Study On The Protective Mechanism Of Total Glycosides Of Cistanche Deserticola Against Alcohol Induced Injury in Primary Cultured Mouse Liver Cells
Jun 04, 2025
Objective:
To study the protective effect of total glycosides of Cistanche deserticola on primary cultured hepatocytes. Methods: The primary hepatocytes were collected by in situ perfusion method, and the effect of total glycosides of Cistanche deserticola on the survival rate of primary cultured hepatocytes injured by alcohol was evaluated by MTT method; The effect of total glycosides of Cistanche deserticola on the morphology of primary cultured hepatocytes and nuclei injured by alcohol was evaluated by fluorescence microscopy; The effect of total glycosides of Cistanche deserticola on apoptosis of primary cultured hepatocytes injured by alcohol was detected by flow cytometry; Immunocytochemical staining was used to detect the effect of total glycosides of Cistanche deserticola on the expression of bcl-2 and c-fos. Results: After alcohol injury, the survival rate of primary hepatocytes decreased, and there were obvious changes in apoptosis and necrosis. The expression of the apoptosis-inhibiting gene bcl-2 decreased, and the expression of the apoptosis-promoting gene c-fos increased. Total glycosides of Cistanche deserticola can significantly improve cell survival rate, improve apoptosis and necrosis, enhance the expression of bcl-2, and inhibit the expression of c-fos. The effect was dose-dependent. Conclusion: Cistanche deserticola glycosides can protect primary cultured hepatocytes by increasing the expression of apoptosis-inhibiting gene bcl-2, reducing the expression of apoptosis-promoting gene c-fos, reducing apoptosis and necrosis, and increasing cell survival rate.
Keywords: total glycosides of Cistanche deserticola; Primary culture; Hepatocyte; Alcoholic liver injury; Apoptosis
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In recent years, with the improvement of people's living standards, the consumption of alcohol has increased rapidly, accompanied by the increase of various alcohol-related diseases, and alcoholic liver injury is one of the important problems. Studies have shown that Cistanche deserticola has the protective effects of enhancing immune function, anti-aging, anti-radiation, anti-oxidation, anti-lipid peroxidation, and alcohol-induced liver injury [1]. Glycosides are the main active substances of Cistanche deserticola. In previous experiments, it was verified by animal experiments that the total glycosides of Cistanche deserticola may reduce the production of free radicals, enhance the scavenging ability of free radicals and their metabolites, thereby inhibiting lipid peroxidation, and play a protective role in alcohol-induced liver injury [2]. This experiment will explore the protective effect of total glycosides of Cistanche deserticola on alcoholic liver injury at the cellular level through the primary culture of hepatocytes.
1. Materials and methods
1.1 Reagent and instrument The Total glycosides of Cistanche deserticola, dark brown powder, provided by the School of Pharmacy of Lanzhou University, contain 88.6% of total glycosides of phenyl ethanol. Dissolve in double-distilled water and dilute the culture solution to the required concentration. Enzyme marker (Beckman Coulter AD340), fluorescence microscope (Olympus, BX51), inverted phase contrast microscope (Leica DMI3000B), flow cytometry (Beckman colter cell, CellLabQuanta SC), etc.
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1.2 The primary hepatocytes were isolated and cultured by the in situ perfusion method [3].
One Kunming mouse was anesthetized by a 0.5% pentobarbital 0.5 mL intraperitoneal injection. After routine skin disinfection, open the abdomen, move the gastrointestinal tract to the left, expose the portal vein, carefully puncture the infusion needle of the infusion device from the far end of the portal vein, and ligate and fix it. Turn the flow of the infusion set to the maximum (about 4mL/min), and drop the calcium-free pre-perfusion solution preheated at 37 ℃. When the liver is swollen, cut off the distal end of the inferior vena cava quickly, and compress the proximal end of the inferior vena cava intermittently so that the liver alternately retracts and expands. When the residual blood is washed out and the liver is uniformly yellow and white, the warm 0.05% collagenase (Sigma) perfusion solution is used for digestion perfusion. Digestion stops when the liver becomes soft and inelastic, small vacuoles appear under the liver capsule, or even cracks appear in the liver tissue. Carefully dissociate the liver, transfer it to a sterile plate containing a proper amount of DMEM high-sugar culture medium (Sigma), peel off the liver envelope and gently shake it, then scatter the hepatocytes in the culture medium, collect them in a conical tube, shake and culture for 10 min (vibration frequency is 100 times/min), centrifuge for 3 min (4 ℃, 1000r/min), and discard the supernatant. The cells were suspended with the culture medium, filtered with a 200 mesh nylon mesh, and the filtrate was centrifuged repeatedly 3 times (4 ℃, 1000 r/min) to obtain liver parenchyma cells. Adjust the cell concentration to 5 × 106 cells/mL, inoculate them into a 25 mL culture flask pretreated with rat tail collagen, and then incubate them in a 37 ℃, 5% CO2 incubator. After 24h, change the solution, discard the non-adherent cells, and continue to culture for use in subsequent experiments.
