CN113599412A - Application of radix codonopsis and radix astragali composition in preparation of medicine for preventing and treating symptoms related to advanced tumor - Google Patents

Abstract

The invention relates to an application of a codonopsis pilosula-astragalus root composition in preparing a medicine for preventing and treating symptoms related to advanced tumors, in particular to an application of a codonopsis pilosula-astragalus root strengthening injection in preparing a medicine for preventing and treating cancer-induced fatigue and/or weight loss of patients with advanced tumors.

Description

Application of radix codonopsis and radix astragali composition in preparation of medicine for preventing and treating symptoms related to advanced tumor

Technical Field

The invention relates to an application of a codonopsis pilosula-astragalus root composition in preparing a medicine for treating symptoms related to advanced tumors. More particularly, the invention relates to application of ginseng and astragalus strengthening injection in preparing a medicament for treating cancer-induced fatigue and/or weight loss of patients with advanced tumors.

Background

Cancer is currently the major disease that clinically endangers health and causes death. Cancer-related fatigue is one of the most common subjective symptoms of cancer patients. Cancer-related fatigue (CRF) is defined as a persistent, subjective feeling of exertion associated with cancer or its treatment, disturbing the body's normal function, disproportionate to activity, and often accompanied by dysfunction. Compared with common fatigue, cancer-induced fatigue has the characteristics of fast occurrence, heavy degree, long duration, incapability of relieving through rest and the like. The treatment of the traditional Chinese medicine composition currently involves two aspects of etiology and symptomatology treatment, wherein the etiology and the symptomatology treatment aim at potential factors related to the traditional Chinese medicine composition, and influence results are reversed through factor intervention; the latter mainly aims at the self-fatigue symptom of the patient, reduces the discomfort of the patient and improves the comfort level.

At the same time, approximately more than 80% of patients with advanced cancer experience weight loss or cachexia, and the continuous loss of weight leads to a reduction in the ability of the cancer patient to move independently and the quality of life, and even to a reduction in the life expectancy of the patient. Anorexia and weight loss are therefore also common and significant clinical problems in oncology. Although nutritional replacement, proper exercise, appetite-promoting drugs, non-steroidal anti-inflammatory drugs, etc. can be used to improve weight loss in patients with advanced cancer, there is currently no standard treatment or intervention regimen.

The Shenqi Fuzheng injection is an injection prepared by extracting effective components from astragalus and codonopsis pilosula which are used as main raw materials, belongs to a clear and transparent pure Chinese medicinal infusion originated in China, has low toxic and side effects (Song Chong Shu, et al, St.Shenqi Fuzheng injection paper compilation 1999:96-99), and is mainly used for the auxiliary treatment of lung cancer and gastric cancer.

There is an urgent need for a drug effective in treating cancer-induced fatigue and/or weight loss in patients with advanced tumors.

Disclosure of Invention

The inventor surprisingly finds that the codonopsis pilosula-astragalus membranaceus composition, in particular the codonopsis pilosula-astragalus membranaceus body resistance strengthening injection can be used for treating symptoms related to advanced tumors. Therefore, the codonopsis pilosula-astragalus root composition, in particular to the codonopsis pilosula-astragalus root injection for strengthening the body resistance, can be used for preparing a medicine for treating cancer-induced fatigue and/or weight loss of patients with advanced tumors.

Therefore, the invention provides the application of the codonopsis pilosula-astragalus root composition in preparing a medicine for preventing and/or treating symptoms related to advanced tumors.

In one embodiment, the advanced tumor-related symptom is cancer-related fatigue.

In a preferred embodiment, said cancer-associated fatigue is cancer-associated fatigue caused by an increase in the level of inflammatory factors resulting from oxidative damage to the body.

In another preferred embodiment, the cancer-causing fatigue is cancer-causing fatigue associated with the integrity of skeletal muscle mitochondrial structure and function.

In yet another preferred embodiment, the cancer-causing fatigue is cancer-causing fatigue associated with mitochondrial membrane potential of C2C12 myoblasts leading to oxidative stress damage and apoptosis.

In another embodiment, the advanced tumor-associated symptom is weight loss.

In the application of the invention, the codonopsis pilosula-astragalus membranaceus composition is prepared from codonopsis pilosula and astragalus membranaceus which are used as active ingredients in a weight ratio of 0.5:1 to 1:0.5, preferably 1: 1.

