CN114028432A - Novel application of bifidobacterium in medicament for improving sensitivity of platinum chemotherapeutic medicament - Google Patents

Abstract

The invention relates to a new application of bifidobacterium in the pharmaceutical field, namely a new application of bifidobacterium in improving the sensitivity of non-small cell lung cancer to platinum chemotherapeutic drugs. According to the invention, the platinum chemotherapeutic drugs can cause the lung cancer to resist the platinum chemotherapeutic drugs by destroying intestinal mucosa barriers and changing flora structures and functions, and the bifidobacterium can reduce the activity of lung cancer cell mitochondria ClpP by regulating histidine metabolism, so that the sensitivity of the non-small cell lung cancer to the platinum chemotherapeutic drugs is improved.

Description

Novel application of bifidobacterium in medicament for improving sensitivity of platinum chemotherapeutic medicament

Technical Field

The invention relates to a new application of a bifidobacterium medicament, in particular to a new application of the bifidobacterium medicament in improving the sensitivity of non-small cell lung cancer to platinum chemotherapeutic medicaments.

Background

Lung cancer is the most common and most mortality malignant tumor in the world at present. Early stage deficiency of non-small cell lung cancer (NSCLC) results in about two-thirds of patients diagnosed with advanced lung cancer at the time of initial diagnosis and are not treated by surgery. Targeted and immunotherapy can improve the 5-year survival of patients, but is only suitable for less than 20% of the population with advanced non-small cell lung cancer.

Therefore, despite the diversity of new therapeutic approaches, platinum-containing chemotherapy remains the most important first-line treatment for advanced NSCLC. Unfortunately, the median survival time of NSCLC patients receiving chemotherapy is only 8-10 months, and the resistance of platinum chemotherapeutic drugs is an important reason for the short survival time of patients. The problem of the continuous decrease in sensitivity of chemotherapy drugs caused by multiple chemotherapies has not been solved due to the lack of mechanistic studies on their sensitivity.

Therefore, an effective treatment scheme is urgently needed in clinic to reduce the incidence rate of drug resistance of NSCLC patients and improve the curative effect of chemotherapy.

The key point for solving the problem of clinical medication is to explore the mechanism of drug resistance of lung cancer cells to platinum chemotherapeutic drugs. The target point aiming at the tumor cell tolerance chemotherapeutic drugs mainly focuses on the enrichment degree and the activity of the platinum chemotherapeutic drugs in the cells, the configuration of the drugs and the like. However, there are various problems such as individual variability and tumor heterogeneity, and no breakthrough progress has been made.

Therefore, there is a need to focus and explore more deeply on common problems of tumor pathogenesis and drug resistance, such as intestinal dysbacteriosis and mitochondrial dysfunction.

The concept of the "gut-lung axis" was first proposed in the 2017 Nature Medicine journal, Robert Dickson et al found in studies on patients with Acute Respiratory Distress Syndrome (ARDS) that the migration of Bacteroides to the lung could be one of the causes of pneumonia-induced lung injury, and this was long overlooked by clinicians. Kate L Bowerman found in the analysis of the intestinal flora and its metabolites of patients with Chronic Obstructive Pulmonary Disease (COPD) that the abundance of the intestinal flora of COPD patients is significantly lower than that of healthy people, wherein streptococcaceae are significantly enriched in COPD patients, so that it can be speculated that the bacteria themselves or their intestinal metabolites transport through the systemic circulation or affect M lymphocytes, linking the intestinal mucosa microenvironment with the lung disease. However, current studies based on the "gut-lung axis" theory are mostly performed in the context of allergic and inflammatory diseases.

The intestinal flora influences the occurrence and development of tumors and is closely related to the sensitivity of the tumors to chemotherapeutic drugs. On one hand, chemotherapy drugs such as cisplatin and the like can damage the intestinal flora structure and the mucous membrane barrier and seriously damage the intestinal function; on the other hand, the damage and unbalance of the intestinal flora greatly reduce the sensitivity of the human body to chemotherapeutic drugs such as oxaliplatin and the like. Therefore, the treatment method for restoring the intestinal flora imbalance caused by the cisplatin and other chemotherapeutic drugs and reducing the drug resistance of the platinum chemotherapeutic drugs becomes an important treatment scheme for improving the tumor chemotherapy sensitivity.

