CN113559116A - Pharmaceutical use of lithium chloride - Google Patents

Abstract

The pharmaceutical application of lithium chloride belongs to the field of hypertension treatment, and specifically provides the application of lithium chloride in preparing a medicine for antagonizing hypertension caused by angiotensin II. Wherein the dosage of the lithium chloride is 100 mg/kg/day. The lithium chloride is injected into the abdominal cavity, so that the hypertension caused by the angiotensin II can be effectively reduced, the blood pressure of a mouse is reduced to a level close to that of a healthy mouse, and the endothelium-dependent diastolic function damage caused by the angiotensin II can be obviously improved. In addition, the mice were monitored in the experiment and no adverse reaction was observed after intraperitoneal injection of lithium chloride.

Description

Pharmaceutical use of lithium chloride

Technical Field

The invention belongs to the field of hypertension treatment, and particularly relates to effects of lithium chloride (LiCl) on blood pressure reduction and endothelium protection of a hypertension mouse induced by angiotensin II (Ang II).

Background

Lithium chloride is a lithium salt with a molecular formula of LiCl and a molecular weight of 42.39. Is white crystal, and has deliquescence. Salty taste, and easy dissolution in water, ethanol, acetone, and other organic solvents. Lithium salts have a sedative effect and are widely used in the treatment of bipolar disorder. And, at present, it is difficult to determine the normal function of lithium in animals, but lithium does affect many biological systems and can therefore be considered as a regulatory molecule. Hypertension is a common chronic disease and is currently the leading risk factor for disability and leading to premature death in the world. According to WHO statistics, 940 million worldwide deaths each year by 2015 were attributed to hypertension. In addition, the Chinese cardiovascular health and disease report in 2019 shows that 2.9 billion patients with Chinese cardiovascular diseases have hypertension of up to 2.7 billion. Angiotensin converting enzyme inhibitors (ACE-Is), angiotensin-II receptor blockers (ARBs), calcium antagonists (CCBs), diuretics and beta-blockers are currently used as antihypertensive drugs, and are combined or combined with other drugs when blood pressure Is poorly controlled. Even so far, nearly half of hypertensive patients do not lower their blood pressure to the target blood pressure after taking antihypertensive drugs, and these drugs have many different side effects. At present, no medicine which is very effective and has small side effect exists for treating hypertension. Lithium chloride has become a new hot spot for the targeted treatment and research of major diseases by virtue of the cytoprotective effect of the lithium chloride, and has extremely important theoretical research and clinical application prospects.

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims at the technical problems and provides a pharmaceutical application of lithium chloride, which can reduce blood pressure and protect endothelium of a hypertensive mouse induced by angiotensin II (Ang II).

The technical scheme is as follows: application of lithium chloride in preparation of medicine for antagonizing hypertension caused by angiotensin II.

The dose of lithium chloride was 100 mg/kg/day.

Has the advantages that: the lithium chloride is injected into the abdominal cavity, so that the hypertension caused by the angiotensin II can be effectively reduced, the blood pressure of a mouse is reduced to a level close to that of a healthy mouse, and the endothelium-dependent diastolic function damage caused by the angiotensin II can be obviously improved. In addition, the mice were monitored in the experiment and no adverse reaction was observed after intraperitoneal injection of lithium chloride.

Drawings

FIG. 1 is a graph showing 2-cycle blood pressure monitoring in each group of mice;

FIG. 2 is a statistical chart of blood pressure monitoring phases of mice in each group;

FIG. 3 is a statistical chart of the vascular endothelium-dependent diastolic function of mice in each group;

FIG. 4 shows the expression patterns of Western Blot for detecting p-GSK3 beta (ser9), p-AKT (ser473), p-AKT (thr308) and p-eNOS in HUVEC cells;

FIG. 5 is a statistical chart of Western Blot detection of expression of p-GSK3 beta (ser9), p-AKT (ser473), p-AKT (thr308) and p-eNOS in HUVEC cells.

Detailed Description

The following examples are given to enable those skilled in the art to fully understand the present invention, but are not intended to limit the present invention in any manner.