The Codonopsis Pilosula (Codonopsis Pilosula (Franch.) Nannf) is a dried root of a plant in the Campanulaceae family. Preferably, the Astragalus membranaceus is a dried root of Astragalus membranaceus (Fisch.) Bge.) or Astragalus membranaceus (Fisch.) Bge.

In an embodiment of the present invention, the codonopsis pilosula-astragalus membranaceus composition can be in any pharmaceutically acceptable dosage form, including but not limited to one or more of oral liquid, tablets, capsules, granules and injections.

In a preferred embodiment of the invention, the codonopsis pilosula-astragalus membranaceus composition is an injection, such as a codonopsis pilosula-astragalus membranaceus body resistance strengthening injection.

In one embodiment, the method for preparing the injection comprises:

(1) adding water into radix Codonopsis and radix astragali, and heating and extracting for 1-3 times to obtain extractive solution;

(2) adding ethanol into the extracting solution, precipitating, and filtering to obtain filtrate;

(3) adding water for injection into the filtrate, adjusting pH, filtering, and sterilizing to obtain injection.

In a preferred embodiment, the injection is prepared by a method comprising:

(1) adding deionized water into radix Codonopsis and radix astragali, and heating and extracting for 1-3 times to obtain extractive solution;

(2) concentrating the extractive solution, adding ethanol, precipitating, filtering, and concentrating the filtrate to obtain concentrated filtrate;

(3) adding water for injection into the concentrated filtrate, adjusting pH, filtering, and sterilizing to obtain injection.

First, the present inventors constructed a mouse model of systemic metastasis of tumors and confirmed the success of model construction by PET/CT and HE staining. Then, on the basis of the establishment of the model, the induction of cancer-induced fatigue and the intervention of the ginseng and astragalus body resistance strengthening injection are carried out. The results show that the codonopsis pilosula-astragalus root composition, particularly the codonopsis pilosula-astragalus root body resistance strengthening injection does not have the effects of delaying the growth of tumors and prolonging the survival time, but has the effect of better maintaining the weight of a mouse. Through exhaustive swimming and autonomous activity experiments, the ginseng and astragalus body resistance strengthening injection is found to have the obvious effect of relieving cancer-induced fatigue. Meanwhile, the tail suspension experiment result proves that the curative effect of the ginseng and astragalus injection for strengthening the body resistance is irrelevant to the depression mood of the mouse.

In a further study, mouse serum and gastrocnemius related assays were performed. The gastrocnemius detection finds that the ginseng-astragalus body resistance strengthening injection can obviously improve the weight of gastrocnemius and increase the cross section area of the gastrocnemius. The transmission electron microscope shows that the Shenqi injection for strengthening the body resistance can maintain the high level of the number of the muscle mitochondria, protect the integrity of the structure of the muscle mitochondria, increase the expression level of mtDNA (mitochondrial deoxyribonucleic acid) and the content of ATP (adenosine triphosphate) of the muscle mitochondria and reduce oxidative stress injury. The detection of mouse serum shows that the Shenqi injection for strengthening body resistance can obviously reduce the expression levels of serum inflammatory factors IL-6, IL-1 and CRP.