The intestine-lung axis, as a bridge connecting the intestine and the lung, plays an important role in the occurrence and development of various lung diseases. The classical theory of the enteropulmonary axis considers that the intestinal microecology can regulate immunity or release of inflammatory factors through metabolic small molecules related to inflammation to influence various lung diseases such as asthma, ARDS and the like; in addition, intestinal micro-ecology can also regulate lung microbiome to influence the occurrence and development of lung cancer.

However, it is not clear whether intestinal microorganisms can pass flora metabolites such as amino acids, and the like, so that the curative effect of the platinum chemotherapeutic drugs on the lung cancer is influenced.

Disclosure of Invention

The invention aims to provide a new application of a bifidobacterium medicament to improve the sensitivity of non-small cell lung cancer to platinum chemotherapeutic drugs.

In fact, the present invention provides the use of bifidobacteria for the manufacture of a medicament for increasing the sensitivity of non-small cell lung cancer to platinum-based chemotherapeutic agents.

The invention particularly provides application of bifidobacterium in preparing a medicament for improving the sensitivity of non-small cell lung cancer to cisplatin.

Preferably, the medicament bifidobacterium is administrated by an oral administration mode.

Compared with a cisplatin-containing double-drug chemotherapy group (DDP group) alone, the bifidobacterium combined with the cisplatin-containing double-drug chemotherapy scheme (BIF + DDP group) can obviously reduce the tumor diameter of a patient with advanced NSCLC, obviously improve the intestinal flora structure and abundance of the patient and improve the chemotherapy curative effect of the patient with advanced lung cancer.

In order to clarify the mechanism, the invention designs a series of animal and cell experiments, and discovers that the bifidobacterium inhibits the activity of casein degrading protease P (ClpP), which is a mitochondrial quality control protein in a tumor body, and reduces the activity of the casein degrading protease P (ClpP) in a tumor body cell by regulating the metabolic level of intestinal metabolite histidine through multiomic combined analysis and WB (WB) verification and the like, so that the mitochondrial protein quality control of the tumor cell is reduced, and the sensitivity of lung cancer to cisplatin is restored.

Chemotherapy increases intestinal inflammation, impairs barrier function, and is accompanied by changes in the composition of intestinal flora and decreased diversity of flora, which are mainly manifested by reduction of probiotics such as bifidobacteria and lactic acid bacteria and increase of escherichia coli, staphylococcus and bacteroides. According to the invention, by analyzing the intestinal flora of lung cancer population and subcutaneous tumor-forming mice, the regulation effect of the bifidobacterium combined with cisplatin on the intestinal microecology while obviously reducing tumors is determined, the intestinal flora imbalance caused by chemotherapy can be reversed, the microbial disturbance of clostridium and the like is inhibited, the bifidobacterium can repair the intestinal mucosa barrier damaged by the cisplatin, the flora structure and function changed by the cisplatin are reversed, and the drug resistance of lung cancer cells to the cisplatin is reduced.

Mitochondria are the first line of defense to sense DNA stress. Platinum chemotherapeutic drugs destroy tumors by inhibiting cell replication by damaging the double strands of the tumor nucleus DNA. However, the mitochondrial stress mechanism is ingeniously utilized by tumor cells, and the high-expression ClpP protein helps the tumor cells to avoid the damage of the chemotherapy drugs to the nuclear genes of the tumor cells, so that the chemotherapy drugs cannot play the maximum killing effect in the shortest time, and finally the drug resistance of the tumor cells to the chemotherapy drugs is caused.

ClpP performs protein quality control by providing sufficient target substrates, which are key components of protein performance, to interact with, including amino acids and lipid metabolites. The multi-group chemical analysis of the invention finds that the histidine of the bifidobacterium and chemotherapy combination group is highly expressed and the ClpP protein is lowly expressed, which prompts that intestinal flora change caused by the bifidobacterium can reduce the activity of the ClpP protein by improving the metabolism level of histidine in intestinal tracts and tumor tissues, thereby reducing the cisplatin resistance and improving the curative effect of chemotherapy.