The method for researching the influence of lithium chloride on the function of blood vessels comprises the following steps:

1) a1.008 mg/kg/day Ang II micro osmotic pump is buried under the skin, the model is built for 14 days, and the normal saline is pumped into the control group. During the molding period, the mice of the control group and the molding group are injected with normal saline or lithium chloride of 100mg/kg and 200mg/kg in the abdominal cavity, the blood pressure is continuously monitored during the period, and the blood pressure value is recorded every two days;

2) two weeks after Ang II modeling, collecting aorta of each group of mice, and detecting Acetylcholine (ACH) mediated endothelium-dependent relaxation function;

3) human Umbilical Vein Endothelial Cells (HUVEC) are extracted, glycine, lithium chloride and glycine and lithium chloride are given for stimulation, glycine stimulation is carried out for half an hour, cellular proteins are extracted after lithium chloride stimulation is carried out for one hour, and the expression conditions of p-GSK3 beta (ser9), p-AKT (ser473), p-AKT (thr308) and p-eNOS are detected by a western blot method.

Example 1

Taking blood pressure monitoring of a hypertensive modeled mouse after injecting lithium chloride into an abdominal cavity of a body as an example, detailed experimental steps are as follows:

1. mouse model for inducing hypertension

(1) 36 healthy C57BL/6j male mice aged 8 weeks were selected and randomized into 4 groups. The mice were kept in an environment of constant temperature (23-25 ℃) and constant humidity (50-65%) distributed for 12h/12h in the day and night time, and water and feed were freely taken in.

(2) The Ang II solution is prepared by using normal saline, the concentration is 10mg/mL, and the Ang II solution is stored in a dark place. The mice establish a hypertension model with an Ang II dose of 1.0mg/kg/day, the volume of Ang II used in the micro-pump was calculated from the body weight of the mice, and the total volume of the micro-pump was made up with physiological saline to activate its release. The control group was perfused with a micro pump using physiological saline.

(3) Anaesthetizing the mouse and weighing the weight, adding a proper amount of Ang II into the micro pump according to the weight, filling the total volume with normal saline, and then sealing the micro pump. Simultaneously, depilating at the neck of the back of the mouse, sterilizing with iodophor, slightly lifting the skin of the neck with ophthalmologic forceps, and transversely cutting an incision about 1 cm. Then, the back skin was separated from the muscle by blunt scissors, and a micro pump was buried under the skin of the back of the mouse through the incision, the wound was closed, and iodophor was sterilized. Finally, the mice were placed on a 37 ℃ heat blanket until the mice were awakened.

2. Non-invasive blood pressure monitoring

(1) Before treatment by injecting glycine or normal saline into the abdominal cavity, the blood pressure of the mice is measured by using a BP2000 noninvasive blood pressure meter. The pre-measurement was performed three consecutive days before the official measurement to let the mice stable and familiar with the environment.

(2) After molding, four groups of mice were injected with an equal volume of physiological saline, lithium chloride (100mg/kg) and lithium chloride (200mg/kg) at a fixed time each day. The blood pressure change of the mice is continuously monitored for 2 weeks by using a BP2000 noninvasive blood pressure monitor, and the experimental components are as follows: WT + Saline; (vii) WT + Ang II + Saline; ③ WT + Ang II +100mg/kg lithium chloride; fourthly, four groups of WT + Ang II +200mg/kg lithium chloride.

3. Mouse aortic endothelial dependent diastolic function assay

After two weeks of modeling, isoflurane is adopted for inhalation anesthesia, blood is taken from the posterior artery of an eyeball, and the aorta of a mouse is harvested for monitoring the change of the acetylcholine-mediated endothelium-dependent relaxation function.

(1) The prepared solution was: kreb's (ready for use), 800mmol/L potassium chloride, norepinephrine (NE, 10)^-4mmol/L) of ACH (10) in different concentrations^-6、9×10^-6、9×10^-5、9×10^-4、9×10^-3mmol/L)；

(2) After weighing, the mice were anesthetized, fixed on a mouse plate, and the thoracico-abdominal part was wiped with an alcohol cotton ball;

(3) opening the chest cavity of the mouse, opening the chest from the sternum part, exposing the heart, shearing the right auricle, slowly injecting precooled kreb's solution into a scalp needle through the left apex, and completely perfusing the blood in the aorta;