1.3 Determination of the growth curve of primary cultured mouse hepatocytes
Adjust the purified hepatocytes to 5 × 106 cells/mL, inoculate them in the 24-well plate, and 1 mL of cell suspension was inoculated in each well. A total of 21 wells were inoculated, and DMEM high-sugar culture medium containing 10% fetal bovine serum (Sigma) was cultured for 7 days. Take 3 holes at random every day, digest and blow 0.25% trypsin evenly, count on the counting board, and draw the growth curve
1.4 Establishment of alcoholic hepatocyte injury model.
The logarithmic hepatocytes obtained by the above method were inoculated into the 96-well plate pretreated with rat tail collagen, and 100 cells were inoculated into each well with 50 μL. The DMEM culture medium with 10% fetal bovine serum was cultured for 24 hours and then changed for another 24 hours. A blank group was set up, and five alcohol intervention groups (50, 75, 100, 125, 150 mmol/L) were set up, and each group was set up with five repetitions. After hepatocytes were cultured with different concentrations of alcohol for 24h, 0.5% MTT (Sigma) 20 was added to each well μ 50 μL. Continue to culture for 4h, discard the supernatant, and add DMSO 150 to each well μ 50. Put it in a shaking table and mix it for 15 minutes, measure the absorbance value at 570nm, and calculate the cell survival rate. Cell survival rate value of the administration group/control group × 100%
1.5 Effect of total glycosides of Cistanche deserticola on the survival rate of primary cultured hepatocytes
Injured by alcohol. Inoculate the hepatocytes obtained by the above methods on the 96-well plate pretreated with rat tail collagen and inoculate each well with 100 μL or 50 μL. The DMEM culture medium with 10% fetal bovine serum was cultured for 24 hours and then changed. After another 24 hours, GCs with the final concentration of 0.2, 0.4, and 0.8 g/L were added to the administration group, respectively. The ethanol model group and the blank group continued to culture without adding drugs, and each group was set with five multiple wells. After 24 hours, the model group and the GCs intervention group were added with alcohol with the final concentration of 100 mmol/L, respectively. After 12 hours of culture, the cell survival rate was measured by the MTT method.
1.6 Effect of total glycosides of Cistanche deserticola on the morphology of primary cultured hepatocytes and nuclei injured by alcohol
Place the pre-sterilized cover glass in a 6-well plate with the inoculation concentration of 5 × 106 cells/mL cell suspension, model group, Cistanche deserticola group(0.2, 0.4, 0.8 g/L), and the blank group. After 24h of culture, add 100 mmol/L alcohol to the final concentration. After 24h of culture, take out 95% ethanol from the glass slide and fix it for 15 minutes. PBS is washed twice and then re-dispersed in PBS. Adjust the cell concentration to 5 × 104 cells/mL, add 10 μ L of acridine orange - ethylidene bromide mixture (Sigma) (0.01 μ G/mL acridine orange solution, 0.02 μ G/mL promethazine solution, mixed volume ratio: 1/1), incubated for 30min, observed and photographed under the fluorescence microscope.
1.7 Effect of total glycosides of Cistanche deserticola on apoptosis of primary cultured hepatocytes injured by alcohol
Adjust the density of the digested cell suspension to 5 × 106 cells/mL, inoculate in a six-well plate, and culture for 24 hours. Set up model group, cistanche deserticola group (0.2, 0.4, 0.8 g/L), and blank group. After 24 hours of culture, add alcohol with the final concentration of 100 mmol/L, incubate for 24 hours, digest and collect cells, wash them with PBS twice, fix them at 4 ℃ overnight with 70% precooled ethanol, wash them with PBS solution twice, add RNA enzyme (Sigma), PI (100 μ G/mL) (Sigma), and put it into 37 ℃ water bath for 30min. The apoptosis rate was measured by flow cytometry.