Finally, the invention further verifies that the combination of histidine and ClpP protein promotes the effect of cisplatin on lung cancer through in vitro experiments. Cell experiments show that histidine can improve the killing effect and the apoptosis promoting effect of cisplatin on tumor cells A549, PC9 and LLC. Further researching the mechanism, the histidine combined with the cisplatin can reduce the expression of the ClpP protein of the mitochondria of the tumor cells. Histidine has better binding force to the ClpP protein, the configuration of the histidine is changed by combining with the ClpP protein, the activity of the histidine for phagocytizing damaged tumor cell protein is reduced, the killing of apoptosis protein or misfolded protein to tumor cells after chemotherapy is increased, and the chemotherapy curative effect of cisplatin is enhanced. This is consistent with the conclusions of clinical and animal studies.

Therefore, the research of the invention in the field of lung cancer chemotherapy for the first time proves that cisplatin causes the resistance of lung cancer to cisplatin by destroying intestinal mucosa barrier and changing flora structure and function; and the bifidobacterium regulates histidine metabolism through a brand-new intestinal-pulmonary axis mechanism and regulates the activity of lung cancer cell mitochondria ClpP so as to improve the sensitivity of the non-small cell lung cancer to platinum chemotherapeutic drugs.

The invention utilizes the non-toxic common bifidobacterium to combine chemotherapy, solves the difficult problem of drug resistance of clinically intractable platinum chemotherapeutic drugs, and provides an important direction for the research of tumor chemotherapeutic drug resistance and the development of drugs.

Drawings

Figure 1 is a representative lung CT image of each group of advanced NSCLC patients at time of enrollment and after 4 treatment cycles.

a) CT images at BIF + DDP incorporation (1) and 4 cycles later (2).

b) CT images at the time of DDP grouping (1) and 4 cycles later (2).

FIG. 2 is a comparison of mean tumor tissue diameter and diameter before and after 4 treatment cycles for each group of advanced NSCLC patients.

a) Mean tumor tissue diameter before and after treatment in BIF + DDP and DDP groups, n =13 per group.

b) Tumor tissue diameters before and after treatment in BIF + DDP and DDP groups were compared, with n =13 per group.

FIG. 3 is an NMDS analysis of DDP and BIF + DDP group samples based on fecal 16S rRNA profiles.

Fig. 4 is a comparison of gut microbiota alpha diversity before and after treatment with BIF + DDP and DDP groups (:pless than 0.05; ns: without statisticsDifference).

FIG. 5 is the relative abundance of Bacteroides and Clostridia in individual fecal samples before and after treatment with DDP and BIF + DDP groups.

FIG. 6 shows the experimental design process of Lewis lung cancer tumor-bearing mice, wherein Day of DDP application is Day of Day 0.

FIG. 7 shows the results of animal experiments in which BIF effectively inhibited tumor growth and increased the efficacy of cisplatin chemotherapy.

a. Subcutaneous neoplasia at day 28 of tumor implantation; b. tumor growth curves for each group of mice; c. tumor volume in each group of mice; d. the relative tumor volumes on day 28 and day 7 of tumor implantation in each group of mice (:p＜0.05；**：pless than 0.01; ns: no statistical difference).

FIG. 8 is a NMDS analysis of the intestinal flora of each group of mice.

Fig. 9 is a comparison of the α -abundance differences in the intestinal flora α diversity of each group of mice (.:pless than 0.05; ns: no statistical difference).

FIG. 10 shows Bacteroides murinus (B.sp.) (Bacteroidetes) And Clostridium (Clostridia) Histogram of relative abundance ratio change.

FIG. 11 is a graph showing HE staining of pathological structures of intestinal tracts of various groups of mice.

FIG. 12 is a KEGG differential pathway for mouse tumor tissue metabolites after DDP and DDP + BIF treatment.

FIG. 13 is a graph of the differential pathway of mouse tumor tissue proteins following DDP and DDP + BIF treatment.

FIG. 14 is a representative image of DDP and L-histidine treated PC9 and LLC cells.

FIG. 15 shows the result of flow cytometry for detecting apoptosis, and the LLC apoptosis ratio after combined culture of 10 μ M cisplatin and 1mM L-histidine for 48h is observed.

FIG. 16 is a result of flow cytometry for detecting apoptosis, and the LLC apoptosis ratio after combined culture of 10 mu M cisplatin and 2mM L-histidine or 20 mu M ClpP inhibitor A2-32-01 for 48h is observed.

FIG. 17 shows the results of Western Blot detecting the expression level of Caspase-3 protein in PC9 cells and A549 cells.