(4) carefully cutting off redundant tissues, slightly separating and cleaning the aorta from the root of thoracic aorta to the branch of common iliac artery after finding out the aorta at the position tightly attached to the spine under a body microscope, and removing clean connective tissues and adipose tissues around blood vessels;

(5) rapidly separating aorta from heart, and placing into a dish containing precooled kreb's buffer solution for standby;

(6) further separating and cleaning residual adipose tissues around the aorta in a plate, and cutting the stripped aorta into small segments with the length of about 3mm in length;

(7) adding prepared kreb's buffer solution into the bath of DMT vascular tensiometer, preheating to 37 deg.C, and introducing 95% O₂With 5% CO₂Mixing gas;

(8) carefully hanging the segmented aorta segment on a DMT (discrete multi-tone) vascular tensiometer probe, setting the initial tension of the blood vessel to be 3.0mN, and stabilizing for 10 minutes;

(9) maintaining the initial tension of each segment of the vessel for 1 hour, during which 37 ℃ preheated kreb's buffer is replaced every 20 minutes without touching the probe and vessel;

(10) after balancing for 1 hour, giving 80mmol/L potassium chloride for pre-stimulation, vasoconstriction for 15 minutes, after the vasoconstriction curve tends to balance, washing the bath tank for 3 times with kreb's buffer solution preheated at 37 ℃ to restore the blood vessel tension to the initial tension, and avoiding touching the probe and the blood vessel during replacement;

(11) keeping the blood vessel balanced for 30 minutes, and replacing preheated kreb's buffer solution once every 15 minutes;

(12) after 30 minutes, 10 was administered^-7The concentration of the MMo/L NE shrinks blood vessels, and after the blood vessels reach a plateau, the blood vessels are treatedAdministered once 10 times^{- 9}mmol/L、10^-8mmol/L、10^-7mmol/L、10^-6mmol/L、10^-5mmol/L of acetylcholine at a concentration of 10^-5When mmol/L, the blood vessel tension reaches 80 percent, which is the evaluation standard of the integrity of the blood vessel endothelium;

(13) after the experiment, the DMT vascular tensiometer is used, the bath is soaked in 80% acetic acid for 3 minutes, and is washed with double distilled water for 3 times, and finally, the residual liquid is sucked dry by absorbent paper, and the mixed gas is closed.

(14) kreb's formulation (pH:7.4)

The experimental results are as follows:

1. to verify the hypotensive effect of lithium chloride on Ang ii-induced hypertension, we performed rat tail pressure monitoring on four groups of experimental mice. The experimental result shows that compared with mice injected with normal saline intraperitoneally after Ang II modeling, the mice injected with 100mg/kg lithium chloride intraperitoneally after Ang II modeling can obviously reduce the blood pressure to a normal value range, and the mice injected with 200mg/kg lithium chloride intraperitoneally after Ang II modeling have a more obvious blood pressure reducing effect in the early stage, but the protective effect on the hypertension in the later stage disappears (fig. 1 and 2).

2. To validate the protective effect of lithium chloride on Ang ii-induced impaired endothelial dependent diastolic function, we examined endothelial dependent diastolic function in four groups of experimental mice. The results show that 100mg/kg and 200mg/kg lithium chloride both significantly improved the endothelium-dependent diastolic function impairment induced by Ang II (FIG. 3).

The results of the animal experiments fully prove that 100mg/kg of lithium chloride can reduce the increase of blood pressure of mice induced by Ang II and improve the endothelium-dependent relaxation function of the mice.

Example 2

Taking the detection of protein expression level after lithium chloride stimulation of HUVEC as an example, the detailed steps are as follows:

HUVEC isolation and culture:

the sample collection method comprises the steps of extruding blood in umbilical cord blood vessels by using a newborn umbilical cord which is taken out immediately after a healthy parturient delivers under an aseptic condition, putting the umbilical cord into a wide-mouth bottle filled with a PBS buffer solution containing the streptomycin dual-antibody, placing the wide-mouth bottle in a prepared ice bucket for preservation, and separating and culturing human umbilical vein endothelial cells within 6 hours.

(1) The umbilical cord of the newborn is taken out under the aseptic condition, and the forceps marks and the blood clot blocking part are cut off by a dissecting scissors to find the umbilical vein.

