group were determined by radioimmunoassay (RIA). . The levels of free triiodothyronine (FT ₃ ), free thyroxine (FT ₄ ) and thyrotropin (TSH) in the serum of each group were determined by radioimmunoassay (RIA). Kit. Experimental data using mean and standard deviation

It is indicated that the comparison of multiple sample means is performed by one-way ANOVA, and the mean is compared with the q-test. The test results are all taken as p<0.05 as a difference to investigate the statistical significance.

3. Experimental results

3.1 General status

The rats in the normal control group were active and active, the diet was normal, the body weight was significantly increased, the hair was bright and dense, and the skin elasticity was good. After modeling, the rats in the model group gradually showed weakness in the limbs, slow movement, and burnout. The skin of the rats was yellow and yellow, tarnished, and dried and shed. Rats in each treatment group improved in terms of action and hair status compared to the model group.

3.2 Weight changes

During the experiment, the body weight of the rats in the normal control group gradually increased. The body weight of the rats in the model group and each administration group decreased before administration, and the body weight increased after 8 weeks of administration. Compared with before administration, rLZ The body weight of the -8 low- and medium-dose groups increased, but there was no statistical significance, while the weight gain of the rLZ-8 high-dose group was significant (p<0.05), which was statistically significant.

Table 2 Changes in body weight of rats in each group (unit: g)

	normal group	Negative control group	Vitamin E group	rLZ-8 high dose group	rLZ-8 medium dose group	rLZ-8 low dose group
							Before modeling	210.4±21.09	203.08±12.00	208.36±7.16	205.46±14.55	204.00±15.62	208.93±9.57
Before administration	312.2±73.94	201.62±47.10	195.86±31.52	203.85±42.95	199.31±46.12	197.29±34.19
							After administration	371.9±115.15 **	215.7±41.59	241.3±54.64 *##	248.6±66.95 *#	232.7±52.34 *	217.70±54.94

Compared with the previous ^{modeling: * p <0.05, ** p} <0.01; compared with before ^{administration: # p <0.05, ## p} <0.01.

3.3 Effect of rLZ-8 on total protein content in rats of each group

The results of rLZ-8 on the total protein content of rats in each group are shown in Table 3. Compared with the normal control group, the total protein content in the serum of the negative control group was significantly reduced (p<0.05); compared with the negative control group, the vitamin The total protein content in serum of group E and rLZ-8 high dose group increased (p<0.05), with statistical difference.

Table 3 Total protein content in serum of each group of rats

Group	Total protein content (g/L)
		Normal control group	67.12±11.53
Negative control group	47.03±8.23 *
		Vitamin E group	59.68±9.03 #
rLZ-8 high dose group	58.71±7.13 #
		rLZ-8 medium dose group	51.14±11.52
rLZ-8 low dose group	53.45±10.02

Compared with the normal control group: ^* p <0.05; compared with the negative control group: ^# p < 0.05.

3.3 Effect of rLZ-8 on growth hormone content in rats

The levels of growth hormone in the serum of rats were measured by radioimmunoassay, as shown in Table 4. The results showed that compared with the normal control group, the serum GH content of the negative control group was decreased (p<0.05); compared with the negative control group, The serum GH content in the vitamin E group and each administration group increased, and increased significantly in the high dose group of rLZ-8 (p<0.01).

Table 4 Serum growth hormone content (unit: ng / mL)

Group	Growth hormone (GH) content
		Normal control group	3.82±0.48
Negative control group	2.86±0.99 *
		Vitamin E group	3.99±0.93 #
rLZ-8 high dose group	4.44±0.79 *##

rLZ-8 medium dose group	3.90±1.01 #
		rLZ-8 low dose group	3.92±0.94 # #

Compared with the normal control group: ^* p <0.05; compared with the negative control group: ^# p < 0.05.