Wherein: a is PC9 cells, and b is A549 cells.

FIG. 18 shows the result of detecting the expression level of ClpP protein in A549 cells by Western Blot.

FIG. 19 shows the results of non-denaturing gel electrophoresis to detect the structural change of ClpP protein in A549 cells under the intervention of L-histidine at different concentrations.

FIG. 20 is a molecular docking analysis between ClpP and L-histidine.

In the figure, L-histidine is shown as a rod, ClpP is shown as a computer-simulated protein structure, and H-bond interactions are indicated by dashed lines.

Detailed Description

Embodiments of the present invention are described in further detail below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical solutions of the present invention so as to enable those skilled in the art to better understand and utilize the present invention, and do not limit the scope of the present invention.

The names and the abbreviations of the experimental methods, experimental instruments and equipment related to the embodiments of the present invention are all conventional names in the art, and are clear and definite in the related fields of use, and those skilled in the art can understand the conventional experimental procedures and apply the corresponding instruments and equipment according to the names, and implement the procedures according to the conventional conditions or the conditions suggested by the manufacturers.

The reagents used in the examples of the present invention are not particularly limited in their sources, and are all conventional products commercially available.

Example 1: the bifidobacterium improves the intestinal flora of a human body and improves the curative effect of platinum-containing double-drug chemotherapy of patients with advanced NSCLC.

During the period from 3 months in 2019 to 11 months in 2020, 26 patients with advanced NSCLC who are mutation-negative for EGFR, Kras, ROS1 were included in the study.

26 patients were randomized into two groups, of which 13 patients received platinum-containing dual-drug chemotherapy in combination with bifidobacterium as the bifidobacterium-in-cisplatin treatment group (BIF + DDP group): the cisplatin-based double-drug chemotherapy is cisplatin 75mg/m²And 500mg/m pemetrexed²Or taxol 175mg/m²Once every 3 weeks for 4 to 6 cycles and receiving a maximum ofSix-cycle oral Bifidobacterium capsule (3 capsules 3 times per day, each capsule containing 2 × 10%⁵CFU bifidobacterium). 13 patients served as a control group (DDP group) and received platinum-containing dual-drug chemotherapy and were orally administered placebo daily.

The specific grouping conditions are shown in Table 1, the average age of BIF + DDP group is 63.92 years (48-74 years), the average age of DDP group is 65.69 years (53-75 years); the BIF + DDP group and DDP group had 2 women (15.38%) and 9 smokers (69.23%). Basic information such as sex, age, smoking history, pathological type, TNM stage and the like of two groups of patients are basically matched, and no statistically significant difference exists.

This example shows the remission of the disease before and after 4 chemotherapy treatments in two groups of patients.

Figure 1 shows CT images of a typical case taken from each of the two groups. Wherein, 1 and 2 of the picture a) are CT pictures before and after four times of chemotherapy of patients in BIF + DDP group, respectively, and 1 and 2 of the picture b) are CT pictures before and after four times of chemotherapy of patients in DDP group, respectively.

As can be seen from fig. 1, the lesions were significantly reduced in patients of BIF + DDP group in a) and not significantly changed in patients of DDP group in b) after four chemotherapy treatments.

Based on CT images, FIG. 2a shows that the sizes of the baseline lung lesions (longest diameter of CT image) of patients in BIF + DDP group and DDP group are 4.32cm and 4.46cm, respectively, and there is no statistical difference between the two groups ((p= 0.835); the contrast of the focus after four times of chemotherapy shows that the focus diameters of patients in BIF + DDP group and DDP group are respectively 3.00cm and 4.02cm, and the focus diameter of BIF + DDP group is obviously smaller than that of DDP group: (A)p= 0.041). Meanwhile, the comparison in the group of FIG. 2b also shows that the lesion size is significantly reduced from 4.46cm to 3.00cm before and after the treatment in the BIF + DDP groupp=0.006), but the lesion size before and after DDP treatment was 4.32cm and 4.02cm, respectively, with no significant change (p=0.184)。

Two groups of feces from 26 patients before and after treatment were also collected in this example and subjected to NMDS analysis based on Bray-Curtis distance of 16S rRNA spectra of intestinal flora. The detection results are shown in fig. 3, which indicates that the intestinal flora of the two groups of patients in the group is not obviously different, but after four weeks of chemotherapy, not only the total intestinal flora structure of the patients is obviously different from that before chemotherapy, but also the flora structure of the patients in the DDP group is obviously separated from that after treatment of the patients in the BIF + DDP group.