To evaluate the effect of colon cancer cells (CT-26) on the degree of apoptosis and necrosis of skeletal muscle cells in mice, the cells were co-cultured for 6H, 12H or 24H in CT-26 cells and C2C12 cells, and in hydrogen peroxide (H)₂O₂) Apoptosis of C2C12 myoblasts was examined 2h after the effect. The results showed that CT26 cells were time-dependent on C2C12 mouse myoblast injury, with percentages of total apoptotic events reaching approximately 11.2%, 23.4% and 17.9% (6H, 12H, 24H), respectively, while H was₂O₂The total apoptosis rate in the middle 2h is 19.1%. And after destroying myoblasts of the C2C12 mouse for 12 hours in a cell co-culture microenvironment, the percentage of apoptotic cells is reduced to about 15.3%, 11.6% and 10.8% by administering different doses (5mg/ml, 10mg/ml and 20mg/ml) of the Shenqi strengthening injection. Changes in mitochondrial membrane potential (Δ Ψ Cm) are indicative of apoptosis, and dissipation Δ Cm directly indicates mitochondrial membrane disruption. In addition, the effect of the ginseng-astragalus strengthening injection on Δ Ψ Cm was measured using a fluorescent probe JC-1. The results show that exposure to CT-26 or H₂O₂Under these conditions, the ratio of JC-1 red fluorescence to green fluorescence in C2C12 myoblasts was significantly reduced, indicating that the co-culture microenvironment resulted in Δ Ψ Cm injury. However, the shenqi injection can significantly reduce the injury of Δ Ψ Cm in C2C12 myoblasts caused by tumor cells. Meanwhile, the mitochondrial Electron Transport Chain (ETC) generates Reactive Oxygen Species (ROS) during activity, which is the largest source of skeletal muscle oxidants. Further, treatment with the ginseng and astragalus strengthening injection will alter ROS levels. The activity of the cytoplasmic oxidant is detected by a DCFH-DA fluorescence detection method, and the result shows that the increase of ROS is reduced by the treatment of the ginseng and astragalus strengthening injection. Mn-SOD protein is continuously measured, so that the mitochondrial oxidation activity of the ginseng and astragalus strengthening injection is verified through Western blot analysis, and the function of mitochondria in myoblasts of C2C12 mice is protected by the ginseng and astragalus strengthening injection.

From the above experimental results, the advantages of the present invention are:

1. the invention discovers the new medical treatment application of the codonopsis pilosula-astragalus root composition, develops a new application field and has good application prospect;

2. the codonopsis pilosula and astragalus composition has rich raw materials, simple preparation method, wide application range and can be prepared into various dosage forms, particularly injection;

3. the radix codonopsitis and astragalus composition has the obvious effect of relieving cancer-caused fatigue, can maintain the high level of the number of muscle mitochondria, protect the integrity of the structure of the muscle mitochondria, increase the expression level of mtDNA (mitochondrial deoxyribonucleic acid) and the content of ATP (adenosine triphosphate) of the muscle mitochondria, reduce oxidative stress injury and obviously reduce the expression levels of serum inflammatory factors IL-6, IL-1 and CRP (C-reactive protein);

4. the codonopsis pilosula-astragalus composition remarkably relieves the damage of a mitochondrial membrane in a C2C12 myoblast caused by tumor cells, reduces the increase of ROS, and further protects the function of mitochondria in the myoblast;

5. the codonopsis pilosula-astragalus root composition has the effects of maintaining weight and preventing weight loss.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

figure 1 shows the results of exhaustive swimming time testing experiments.

Figure 2 shows the results of the open field experiment.

FIG. 3 shows the results of general observations (body weight) of mice.

Figure 4 shows the results of a gastrocnemius mitochondrial mtDNA detection experiment.

FIG. 5 shows the results of a gastrocnemius total ATPase content assay experiment.

FIG. 6 shows the results of a transmission electron microscopy observation experiment of gastrocnemius mitochondria.

FIG. 7 shows the results of myoblast apoptosis experiments.

FIG. 8 shows the results of myoblast Reactive Oxygen Species (ROS) level detection experiments.

FIG. 9 shows the results of the myoblast mitochondrial membrane potential (JC-1) assay.

Detailed description of the preferred embodiments

The present invention will be described in further detail below with reference to specific embodiments, and the present invention will be better understood from the following examples. However, it should be readily understood by those skilled in the art that the following examples are illustrative only and are not intended to limit the present invention to these specific examples. It will be appreciated by those skilled in the art that the present invention encompasses all modifications, alternatives, and equivalents as may be included within the scope of the claims.

In order to better understand the invention, the following in vivo animal experiments and in vitro cell molecular experiments are adopted in the examples to verify and illustrate the new application of the radix codonopsitis and astragalus membranaceus composition in preparing the medicine for treating symptoms related to advanced tumors.

