阅读说明：本技术 6α-乙基-23(S)-甲基胆酸在制备治疗周围神经髓鞘损伤类疾病的药物中的应用 (Application of 6 alpha-ethyl-23 (S) -methyl cholic acid in preparation of medicine for treating peripheral nerve myelin sheath damage diseases ) 是由 刘晓宇 孙诚 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种6α-乙基-23(S)-甲基胆酸在制备治疗周围神经髓鞘损伤类疾病的药物中的应用。本发明提供了激动剂6α-乙基-23(S)-甲基胆酸的新用途,且效果好；研究机理的方法简便,准确。(The invention discloses application of 6 alpha-ethyl-23 (S) -methyl cholic acid in preparation of a medicine for treating peripheral nerve myelin sheath injury diseases. The invention provides a new application of an agonist 6 alpha-ethyl-23 (S) -methyl cholic acid, and the effect is good; the method for researching the mechanism is simple, convenient and accurate.)

1. Application of 6 alpha-ethyl-23 (S) -methyl cholic acid in preparing medicine for treating peripheral nerve myelin sheath damage diseases is disclosed.

2. The use of 6 α -ethyl-23 (S) -methylchollic acid according to claim 1 for the preparation of a medicament for the treatment of diseases of the peripheral nerve myelin sheath damage type, characterized in that: the mechanism research method comprises the following steps:

(1) establishment of sciatic nerve clamp injury model

Taking injured sciatic nerve of 12-week-old male wild type C57BL/6 mouse injected with 6 alpha-ethyl-23 (S) -methyl cholic acid;

(2) transmission electron microscopy morphological analysis

Transecting sciatic nerve tissues of different groups for 1-2mm, and soaking in mixed solution containing 4% paraformaldehyde and 2.5% glutaraldehyde for 24 hr; tissue samples were fixed in 1% osmium tetroxide for 1 hour and embedded in Epon812 epoxy; staining the slices with lead citrate and uranyl acetate, and observing under a transmission electron microscope of 80 KV; g ratio is the ratio of the average inner diameter of axons to the average diameter of fibers, and is analytically calculated using Image J software;

(3) behavioral detection analysis

The rod rotating experiment adopts an accelerated rod rotating device, and before the experiment, a mouse is habituated for 30 minutes in a laboratory; by swinging the mice, the mice were placed on the twill horses and trained in an accelerated mode for 3 days at intervals of 3 minutes and 5 minutes on the twill horses; repeating the training at a constant speed until the mouse is able to rest on the bar for at least 300 seconds; for the official test, the mouse was placed on a rotating drum and the carousel was set to acceleration mode; recording the time of movement on the rod before the drop is measured;

gait experiment the gait dynamics of the quadruped animals are evaluated by analyzing the footprints of the mice through a Digigate imaging system, and the stride of the hind limb is evaluated at the speed of 8 cm/s; footprints were analyzed by four measurement methods: distance from the opposite foot, footprint length, maximum toe extension between the first and fifth toes, and paw extension between the centers of the second and fourth toes;

(7) results

The results show that treatment with 6 α -ethyl-23 (S) -methylcholic acid promotes the progression of sciatic nerve remyelination in mice post-bruising compared to wild type mice post-bruising; the transmission electron microscope result indicates that the myelin sheath thickness of sciatic nerve of the mouse, the remyelination process is accelerated, and the movement balance capability is enhanced; gait experiment results indicate that the exercise recovery capacity of the mice in the 6 alpha-ethyl-23 (S) -methyl cholic acid treatment group is far better than that of wild mice after sciatic nerve bruise.

Technical Field

The invention relates to a new application of 6 alpha-ethyl-23 (S) -methyl cholic acid.

Background

The existing drugs for treating peripheral nerve demyelinating diseases have certain defects in the aspects of treatment effect and the like, and new drugs need to be further searched and developed. The effect of 6 alpha-ethyl-23 (S) -methyl cholic acid is not reported to be capable of preparing a medicament for treating peripheral nerve myelin sheath damage diseases.

Disclosure of Invention

The invention aims to provide application of effective 6 alpha-ethyl-23 (S) -methyl cholic acid in preparing a medicament for treating peripheral nerve myelin sheath damage diseases.

The technical solution of the invention is as follows:

application of 6 alpha-ethyl-23 (S) -methyl cholic acid in preparing medicine for treating peripheral nerve myelin sheath damage diseases is disclosed.