Furthermore, an alpha diversity test (based on the Chao1 index) was performed on the population of flora, and the statistical analysis used a Wilcoxon rank sum test. As can be seen from FIG. 4, there was a significant decrease in the abundance of the flora before and after treatment in patients with DDP group: (p< 0.05), but there was no overall difference in the abundance of the flora before and after treatment in patients in the BIF + DDP group.

Further, FIG. 5 shows that Bacteroides (Bacteroides) are found after treatment of DDP group by observing changes in single genus in floraBacteroidetes) The relative abundance is improved, but the increase ratio of bacteroides after the BIF + DDP group is combined with the bifidobacteria is far higher than that of the DDP group (a). Also, the two groups of patients after the treatment had intestinal clostridia (C. coli) ((C. coli))Clostridia) The ratio of clostridium in the BIF + DDP group was also found to be significantly lower than that in the DDP group (b) with cisplatin alone.

The results of this example show that bifidobacteria improve the intestinal microbiota composition in patients with advanced NSCLC and increase the efficacy of cisplatin-based duplex chemotherapy.

Example 2: animal experiments of bifidobacterium to inhibit the growth speed of tumor and improve the chemotherapy effect of cisplatin.

To further investigate the effect of Bifidobacterium on the efficacy of lung cancer chemotherapy, this example utilized C57BL/6J mice, using Lewis lung cancer cells (1X 10)⁶One cell/mouse) to construct a Lewis lung cancer tumor-bearing mouse model.

Mice were set with DDP treatment group alone (DDP group), DDP in combination with bifidobacterium treatment group (BIF + DDP group), and bifidobacterium treatment group alone (BIF group) and drug-free control group (Con group), for 4 groups. The specific administration time is shown in fig. 6, and the Con group is administered 0.9% physiological saline per day for intragastric administration; 3X 10 mice in BIF and BIF + DDP groups, starting one week before tumor bearing¹⁰Perfusing CFU/Bifidobacterium; for the DDP and BIF + DDP groups, 4mg/kg D was received every 3 days, one week after tumor bearingDP intraperitoneal injection treatment.

After the administration is started, the tumor volume is measured once every 3 days, a tumor body growth curve is drawn, the tumor inhibition rate is calculated, the tumor body is taken out on the 28 th day after tumor inoculation, the experiment is stopped, and the tumor sizes of the mice of different experimental groups are analyzed.

Fig. 7a is subcutaneous tumor formation at day 28 after tumor implantation, fig. 7b is the tumor growth curve for each group of mice, and fig. 7c is the tumor volume statistics for each group of mice at day 28. It can be seen that tumor growth was significantly inhibited in both the DDP and BIF + DDP mice compared to the BIF and Con groups.

Furthermore, as can be seen from the comparison of the relative tumor volumes (tumor body growth fold at day 28 and day 7 after DDP treatment) in FIG. 7d, although the tumor-inhibiting effect of DDP group was significant compared to Con group: (pLess than 0.05), but the tumor inhibiting effect of the BIF + DDP group is more obvious than that of the DDP group (p< 0.01), and this inhibitory advantage is more pronounced with longer dosing times.

The animal test results show that the BIF + DDP treatment of the mouse has very obvious inhibition effect on the lung cancer, and the BIF strengthens the inhibition effect of the DDP on tumor bodies, thereby proving that the bifidobacterium effectively inhibits the tumor growth speed and improves the curative effect of cisplatin chemotherapy in a mouse lung cancer model.

Example 3: the bifidobacterium remarkably repairs intestinal metabolite disorder and intestinal structure damage caused by cisplatin through up-regulating the abundance of intestinal probiotic flora, and analyzes the composition of the mouse intestinal flora.

In this example, NMDS analysis of intestinal flora 16S rRNA was performed on the feces of the bifidobacterium-only group (BIF group), cisplatin-only group (DDP group), bifidobacterium and cisplatin-combined group (BIF + DDP group) and control group (Con group) mice in example 2. The NMDS analysis results in fig. 8 show that there is a difference in the distribution of intestinal flora in the four groups of samples (Stress = 0.061).