1. In vivo animal experiments: a metastatic tumor mouse model with cancer-related fatigue is constructed, the general condition, the tumor growth condition, the survival curve and the gastrocnemius weight of a mouse after administration are observed, an open field experiment, a tail suspension experiment and an exhaustion swimming experiment related to fatigue are carried out, an ELISA method and an RT-PCR (enzyme-linked immuno sorbent assay) method are applied to detect the expressions of mouse serum inflammatory factors TGF-alpha, IL-6, IL-3, IL-1 beta, skeletal muscle oxidative stress protein and mitochondrial mtDNA (mitochondrial dna), HE (human immunodeficiency Virus-bovine serum) staining and a transmission electron microscope are applied to observe the morphology and the mitochondrial structure of mouse skeletal muscle cells, and the fact that the Shenqi strengthening injection has the prevention and treatment effect on cancer-related fatigue is verified, and the action mechanism of the Shenqi strengthening injection is related to the reduction of the level increase of the inflammatory factors caused by the oxidative damage of an organism and the integrity of the mitochondrial structure and function of the skeletal muscle is protected.

2. In vitro cell experiments: flow cytometry and metabonomics detection prove that the ginseng-astragalus strengthening injection can protect the mitochondrial membrane potential of C2C12 myoblasts and reduce oxidative stress injury and apoptosis.

3. In-vivo animal experiments show that the ginseng and astragalus body resistance strengthening injection has the effects of maintaining weight and preventing weight loss.

Example 1 preparation of an injection of ginseng and astragalus for strengthening the body resistance

Removing impurities from radix Codonopsis and radix astragali, and processing into decoction pieces. Accurately weighing 400 g of each of radix codonopsitis and radix astragali, washing with deionized water, adding 6400ml of deionized water, heating, boiling and extracting for 1 hour, and discharging the extracting solution; adding 3600ml deionized water into the residue, heating, boiling and extracting for 1 hour, and discharging the extracting solution; adding 3600ml deionized water into the residue, heating, boiling and extracting for 0.5 hour, and discharging the extracting solution; mixing the three extractive solutions, filtering, concentrating to 520ml, adding ethanol with concentration of above 85% under stirring to make ethanol content 75%, standing for over 24 hr, filtering to recover ethanol to 440ml, adding ethanol with concentration of 95% under stirring to make ethanol content 85%, standing for over 24 hr, filtering to recover ethanol, and concentrating to 240 ml. Adding 440ml of water for injection into the concentrated solution, heating and boiling for 20 minutes, adding 0.8g of active carbon while stirring, continuously boiling for 10 minutes, taking out and cooling, filtering to remove carbon, adding water for injection to nearly 400ml, adjusting the pH value to 6.5 by using 10% NaOH solution, roughly filtering, finely filtering, filling into a 20ml ampoule, sterilizing at 100 ℃ for 30 minutes, and checking to be qualified to obtain the ginseng and astragalus strengthening injection.

Example 2 animal experimental part:

(1) model construction

Modeling of a fatigue mouse model: before inoculating CT-26 cells, all nude mice are placed in a swimming box (height 30cm, diameter 25cm) for adaptive swimming training for 2 days, 5 min/day, and the water temperature is 25 +/-1 ℃. Mice with too long and too short swimming time and nude mice with too much excitement and too much silence were culled. Digesting and centrifuging cultured CT-26 cells to obtain about 1 × 10⁷Individual cells/ml suspension. Disinfecting abdominal skin of mouse, and collecting 0.2ml (about 1 × 10)⁶Individual tumor cells) are inoculated in the abdominal cavity, the tumor metastasis condition is observed by PET/CT imaging after 1 week of inoculation, the model nude mouse is continuously raised for 2-3 weeks after the colon cancer abdominal cavity metastasis model succeeds, the nude mouse fatigue condition is observed by behavior detection after the nude mouse has slow whole body activity, diet reduction, emaciation and the like, and the cancer-induced fatigue mouse model is prepared.

(2) Drug grouping and intervention

The random grouping principle is followed to divide the mixture into 3 groups of 20 and 60. The test groups are respectively a normal control group, a CRF model group and an SFI intervention group, and the specific test groups are as follows:

normal control group: the corresponding volume of normal saline is given for intraperitoneal injection;

CRF model group: the corresponding volume of normal saline is given for intraperitoneal injection;

SFI intervention group: SHENQIFUZHENG injection (SFI) 3.0g/kg (0.75ml/kg) is administered by intraperitoneal injection once a day for 21 days.