The mechanism research method comprises the following steps:

(1) establishment of sciatic nerve clamp injury model

Taking injured sciatic nerve of 12-week-old male wild type C57BL/6 mouse injected with 6 alpha-ethyl-23 (S) -methyl cholic acid;

(2) transmission electron microscopy morphological analysis

Transecting sciatic nerve tissues of different groups for 1-2mm, and soaking in mixed solution containing 4% paraformaldehyde and 2.5% glutaraldehyde for 24 hr; tissue samples were fixed in 1% osmium tetroxide for 1 hour and embedded in Epon812 epoxy; staining the slices with lead citrate and uranyl acetate, and observing under a transmission electron microscope of 80 KV; g ratio is the ratio of the average inner diameter of axons to the average diameter of fibers, and is analytically calculated using Image J software;

(3) behavioral detection analysis

The rod rotating experiment adopts an accelerated rod rotating device, and before the experiment, a mouse is habituated for 30 minutes in a laboratory; by swinging the mice, the mice were placed on the twill horses and trained in an accelerated mode for 3 days at intervals of 3 minutes and 5 minutes on the twill horses; repeating the training at a constant speed until the mouse is able to rest on the bar for at least 300 seconds; for the official test, the mouse was placed on a rotating drum and the carousel was set to acceleration mode; recording the time of movement on the rod before the drop is measured;

gait experiment the gait dynamics of the quadruped animals are evaluated by analyzing the footprints of the mice through a Digigate imaging system, and the stride of the hind limb is evaluated at the speed of 8 cm/s; footprints were analyzed by four measurement methods: distance from the opposite foot, footprint length, maximum toe extension between the first and fifth toes, and paw extension between the centers of the second and fourth toes;

(7) results

The results show that treatment with 6 α -ethyl-23 (S) -methylcholic acid promotes the progression of sciatic nerve remyelination in mice post-bruising compared to wild type mice post-bruising; the transmission electron microscope result indicates that the myelin sheath thickness of sciatic nerve of the mouse, the remyelination process is accelerated, and the movement balance capability is enhanced; gait experiment results indicate that the exercise recovery capacity of the mice in the 6 alpha-ethyl-23 (S) -methyl cholic acid treatment group is far better than that of wild mice after sciatic nerve bruise.

The invention provides a new application of an agonist 6 alpha-ethyl-23 (S) -methyl cholic acid, and the effect is good; the method for researching the mechanism is simple, convenient and accurate.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a schematic representation of INT777 promoting sciatic nerve remyelination after a pinch injury.

Figure 2 is a statistical representation of the effect of INT777 on sciatic nerve myelin thickness after a pinch.

FIG. 3 is a graphical representation of the effect of INT777 on the motor balance ability of sciatic nerve-clipped mice.

FIG. 4 is a schematic representation of the effect of INT777 on the recovery of motor function in sciatic nerve-pinched mice.

Detailed Description

Application of 6 alpha-ethyl-23 (S) -methyl cholic acid in preparing medicine for treating peripheral nerve myelin sheath damage diseases is disclosed.

The mechanism research method comprises the following steps:

firstly, experimental steps

1. Establishment of sciatic nerve clamp injury model

6 alpha-ethyl-23 (S) -methyl cholic acid (INT 777, CAS: 1199796-29-6) (the concentration is 10 microgram/g) is injected into the abdominal cavity of a 12-week-old male wild type C57BL/6 mouse, the injection is performed once every other day, sciatic nerve clamping injury is performed after 4 days, and follow-up study is performed after 14 days. The process of establishing the clamp wound model is as follows: anesthesia was performed by subcutaneous injection of 3% sodium pentobarbital (1 ml/kg). The skin below the hip is cut and the muscles are blunt cut with fine surgical scissors and forceps to expose the right sciatic nerve in the middle of the thigh. The nerve was crushed 0.5 cm above the fracture of the sural, common fibular and tibial branches. For compression, the sciatic nerve was first compressed with straight-tipped serrated 5.0 fine forceps for 15s until a clear translucent area appeared. The right hind limb uninjured sciatic nerve was used as a control. All animal experimental protocols were approved by the university of southeast university committee on animal protection and utilization and the ethical committee on animal protection in Jiangsu province. The establishment of a mouse sciatic nerve injury model was performed according to approved guidelines.