In addition, fig. 9 analyzes α diversity of intestinal flora of each group of mice based on Chao1 index, and the result indicates that bifidobacterium reverses intestinal flora disturbance caused by DDP.

FIG. 10 shows the composition of the intestinal flora of each group of miceDiscovery of Bacteroides (A), (B)Bacteroidetes) The content of the DDP group is the least and is obviously lower than that of the other three groups, and the content of the BIF group and the BIF + DDP group are similar to that of the Con group; meanwhile, after mice received DDP treatment, Clostridium bacteria (C.sp.) (Clostridia) The content of the bacillus bifidus is increased, the content of the DDP group is obviously higher than that of the Con group, and the content of the BIF + DDP group is obviously reduced due to the combined use of the bifidobacterium and the cis-platinum.

On day 28 post-tumor loading, the mid-jejunum of each group of mice was fixed and pathologically stained with hematoxylin and eosin (H & E) for visualization of the lamina propria thickness. The endoscopic observation in FIG. 11 shows that DDP group caused a thinning of the intestinal lamina propria and a disturbance of the glandular structure due to the long-term, continuous intraperitoneal injection of DDP, compared with Con group. However, these changes were not found in the small intestine tissue without DDP. Furthermore, compared to the DDP group, the BIF + DDP group had a significantly thicker mucosa of the small intestine, more intact glands, and the use of bifidobacteria increased the thickness of the indigenous layer of the small intestine.

This result indicates that cisplatin destroys the flora structure in the intestinal tract and the tissue structure of the intestinal lamina propria, while the bifidobacterium reverses the intestinal flora structure imbalance caused by cisplatin and restores the damage of cisplatin to the intestinal mucosal lamina propria.

Example 4: the bifidobacterium promotes intestinal tract and lung cancer tumor body histidine metabolism, and inhibits the mouse tumor body proteomics and metabonomics combined analysis of lung cancer tissue ClpP protein expression.

In this example, tumor metabolites of mice in DDP group and BIF + DDP group were first subjected to omics analysis using a Fold change of 1.2 or more or 0.8 or lesspThe value less than 0.05 is the screening condition, and the differential metabolites among groups of tumor bodies are obtained. By performing KEGG pathway enrichment analysis on lung tumor differential metabolites, it is suggested that the differential metabolic pathways are mainly concentrated on pathways such as amino acid metabolism, lipid metabolism, energy metabolism, transport and catabolism (FIG. 12). Wherein the up-regulated differential metabolites mainly comprise L-Histidine, Citric Acid, Isocitric Acid and the like, and the down-regulated differential metabolites comprise Pyroglutamic Acid, Acetylcholine and the like.

Through the combined analysis of the intestinal metabolite and tumor metabolite enrichment pathways, the bifidobacterium is found to simultaneously up-regulate two histidine metabolic pathways in an omic. The results of the combined analysis suggest that BIF may elevate levels of histidine in the gut and, via the "gut lung axis" pathway, elevate levels of histidine metabolism in lung cancer tissues.

Meanwhile, TMT quantitative proteomics analysis is carried out on tumor body proteins of mice in the DDP group and the BIF + DDP group, 1.2 times and 0.8 time are used as differential expression change threshold values (the Fold change is more than or equal to 1.2 or less than or equal to 0.8),pand < 0.1 is a significance threshold value for screening different proteins, and 136 up-regulated proteins and 135 down-regulated proteins are screened out in total.

The results of the KEGG and GO pathway enrichment analysis of all the different proteins are shown in FIG. 13, which shows that the pathways are mainly focused on endopeptidase regulator activity (endopeptidase activity regulation), free fatty acids oxidation (free fatty acid oxidation), alanine aspartate and glutamate metabolism (alanine-aspartate-glutamate metabolism), and the like. Further analyzing these differential pathways, it was found that proteins significantly altered in the DDP and BIF + DDP groups included down-regulation of the mitochondrial mass control-related proteolytic protease ClpP and proliferation-related Stoml2, and up-regulation of autophagy-related Serpina1 family proteins, and the like.

Based on the above-mentioned omics combined analysis, it was found that the BIF combined DDP increased the L-histidine metabolic level in the intestinal tract and lung cancer tissues and down-regulated the ClpP protein expression in the lung cancer tissues, compared to the DDP group alone.