(3) Evaluation of fatigue index

A. Exhaustive swimming time detection

The time to exhaustive swimming of each group of nude mice was measured before the start of administration, on day 7, day 14 and day 21 of administration. The specific method comprises the following steps: the nude mice were tethered to their tails with a 7% weight lead pendant, placed in a swimming box (height 30cm, diameter 25cm) at 25 ℃. + -. 1 ℃ for exhaustive swimming tests, and the time of exhaustive swimming was recorded. The standard of exhaustive swimming is that the nose tip of the nude mouse is submerged for 10 s. Every 7 days 1 time. Immediately after each experiment, the nude mice were wiped dry with a dry towel.

As shown in fig. 1, it can be seen that the swimming time of the mice in the ginseng and astragalus strengthening injection intervention group is significantly increased at day 10 compared with that in the model group (p < 0.05).

B. Open field experiment

Open field experiments were performed on all nude mice at day 14, day 21 after intervention. Holding the proximal ends 1/2-1/3 of the nude mice' tails, gently placing the nude mice in the median grid of the open field experimental box. After the mice are adapted for 1min, timing is started, 2 persons jointly observe and record the behaviors of the nude mice within 5min, and simultaneously real-time video recording is carried out. And (3) recording the total movement distance, the movement distance of the peripheral lattices, the movement distance of the central lattices, the hair-cleaning times (the times of lifting forelimbs upwards, washing the face, scratching the itch and licking the feet), the stool grain number and the wall climbing times of the nude mice in the experimental box by using the automatic fine behavior identification software of the mice. At the end of each nude mouse experiment, the feces were cleared, sprayed with 70% ethanol onto the bottom of the box and wiped dry with clean gauze. The measurement conditions are that a static ventilating and indoor dark light behavioristic laboratory is kept, and the behavioristic laboratory is reflected to an experimental operation area by illuminating a ceiling with an operating lamp; the experimenter is far away from the open field analysis box (as for the experimental animal in the box, the operation angle is not visible). Prior to the experiment, nude mice were acclimated in the assay room for more than 10 min.

The results are shown in fig. 2, and it can be seen that the mice in the ginseng and astragalus strengthening injection intervention group have significant increases in movement time, movement distance and movement speed (p <0.05) compared with the mice in the model group.

(4) General observations in mice

The nude mice were observed daily for food intake, water intake, mental status. The body weight of the nude mice was weighed every 3 days, and the circumference of the nude mice was observed.

The results are shown in fig. 3, and it can be seen that the body weight of the mice in the Shenqi injection intervention group is obviously increased after day 15 compared with the body weight of the mice in the model group (p < 0.05).

(5) Gastrocnemius mitochondrial mtDNA detection

Extraction of genome

a. Gastrocnemius sample treatment: weighing gastrocnemius tissue 10mg, breaking into cell suspension, centrifuging at 12000g for 1min, discarding supernatant, adding 200 μ l buffer GA, shaking to completely suspend, adding 4 μ l RNase A (100mg/ml) solution, shaking for 15 s, and standing at room temperature for 5 min.

b. Add 20. mu.l proteinase K solution, mix well, place at 56 ℃ until the tissue is dissolved, and centrifuge to remove water droplets on the inner wall of the tube cap.

c. Adding 200 μ l buffer solution GB, mixing thoroughly, standing at 70 deg.C for 10min, and centrifuging to remove water droplets on the inner wall of the tube cover when the solution becomes clear.

d. Adding 200 μ l of anhydrous ethanol, shaking thoroughly and mixing for 15 s, observing whether flocculent precipitate appears, and centrifuging to remove water drops on the inner wall of the tube cover.

e. Adding the solution and flocculent precipitate obtained in the previous step into adsorption column CB3, placing into a collection tube, centrifuging at 12000g for 30 s, removing waste liquid, and placing adsorption column CB3 back into the collection tube.

f. Add 500. mu.l buffer GD to adsorption column CB3, centrifuge at 12000g for 30 seconds, discard the waste, place adsorption column CB3 in the collection tube.

g. 600. mu.l of the rinsing solution PW was added to the adsorption column CB3, 12000g was centrifuged for 30 seconds, the waste liquid was discarded, and the adsorption column CB3 was put into the collection tube and the operation was repeated.

h. And (3) putting the adsorption column CB3 back into the collecting pipe, centrifuging for 2min at 12000g, removing waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to completely dry the residual rinsing liquid in the adsorption material.

i. Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 200 mu l of elution buffer TE into the middle part of the adsorption film, standing for 5min at room temperature, centrifuging for 2min at 12000g, and collecting the solution into the centrifuge tube.