1.8 Total glycosides of Cistanche deserticola can inhibit the apoptosis of primary cultured hepatocytes injured by alcohol, bcl-2, and pro-apoptotic gene c-fos
The effect of fos expression was taken from three six-well culture plates, each of which was a group of three wells. The model group, Cistanche deserticola group (0.2, 0.4, 0.8 g/L), and the blank group were set up. One sterile slide was placed in each hole. The logarithmic growth phase cells were taken, and the trypsin digestion was used to prepare the single cell suspension, and the cell density was adjusted to 5 × 106/mL and inoculated on a 6-well plate, 2 mL/well. After 24 hours of culture, add alcohol with a final concentration of 100 mmol/L, and continue to culture for 24 hours. Take out the slides full of cells, wash them with PBS twice, and dye them with the routine immunohistochemical ABC method. The brief steps are as follows: fix them with 40% paraformaldehyde for 10 minutes, seal them with normal sheep serum for 30 minutes, drop and add bcl-2 polyclonal antibody (Invitrogen) (1:75) or c-fos polyclonal antibody (Invitrogen) (1:50), react at room temperature for 1.5 hours, add ABC complex at room temperature for 2 hours, color DAB, and seal them with gelatin. The expression of Bcl-2 and c-fos protein was compared by light microscopy and photography.
1.9 Statistical processing The experimental data were processed with SPSS 13.5 software. It is expressed as mean ± standard deviation (x ± s), and a t-test is used for comparison between groups. P<0.05 indicates that the difference is statistically significant.
2 Results
2.1 The growth curve of primary cultured mouse hepatocytes is shown in Figure 1. It can be seen that the number of hepatocytes increased with the incubation time, and entered the logarithmic growth phase on the 4th day.
2.2 The effect of different concentrations of alcohol on the survival rate of primary cultured mouse hepatocytes
It can be seen from Table 1 that alcohol has a strong damage effect on primary cultured hepatocytes, and the effect is dose-dependent. According to the needs of the test, 100mmol/L will be used as the working concentration in the subsequent test, and the cell survival rate is 43.40%.
2.3 The effect of total glycosides of Cistanche deserticola on the survival rate of primary cultured hepatocytes injured by alcohol
Can be seen in Table 2. After alcohol injury, the survival rate of hepatocytes is significantly reduced. Total glycosides of Cistanche deserticola can significantly increase the survival rate of hepatocytes (P<0.05), and the effect is dose-dependent (P<0.05).
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2.4 Effect of total glycosides of Cistanche deserticola on the morphology of primary cultured hepatocytes and nuclei damaged by alcohol.
Azidine orange shows green fluorescence after entering the cells, while promethazine can only stain the necrotic cells with an incomplete cell membrane. It was found through observation (Fig. 2) that the hepatocytes in the blank group were uniformly green, and there was no bromine red staining of necrotic cells (A); After treatment with alcohol, the hepatocytes became smaller, and a large number of necrotic cells stained with bromine red and apoptotic cells with chromatin concentration in the nucleus (the nucleus was bright green) (B) appeared. Therefore, it is believed that alcohol can also cause cell necrosis while inducing hepatocyte apoptosis. GCs can significantly reduce necrosis and apoptosis, and the effect is positively correlated with dose (C, D, E).
Fig. 2 Observation of the effect of total glycosides of Cistanche deserticola on the morphology of primary cultured liver nuclei under a fluorescence microscope (acridine orange staining, 400)
A: Blank group; B: Model group; C:0.2g/L-GCs; D:0.4g/L-GCs; E:0.8g/L-GCs
2.5 The effect of total glycosides of cistanche deserticola on the apoptosis of primary cultured hepatocytes injured by alcohol
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As can be seen in Figure 3-B, the apoptosis rate of hepatocytes after alcohol injury is significantly increased (34.7%), and total glycosides of Cistanche deserticola can significantly increase the survival rate of hepatocytes in a dose-dependent manner, which is consistent with the results observed under the fluorescence microscope.
Fig. 3 Effect of total glycosides of Cistanche deserticola on apoptosis of primary cultured hepatocytes injured by alcohol
A: Blank group; B: Model group; C:0.2g/L-GCs; D:0.4g/L-GCs; E:0.8g/L-GCs
By immunocytochemistry, it was found that the expression of apoptosis-inhibiting factor bcl-2 decreased (4-B) and the expression of c-fos increased (5-B) after liver cells were injured by alcohol. Total glycosides of Cistanche deserticola can significantly enhance the expression of bcl-2 (4-C, D, E) and inhibit the expression of c-fos (5-C, D, E). The results are shown in Fig. 4-5.