Example 5: histidine cell experiments for improving the sensitivity of lung cancer cells to cisplatin by down-regulating ClpP expression.

It was first verified whether histidine could increase the sensitivity of cells to DDP using LLC and PC9 cells. CCK-8 experimental results show that histidine enhances the killing effect of DDP on lung cancer cells.

From representative images of PC9 and LLC cells treated with DDP and L-histidine as observed under the microscope of FIG. 14, it was found that no growth inhibition was observed on each tumor cell line with 1mM histidine alone, with 10. mu.M DDP producing less growth inhibition in the different tumor cell lines. However, when the two were added, the proliferation inhibitory effect of 10. mu.M DDP +1mM histidine on PC9 tumor cells was significantly improved. The same effect was seen in LLC cells as well.

The apoptosis result of the flow cytometry detection in fig. 15 shows that histidine does not have the ability of killing tumor cell growth, but can greatly improve the apoptosis ability of DDP to induce LLC. 1mM histidine combined with 10 mu M DDP cultured LLC cells for 48h, so that the proportion of DDP induced apoptosis is improved from 28.7% to 55.7%. Histidine was therefore inferred to have the ability to kill tumor cells synergistically with cisplatin.

A similar trend was also found in PC9 cells (fig. 16). For PC9 cells, 2mM histidine combined with 10 μ M DDP for 48h can increase the apoptosis induction rate of DDP cultured alone from 55.2% to 65.7%. Meanwhile, similar effects of inhibiting the activity of ClpP were also found after replacing histidine with ClpP inhibitor A2-32-01. This further demonstrates that histidine can enhance the killing effect of cisplatin on tumor cells by inhibiting ClpP activity.

Western Blot further proves that histidine promotes the expression level of apoptotic protein Caspase-3 and promotes the apoptosis effect of cisplatin on lung cancer cell lines A549 and PC9 (FIG. 17).

Furthermore, the influence of histidine and cisplatin on the ClpP protein is verified in an A549 cell line, and the result shows that the expression level of the ClpP protein is obviously increased after 10 mu M DDP intervenes for 48 hours when histidine is not added; after addition of histidine, however, it was found that the expression of ClpP protein in tumor cells returned to a lower level as the concentration of histidine increased (fig. 18).

Accordingly, it is considered that histidine may affect the phagocytosis of the ClpP protein and the activity and function of hydrolyzed protein by changing the configuration of the ClpP protein.

To verify this guess, a549 cell protein was extracted and subjected to non-denaturing gel electrophoresis experiments.

Total protein extracted from a549 cells was incubated with different concentrations of L-histidine, electrophoresed through a non-denaturing gel, and probed by western blotting using an antibody specific for ClpP. The arrows highlight the different protein subtypes obtained. Under histidine intervention, the ClpP protein appeared isomeric, and the content of ClpP protein isomeric was gradually increased with the increase of histidine concentration (fig. 19).

Under the Molecular Docking simulation, 5 binding sites which can possibly generate hydrogen bonding effect are obtained, the binding force reaches-2.786 kcal/mol, and the fact that histidine has certain binding force on the ClpP protein is suggested, so that the active structural domain of the ClpP protein is changed, the function of hydrolyzing the pro-apoptotic protein of the ClpP protein is influenced (figure 20), and the chemotherapeutic treatment effect is improved.

The above results indicate that BIF restores cisplatin-damaged intestinal mucosa and disturbed intestinal flora. At the same time, restoration of the flora structure may up-regulate the histidine metabolism level in the intestinal tract and up-regulate histidine in lung cancer tissues by positively regulating the "gut-lung axis". Histidine in cancer tissues will then target the mitochondrial hydrolase ClpP, improving and enhancing the sensitivity of lung cancer cells to cisplatin by altering the structure of ClpP.

The above embodiments of the present invention are not intended to be exhaustive or to limit the invention to the precise form disclosed. Various changes, modifications, substitutions and alterations to these embodiments will be apparent to those skilled in the art without departing from the principles and spirit of this invention.

Claims (3)

1. Application of bifidobacterium in preparing a medicament for improving the sensitivity of non-small cell lung cancer to platinum chemotherapeutic drugs.

2. Application of bifidobacterium in preparing a medicament for improving the sensitivity of non-small cell lung cancer to cisplatin.

3. The use according to claim 1 or 2, wherein the bifidobacterium drug is administered orally.

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