② quantitative PCR reaction

a. Primer: the primers were synthesized by Biotechnology engineering (Shanghai). The primer sequences are shown in Table 1.

b. Taking a 0.2ml PCR tube, preparing the following reaction system:

and c, PCR amplification, and obtaining a melting curve from 55 ℃ to 95 ℃ after circulation is finished.

The results are shown in fig. 4, and the mitochondrial mtDNA content of the intervention group of the ginseng and astragalus strengthening injection is obviously increased compared with the model group (p < 0.05).

(6) Detection of total ATP enzyme content in gastrocnemius

After 21 days of administration, killing the mice, stripping gastrocnemius muscles, weighing about 0.1g of gastrocnemius muscle tissues, adding PBS with the volume 10 times that of the gastrocnemius muscle tissues for grinding, centrifuging to obtain supernatant, adding diluent 50 Xfor dilution, sequentially adding microporous plates according to 100 mul of each hole, removing liquid after incubation reaction at 37 ℃ for 2 hours, not washing, sequentially adding 100 mul of detection reagent A into each hole, incubating for reaction at 37 ℃ for 1 hour, washing, sequentially adding 100 mul of detection reagent B into each hole, incubating for reaction at 37 ℃ for 30 minutes, washing, sequentially adding about 90 mul of substrate into each hole, fully mixing, reacting in a dark place at room temperature for 25 minutes, adding 50 mul of stop solution, fully mixing, and detecting in an enzyme labeling instrument at 450nm within 30 minutes.

The results are shown in fig. 5, and it can be seen that the ATP content of the intervention group of the ginseng and astragalus strengthening injection is significantly increased compared with the model group (p < 0.05).

(7) Transmission electron microscope observation of gastrocnemius muscle mitochondria

Firstly, material taking and fixing: after 21 days of administration, the mice were sacrificed, gastrocnemius tissue sections were taken, the size was not more than 1mm × 1mm × 1mm, and the sections were rapidly fixed for 2-4 hours at 4 ℃ in an electron microscope fixing solution. PBS rinse 3 times, 15min each time.

Secondly, post-fixing: 1% osmic acid-PBS (pH 7.4) was fixed at room temperature (20 ℃ C.) for 2 h. PBS rinse 3 times, 15min each time.

Thirdly, dewatering: the tissue is dehydrated on 50% -70% -80% -90% -95% -100% -100% ethanol-100% acetone in sequence, 15min each time.

And fourthly, infiltration: acetone 812 embedding medium was treated at 1:1 for 2h, acetone 812 embedding medium was infiltrated at 1:2 overnight, pure 812 embedding medium was treated for 5h, pure 812 embedding medium was poured into the embedding plate, the sample was inserted into the embedding plate and then oven was left overnight at 37 ℃.

Embedding: polymerizing for 48h in an oven at 60 ℃.

Sixthly, slicing: and (5) slicing the ultrathin slice with the ultrathin slicer to obtain a 60-80nm ultrathin slice.

And (c) dyeing: uranium lead double staining (2% uranium acetate saturated ethanol solution, lead citrate, each staining for 15min), slicing and drying overnight at room temperature.

Observing under a transmission electron microscope, and collecting and analyzing images.

The result is shown in fig. 6, and it can be seen by transmission electron microscopy that the number of mitochondria in the intervention group of the ginseng and astragalus strengthening injection is significantly more than that in the model group (p <0.05), and the structural integrity is better than that in the model group.

Example 3 cellular experimental part:

(1) cell recovery, culture and passage

Taking out the freezing tube of CT26 cells and C2C12 myoblasts from liquid nitrogen, rapidly placing in a constant temperature water bath, transferring the cell suspension to a preparationIn PBS (5%), 250g was centrifuged for 4min, the supernatant was discarded and resuspended in DMEM complete medium (containing 10% FBS) and then in 5% CO₂And culturing in a constant temperature incubator at 37 ℃. When the cells grew 80% of the bottom of the dish, 0.25% trypsinized and passaged at 1:2 dilution.