Fig. 4 Effect of total glycosides of Cistanche deserticola on the expression of bcl-2 in primary cultured hepatocytes injured by alcohol (400)
A: Blank group; B: Model group; C:0.2g/L-GCs; D:0.4g/L-GCs; E:0.8g/L-GCs
Fig. 5 Effect of total glycosides of Cistanche deserticola on the expression of c-fos in primary cultured hepatocytes injured by alcohol (400)
A: Blank group; B: Model group; C:0.2g/L-GCs; D:0.4g/L-GCs; E:0.8g/L-GCs
3 Discussion
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Berry et al. first established the in-situ perfusion method of using collagenase to perfuse the liver to obtain hepatocytes under physiological conditions, and later developed by Seglen et al. [4] to make this collagenase perfusion technology more perfect. This method has a high cell yield and a high survival rate. In this experiment, primary hepatocytes were obtained by in situ perfusion of mouse hepatocytes and cultured. The method was stable, and the cell activity was strong. Through the growth curve test, it was found that the primary hepatocytes obtained entered the logarithmic growth phase after 4 days.
MTT colorimetric microanalysis is a sensitive method to detect cell growth and survival with good repeatability. Succinate dehydrogenase in the mitochondria of living cells can reduce yellow MTT to blue-violet crystals, while dead cells have no such function [5]. Cell activity can be reflected by measuring optical density. In this experiment, the MTT method was used to detect that the survival rate of hepatocytes after alcohol injury was significantly reduced, and total glycosides of Cistanche deserticola could significantly increase the survival rate of hepatocytes (P<0.05), and the effect was dose-dependent (P<0.05).
Apoptosis is a special form of cell death. When cells undergo apoptosis, special morphological and biochemical changes will occur [6-8]. The experiment confirmed that most of the hepatocytes were apoptotic after alcohol treatment, and the cells showed obvious morphological changes, which were as follows: the cells became smaller, the microvilli on the cell surface disappeared, the cell surface shrunk, there were vacuoles, nuclear condensation, and the formation of apoptotic bodies; It is accompanied by necrosis. After treatment with total glycosides of Cistanche deserticola, the apoptosis rate decreased significantly, and the necrosis situation also eased.
The bcl-2 gene is a proto-oncogene. At present, five bcl-2 gene families (bcl-2, bcl-2 x, bax, mcl-1, and A1) have been found, in which bcl-2 can inhibit programmed cell death (PCD). The work of the mechanism may include: (1) inhibiting the release of Ca2+. Bcl-2 is a transmembrane protein, which is mainly located on the nuclear membrane. The inner and outer nuclear membranes are connected by the lumen of the endoplasmic reticulum. The latter is the main storage site of intracellular Ca2+, which plays an important role in the process of cell apoptosis. Using transgenic methods, it was found that the high expression of bcl-2 could inhibit the release of Ca2+ from the endoplasmic reticulum, so it was speculated that the inhibitory effect of bcl-2 on apoptosis might be related to Ca2+ in the endoplasmic reticulum [9]. (2) Bcl-2 plays an anti-apoptotic role by blocking the signal transmission of pro-apoptotic genes or by blocking these inducible gene products. Studies have shown that bcl-2 protein can inhibit apoptosis induced by P53 and c-fos [10]. (3) Bcl-2 can play an anti-apoptotic role by inhibiting free radicals. Bcl-2 has the role of an antioxidant, which can inhibit the production and action of superoxide, thus inhibiting the occurrence of apoptosis [11].
Early immediate genes (IEGs) are genes that appear early and have a short duration under various harmful stimuli. c-fos is one of the proto-oncogenes, located on chromosome 14q21-q31. It is a 9kb DNA, composed of four exons and three introns [12]. The basic transcription is low, but it can be triggered by different second messenger molecules for rapid and transient expression. The protein product of c-fos is FOS, and the protein encoded by proto-oncogene c-Jun is JUN. FOS contains a leucine zipper (LZ) [13]. FOS and JUN form a heterodimer in the nucleus through the LZ structure, called transcription factor AP-1, which combines with the AP-1 site of the target gene and acts as the third messenger of the nucleus, enabling the expression of the target gene for a long time [14]. Morgan believes that the different expressions of c-fos indicate the diversity and complexity of its functions. The transient expression may be involved in cell protection, repair, remodeling, and regeneration, while sustained overexpression is related to secondary damage. The expression mechanism of the two is different. Therefore, it can be considered that IEGs such as c-fos play a dual role. When the repair defense system is completely inhibited, they participate in apoptosis; When it is not inhibited, it has a protective effect on the cells around the lesion and participates in the process of cell repair and regeneration [15]. WebsterKR uses antisense nucleotides to reduce the transcription of c-fos and prevent the occurrence of apoptosis, indicating that the overexpression of c-fos is inextricably related to the occurrence and development of various diseases [16].
In this experiment, after liver cells were injured by alcohol, the expression of apoptosis-inhibiting factor bcl-2 was significantly reduced, and the expression of c-fos was increased, suggesting that the total glycosides of Cistanche deserticola could protect liver cells by enhancing the expression of bcl-2, inhibiting the expression of c-fos, reducing liver cell injury and apoptosis.