(2) One end of the umbilical vein is inserted with a gastric perfusion needle and fixed by vascular forceps, the syringe is connected through the gastric perfusion needle, and the residual blood is washed by using a sterile PBS solution preheated at 37 ℃.

(3) After the residual blood in the vein is removed, the other end of the blood vessel is clamped by a vascular clamp, 10-15mL of type IA collagenase (1mg/mL) is perfused into the umbilical vein, then the two ends of the blood vessel are clamped, the blood vessel is wrapped by tin foil paper after being sterilized by ultraviolet rays, the mixture is digested at 37 ℃ for 10-15 minutes, and the umbilical cord is massaged every 3 minutes during the digestion process to promote the collagen enzyme in the blood vessel to be uniformly contacted with endothelial cells.

(4) The umbilical cord was removed, the walls were gently squeezed, collagenase containing endothelial cells was collected in a 50mL centrifuge tube, the vessel was then flushed with endothelial cell culture medium, digestion was stopped, and the effluent was likewise collected in a centrifuge tube.

(5) Centrifuging the centrifuge tube for 10 min at 1000 rpm, discarding the supernatant, adding new endothelial cell culture solution to resuspend the cells, inoculating into a culture flask, and placing in 5% CO₂And (4) performing static culture at 37 ℃ in an incubator.

(6) After 24 hours, the cell state was observed, and the culture medium was changed.

2. Glycine and lithium chloride stimulation were given separately in HUVEC:

(1) HUVECs were divided into four groups: a control group, a glycine-treated group, a lithium chloride-treated group, and a glycine and lithium chloride-simultaneously treated group. And (3) when the four groups of HUVECs are cultured to the density of 70% -80%, replacing the original culture solution with a serum-free culture solution.

(2) Glycine (5mmol/L) stimulation was given for 30 minutes after 8 hours in the glycine treated group; the lithium chloride treatment group was given a stimulation of lithium chloride (20. mu. mol/L) for 1 hour; glycine and lithium chloride treatment groups were stimulated with lithium chloride (20. mu. mol/L) for 1 hour, followed by glycine (5mmol/L) for 30 minutes.

3. Extraction of cell proteins:

(1) the cell culture was discarded, the cells were washed 2 times with pre-cooled TBS and an appropriate amount of cell lysate and phosphatase inhibitor mix (9: 1 ratio) was added depending on the cell density.

(2) The cells were scraped off with a cell scraper and transferred to a 1.5mL EP tube, lysed on ice for 30 minutes and centrifuged at 12000rpm for 15 minutes at 4 ℃, and the supernatant was aspirated and the Protein concentration was determined according to the BCA Protein Assay Kit instructions.

(3) Packaging protein after protein quantification, boiling at 100 deg.C for 10 min to allow protein denaturation, and storing in refrigerator at-40 deg.C.

BCA assay for protein concentration:

(1) preparing a protein standard curve, namely adding a protein standard substance with the concentration of 2mg/mL and sterilized double distilled water into the enzyme standard strips according to the following volumes:

(2) mu.L of the protein sample to be detected and 8 mu.L of double distilled water were added to each sample well.

(3) Preparing a BCA working solution, namely taking out the solution A and the solution B in the BCA Protein Assay Reagent Kit, preparing the BCA working solution according to the proportion of 1:50, fully mixing the solution uniformly, and placing the solution in a dark place.

(4) And sucking 200 mu L of the solution, adding the pre-mixed BCA working solution into the standard curve hole and the sample hole, gently shaking uniformly, and then placing the solution at 37 ℃ for incubation for 30 minutes in the dark.

(5) Reading the absorbance value at the wavelength of 550nm by using an enzyme-labeling instrument; and drawing a standard curve according to the concentration and the absorbance value of the standard curve, and calculating the concentration of each protein sample according to the standard curve.

5. Polyacrylamide gel (SDS-PAGE) electrophoresis and immunoblotting:

(1) preparing polyacrylamide gel: firstly, selecting polyacrylamide gel with proper concentration according to the molecular weight of target protein, taking a piece of 10% separation gel with the thickness of 1.5mm as an example, preparing the separation gel according to the formula in the table 1, filling the separation gel into a glass plate, then adding absolute ethyl alcohol to cover the liquid surface, and standing for 30 minutes at room temperature. After the separation gel is solidified, the absolute ethyl alcohol is discarded, then 3% concentrated gel is prepared according to the formula in the table 1, the tooth comb is inserted lightly to avoid air bubbles, and the mixture is kept stand for 20 minutes at room temperature.