(2) Construction of C2C12 myoblast mitochondria damage model

Observe the growth of CT26 colon cancer cells and C2C12 myoblasts to cover 75cm²When the bottom wall of the culture bottle has 70% of basal area, discarding the old culture medium, digesting with 0.25% of pancreatin, centrifuging for 5min at 250g, discarding the supernatant, adding DMEM culture medium, beating uniformly, and adjusting the cell density to 5 × 10⁷The myoblasts of C2C12 were seeded in the lower layer of the Transwell 12 well plate in an amount of 1.2mL of cell suspension per well, while the cells of CT26 colon cancer were seeded in the Transwell chamber in an amount of 1mL of cell suspension per well, and the blank group was seeded with myoblasts of C2C12 in equal density in a culture condition of 37 ℃ and 5% CO₂And (5) culturing.

(3) Cell grouping and drug intervention

The cell experiments are divided into 5 groups, namely a C2C12 cell group, a C2C12 cell + Shenqi body-strengthening injection (SFI) intervention group, a C2C12 cell mitochondrial damage model group, a mitochondrial damage cell model + Shenqi body-strengthening injection (SFI) intervention group and hydrogen peroxide (H)₂O₂) And (4) a positive control group. Hydrogen peroxide (H)₂O₂) The intervention concentration of (2) is 100 mu mol/L, and the intervention time is 2 h; the intervention concentration of the Shenqi Fuzheng Injection (SFI) is determined to be 10mg/mL by the apoptosis experiment screening, the prognosis is started, and the cells of each group are continuously cultured for 6, 12, 24 and 48 hours according to the experiment requirements.

C2C12 cell group (control): conventional culture of C2C12 myoblasts;

C2C12 cell mitochondrial damage model group (C2C12+ CT 26): CT26 colon cancer cells were co-cultured with C2C12 myoblasts;

C2C12 cells + shenqi injection (SFI) intervention group (C2C12+ SFI): adding radix Codonopsis and radix astragali injection (SFI) into C2C12 myoblast, and culturing by conventional method;

mitochondrial injury cell model + shenqi injection (SFI) intervention group (C2C12+ CT26+ SFI): the method comprises the steps of co-culturing CT26 colon cancer cells and C2C12 myoblasts, and adding ginseng and astragalus body resistance strengthening injection (SFI) for intervention;

hydrogen peroxide (H)₂O₂) Positive control group: adding hydrogen peroxide (H) into C2C12 myoblast₂O₂) Intervening and constructing a mitochondria damage positive control.

(4) Apoptosis test of myoblasts

Detecting myoblast apoptosis by using an annexin-V FITC/PI double staining method, digesting and inoculating CT-26 colon cancer cells and C2C12 myoblasts in a logarithmic growth phase to a Transwell 12-pore plate for co-culture for 6h, 12h and 24h respectively to construct a C2C12 myoblast mitochondria damage model, and adding ginseng and astragalus strengthening injection solutions with different concentrations for intervention (5mg/mL, 10mg/mL and 20mg/mL) respectively after determining the optimal apoptosis intervention time so as to determine the optimal intervention concentration of the drug. The specific operation is as follows:

a. after the cells are co-cultured for different time and the action of the drugs with different concentrations, 0.25 percent pancreatin is used for digestion and collection of myoblasts;

b. washing with PBS Buffer solution for 2 times, centrifuging at 12000g for 5min, adding 500 μ l Binding Buffer suspension cells, and adjusting cell concentration to 5 × 10⁵/ml；

c. Adding 5 μ l annexin-V FITC, mixing, adding 5 μ l PI, mixing, reacting at room temperature in dark for 5-15min, and detecting myoblast apoptosis with flow cytometer.

The results are shown in FIG. 7, representative apoptosis analysis of time points and dose of SHENQIFUZHENG injection, and induction of 6, 12, 24H and H in CT-26 culture medium₂O₂2h induced C2C12 mouse myoblasts. After 12h of CT-26 medium stimulation, the ginseng and astragalus strengthening injections (P <0.05 compared to the model group, values expressed as mean ± SD, n ═ 3) were administered at doses of 5, 10, 20 mg/ml.