3.4 Effects of rLZ-8 on sex hormone levels in rats

The serum levels of sex hormones in rats were measured by radioimmunoassay. It can be seen from Table 5:

(1) Compared with the normal control group, the serum follicle stimulating hormone content in the negative control group was significantly increased (p<0.01); compared with the negative control group, the serum follicle stimulating hormone content in each group was decreased (p< 0.05), in the rLZ-8 high-dose group can significantly reduce the content of follicle stimulating hormone (p <0.01), the difference was statistically significant.

(2) The serum levels of luteinizing hormone in the negative control group were significantly higher than those in the normal group (p<0.01). Compared with the negative control group, the serum luteinizing hormone content in each group was decreased. The difference (p<0.05), rLZ-8 high dose and rLZ-8 medium dose group significantly decreased luteinizing hormone content (p<0.01).

(3) Compared with the normal control group, the serum levels of estradiol in the negative control group were significantly lower (p<0.01); compared with the negative control group, the serum in the low, medium and high dose groups of rLZ-8 The alcohol content increased, and the rLZ-8 low and high dose groups increased significantly (p<0.05), and the difference was statistically significant.

(4) Compared with the normal group, the serum progesterone level in the negative control group was significantly lower (p<0.05); compared with the negative control group, the progesterone content in the serum of the vitamin E group and the rLZ-8 group Both increased, and increased significantly in the middle and high dose groups of rLZ-8 (p<0.01), which was statistically significant.

(5) Compared with the normal group, the testosterone content in the serum of the negative control group was significantly decreased (p<0.01); compared with the negative control group, the serum progesterone level in the high dose group of rLZ-8 was significantly increased (p<0.05). ),has statistical significane.

Table 5 serum levels of sex hormones in each group of rats

Compared with the control ^{group: * p <0.05, ** p} <0.01; compared with model ^{group: # p <0.05, ## p} <0.01.

Effects of 3.5 rLZ-8 on thyroid hormone and thyroid stimulating hormone in rats

Radioimmunoassay was used to detect thyroid hormone and thyroid stimulating hormone levels in rat serum. The results are shown in Table 6:

(1) Compared with the normal control group, the FT ₃ content in the serum of the negative control group was significantly decreased (p<0.05); compared with the negative control group, the serum of the vitamin E group and the rLZ-8 group were in the serum. The FT ₃ content increased, and the increase was significantly higher in the rLZ-8 high-dose group (p<0.05), with statistical difference.

(2) Compared with the normal control group, the serum FT ₄ content in the negative control group was decreased (p<0.01); compared with the negative control group, the rLZ-8 high dose and the rLZ-8 low dose group were in the serum. The FT ₄ content increased significantly (p < 0.05) with statistical differences.

(3) Compared with the normal control group, the serum TSH content in the negative control group was significantly decreased. Low (p<0.05); compared with the negative control group, the TSH content of the vitamin E group and each rLZ-8 administration group increased (p<0.05), and the difference was statistically significant.

Table 6 thyroid hormone and thyroid stimulating hormone content (unit: ng / mL)

Compared with the normal control group: ^* p < 0.05, ^** p <0.01; compared with the negative control group: ^# p < 0.05, ^## p < 0.01.

Through the above embodiments, the object of the present invention has been fully achieved. Those skilled in the art will appreciate that the present invention includes, but is not limited to, the drawings and the details described in the Detailed Description. Any modifications that do not depart from the functional and structural principles of the invention are intended to be included within the scope of the appended claims.

Claims (5)

1. The application of rLZ-8 in anti-aging drugs.
2. The use of claim 1 wherein rLZ-8 increases total protein, growth hormone, thyroid hormone, and thyroid stimulating hormone levels in the body.
3. According to the application of claim 1, rLZ-8 lowers the content of follicle stimulating hormone and luteinizing hormone in the body while increasing the contents of testosterone, progesterone and estradiol, and effectively regulating the balance of sex hormones in the body.
4. The use according to claim 1, characterized in that the core component of the pharmaceutical preparation consists of rLZ-8 according to claim 1 and optionally a pharmaceutically acceptable adjuvant.
5. The use according to claim 1, wherein the administration route is oral or parenteral administration, wherein oral administration includes oral liquids, tablets, pills and capsules; and parenteral administration includes external preparations and injections.