TABLE 1 Polyacrylamide gel formulations

(2) Taking out the protein sample, heating at 100 ℃ for 5 minutes, and centrifuging at low speed for later use.

(3) Electrophoresis: taking down the glass plate and fixing the glass plate in an electrophoresis tank, pouring electrophoresis buffer solution into the glass plate to enable the liquid level of the inner tank to be higher than that of the outer tank, firstly washing a lane by using a micro-sampling needle, covering an electrophoresis tank cover after the protein is loaded, adjusting the voltage to be 80V for constant voltage electrophoresis, adjusting the voltage to be 120V after the protein enters the separation gel, and stopping electrophoresis when bromophenol blue reaches the bottom of the separation gel.

(4) Film transfer: preparing protein membrane transferring buffer solution, pouring the protein membrane transferring buffer solution into a membrane transferring disc, placing a membrane transferring clamp, and sequentially placing black sponge and filter paper sheets. After electrophoresis is finished, taking out the gel, cutting off the concentrated gel, carefully transferring the separation gel to a filter paper sheet of a membrane transferring clamp, activating the PVDF membrane by a methanol solution for 15 seconds, taking out the PVDF membrane, placing the PVDF membrane on the separation gel, removing bubbles by using a rolling shaft, clamping the membrane transferring clamp, placing the membrane transferring clamp into a membrane transferring groove, aligning the gel to a negative electrode, aligning the PVDF membrane to a positive electrode, and transferring the PVDF membrane for 2 hours at 350 mA.

(5) And (3) sealing: after the membrane transfer was completed, the PVDF membrane was removed, washed with TBS-T (0.05% Tween-20) for 5 minutes, and then the membrane was immersed in 5% BSA/TBS-T blocking solution and blocked at room temperature for 2 hours.

(6) Incubating the primary antibody: after the sealing is finished, the membrane is placed in a plastic package bag, 2.5% BSA/TBS-T is used as an antibody diluent, a proper amount of antibody is added according to the antibody specification, the mixture is uniformly mixed and then is added into the plastic package bag, the mixture is fully contacted with the membrane, and the membrane is incubated overnight at the temperature of 4 ℃ by shaking.

(7) Incubation of secondary antibody: the next day, the membrane was removed, primary antibody discarded, washed 3 times with TBS-T, and the solution was changed 1 time every 10 minutes. And (3) incubating the secondary antibody, wherein 2.5% BSA/TBS-T is used as an antibody diluent, a proper amount of the secondary antibody is added, and the secondary antibody is incubated for 1.5 hours at room temperature.

(8) Washing the membrane: the secondary antibody was discarded, and the membrane was washed 4 times with TBS-T, and the solution was changed 1 time every 10 minutes.

(9) And (3) developing: and fully and uniformly mixing the solution A and the solution B of the ECL developing solution according to the proportion of 1:1, uniformly dripping the mixture on the surface of the PVDF membrane, observing an experimental result through a fluorescence imaging system, and selecting a clear and proper developing picture for storage.

(10) And (4) carrying out gray level analysis on the picture by using Image Lab software, and counting the experimental result.

The experimental results are as follows:

to investigate the effect of glycine and lithium chloride on endothelial cells, human umbilical vein endothelial cells were first stimulated with glycine, lithium chloride, glycine plus lithium chloride, and cellular proteins were collected and western blots were used to measure the expression levels of p-GSK3 β (ser9), p-AKT (ser473), p-AKTt (thr308), p-eNOS (FIG. 4, FIG. 5). The results show that the expression levels of p-GSK3 beta (ser9), p-AKT (ser473), p-AKT (thr308) and p-eNOS are obviously increased after glycine and lithium chloride are stimulated. The results of the in vitro experiments fully prove that the lithium chloride can activate p-GSK3 beta (ser9), p-AKT (ser473), p-AKT (thr308) and p-eNOS, thereby improving the endothelial function.

Claims (2)

1. Application of lithium chloride in preparation of medicine for antagonizing hypertension caused by angiotensin II.

2. The use according to claim 1, wherein the dose of lithium chloride is 100 mg/kg/day.

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