(5) Myoblast Reactive Oxygen Species (ROS) level detection

CT-26 colon cancer cells and C2C12 myoblasts in logarithmic phase are digested and inoculated to a Transwell 12-pore plate respectively for co-culture, a C2C12 myoblast mitochondria damage model is constructed, and ginseng and astragalus strengthening injection and hydrogen peroxide are added for intervention according to cell experimental groups. The specific operation is as follows:

a. after the medicine drying is finished, digesting and collecting myoblasts by using 0.25% pancreatin;

b. washing with PBS buffer for 3 times, centrifuging at 12000g for 5min, and adjusting cell concentration to 5 × 10⁵Centrifuging at 10000g for 5min, and removing supernatant;

c. preparing DCFH-DA (working solution) with serum-free DMEM medium at a ratio of 1000:1, adding 1ml of working solution, suspending the cells, and standing at 37 deg.C under 5% CO₂Incubating in incubator for 30min, shaking every 5min, and mixing;

d. after incubation, centrifuging at 10000g for 5min, discarding the supernatant, washing with PBS buffer for 3 times, centrifuging at 10000g for 5min, and discarding the supernatant;

resuspending in PBS buffer, detecting the level of Reactive Oxygen Species (ROS) on a flow cytometer, excitation wavelength 488nm, emission wavelength 525 nm.

The results are shown in FIG. 8, and intracellular ROS were reduced after 6h incubation.

(6) Myoblast mitochondrial membrane potential (JC-1) assay

Preparing JC-1 working solution: diluting according to the proportion of 50 mu l of JC-1(200X) and 8ml of ultrapure water to 2ml of JC-1 staining buffer solution (5X), and fully and uniformly mixing to prepare JC-1 staining working solution;

b. cell treatment: treating cells according to experimental design, centrifuging, collecting cells, washing with PBS buffer solution for 3 times, adding 1ml JC-1 staining working solution, mixing, standing at 37 deg.C and 5% CO₂Incubating in incubator for 20 min; preparing JC-1 staining buffer (1X) during the preparation, and storing at 4 ℃; after the incubation was completed, the supernatant was removed, and after washing with JC-1 staining buffer (1X) for 2 times, 1ml of PBS buffer was added and the cells were examined on a flow cytometer.

As a result, as shown in fig. 9, the ginseng and astragalus strengthening injection significantly enhanced JC-1 mitochondrial membranes (P < 0.0001v.s.ct-26, expressed as mean ± SD, n ═ 3).

While various aspects and embodiments of the invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration only and are not intended to be limiting. The scope and subject matter of the present invention are to be determined solely by the claims appended hereto.

Claims (10)

1. Application of radix Codonopsis and radix astragali composition in preparing medicine for preventing and/or treating late stage tumor related symptoms is provided.

2. The use according to claim 1, wherein the advanced tumor-related symptom is cancer-related fatigue.

3. The use according to claim 2, wherein said cancer-associated fatigue is cancer-associated fatigue caused by an increase in the level of inflammatory factors resulting from oxidative damage to the body.

4. The use according to claim 2, wherein said carcinogenic fatigue is carcinogenic fatigue associated with the integrity of skeletal muscle mitochondrial structure and function.

5. The use according to claim 2, wherein said carcinogenic fatigue is carcinogenic fatigue associated with mitochondrial membrane potential of C2C12 myoblasts leading to oxidative stress damage and apoptosis.

6. The use according to claim 1, wherein the symptom associated with advanced tumors is weight loss.

7. Use according to any one of claims 1 to 6, characterized in that the Codonopsis pilosula and Astragalus membranaceus composition is prepared from Codonopsis pilosula and Astragalus membranaceus as active ingredients in a weight ratio of 0.5:1 to 1:0.5, preferably 1: 1.

8. The use according to any one of claims 1 to 7, wherein the Codonopsis pilosula and Astragalus membranaceus composition is in any pharmaceutically acceptable dosage form.

9. The use according to claim 8, wherein the composition of codonopsis pilosula and astragalus membranaceus is an injection, such as a ginseng and astragalus strengthening injection.

10. The use according to claim 9, wherein the injection is prepared by a process comprising:

(1) adding water into radix Codonopsis and radix astragali, and heating and extracting for 1-3 times to obtain extractive solution;

(2) adding ethanol into the extracting solution, precipitating, and filtering to obtain filtrate;

(3) adding water for injection into the filtrate, adjusting pH, filtering, and sterilizing to obtain injection.

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