2. Transmission electron microscopy morphological analysis

The sciatic nerve tissues of the different groups are quickly taken out, transversely cut 1-2mm of the tissues and soaked in a mixed solution containing 4% of paraformaldehyde and 2.5% of glutaraldehyde for 24 hours. Tissue samples were fixed in 1% osmium tetroxide for 1 hour and embedded in Epon812 epoxy. The sections were stained with lead citrate and uranyl acetate and observed under a transmission electron microscope (Japanese, JEO Co., Ltd.) of 80 KV. The g-ratio (g-ratio) is the ratio of the mean axon internal diameter to the mean fiber diameter (myelin sheath) and is calculated analytically using Image J software.

3. Behavioral detection analysis

To further clarify the effect of 6 α -ethyl-23 (S) -methyl cholic acid (INT 777, CAS: 1199796-29-6) on sciatic nerve remyelination and motor recovery, we performed an behavioral rod transfer test and a gait test.

The rotarod experiment used an accelerated rotarod apparatus (LE 8500 model, Pan laboratory) and mice were habituated in the laboratory for 30 minutes prior to testing. Mice were gently placed on the twill horses by gently swinging them and trained in an accelerated mode (4-40 rpm) for 3 days at intervals of 3 minutes and 5 minutes on the twill horses. The training was repeated at a constant speed (16 rpm) until the mice were able to rest on the bar for at least 300 seconds. For the official test, mice were placed on a rotating drum and the carousel was set to acceleration mode, i.e. accelerated at a speed of 4 to 40 rpm over 5 minutes. The time of movement on the rod was recorded before the drop was measured.

Gait experiments gait dynamics of quadruped animals were assessed by footprinting analysis of mice by Digigait imaging system, assessing hindlimb strides at a speed of 8 cm/s. Footprints were analyzed by four measurement methods: distance from the opposite foot, footprint length, maximum toe extension between the first and fifth toes, and paw extension between the centers of the second and fourth toes.

Second, experimental results

TGR5 (G protein-coupled acid receptor 1) is a G protein-coupled receptor, mainly expressed in brown fat, liver and muscle. Previous studies have indicated that TGR5 is able to stimulate energy expenditure and reduce the incidence of diet-induced obesity in obese mice. 6 α -Ethyl-23 (S) -methylchollic acid (INT 777, CAS: 1199796-29-6) is an activator of TGR5, specifically increasing the activity of TGR 5. Research reports indicate that the agonist INT-777 can reduce pancreatic acinar cell necrosis of the acute pancreatitis of mice, and can also play a protective role in cognitive impairment, neuroinflammation, apoptosis and synaptic dysfunction of the mice induced by Abeta 1-42.

The peripheral nervous system is widely distributed in the body and plays a role in signal transmission between target organs and the central nervous system. Myelination of axons is the fundamental guarantee of the functioning of the nervous system, and abnormalities in myelination can lead to the development of a variety of diseases. Peripheral nerve injury is a common clinical sign, and particularly is a disease caused by demyelination of neuron axons at injury positions caused by traffic accidents, wars, earthquakes and other accidental injuries and failure of transmission of nerve impulses. Numerous studies have indicated that remyelination requires a large supply of energy, and TGR5 is a key factor in activating energy metabolism, and therefore, it is speculated that INT777 may contribute to remyelination of sciatic nerve after activation of TGR5 activity.

To investigate whether 6 α -ethyl-23 (S) -methyl cholic acid (INT 777, CAS: 1199796-29-6) could promote sciatic nerve myelin regeneration, a sciatic nerve bruise model was constructed and morphological and behavioral analyses were performed, showing that treatment with 6 α -ethyl-23 (S) -methyl cholic acid (INT 777) promoted sciatic nerve myelin regeneration in mice after bruise compared to wild type mice after bruise. As shown in the figure, the transmission electron microscope results indicate that the sciatic nerve myelin sheath thickness of the mice, the remyelination process is accelerated (figures 1 and 2), the motor balance ability is enhanced (figure 3), and the gait experiment results indicate that the motor recovery ability of the mice in the 6 alpha-ethyl-23 (S) -methyl cholic acid (INT 777) treatment group is far better than that of the wild type mice (figure 4) after sciatic nerve bruising.

The above results indicate that the agonist 6 alpha-ethyl-23 (S) -methyl cholic acid (INT 777) of TGR5 can promote the regeneration and repair of sciatic nerve myelin sheath, and possibly provide a new therapeutic drug for the repair of peripheral nerve injury.