Protective effect and mechanism of endophytic bacteria in licorice against cisplatin induced HEK293 cell damage
Cisplatin (CP), as a commonly used anti-tumor drug in clinical practice, has significant therapeutic effects but strong toxic side effects. The main manifestations are nephrotoxicity, hepatotoxicity, ototoxicity, and cardiotoxicity, with renal injury being particularly severe. It has a time dose dependence. The nephrotoxicity caused by CP may involve multiple pathogenic mechanisms, including mitochondrial dysfunction, induction of cell apoptosis, oxidative stress, etc. Therefore, how to effectively reduce the toxic side effects of CP has become the primary issue in expanding the clinical dosage of CP, and finding suitable treatment plans or drugs to synergistically reduce CP toxicity and increase efficacy has become the main research direction at present.
In recent years, with the development of traditional Chinese medicine, the combination of traditional Chinese medicine to enhance efficacy and reduce toxicity has become a major research hotspot. Licorice, as a “detoxifying holy herb”, has a sweet and mild taste, functions to nourish the spleen and qi, and harmonize various medicines. Its active ingredients can alleviate poisoning symptoms, reduce mortality, and have significant detoxifying effects on drugs and toxins. Endophytic bacteria belong to the category of medicinal microbial resources, which refer to fungi or bacteria living inside healthy plant tissues and organs. During the long-term evolution process, they undergo genetic recombination with the host and obtain the genes of the host plant, producing medicinal active ingredients that are the same or similar to those of the host. Man et al. found that endophytic strains of licorice have the same physiological effects as the host, and can metabolize saponin and flavonoid active ingredients similar to licorice, with pharmacological effects of antibacterial and antioxidant. Yang et al. found that the fermentation broth of endophytic bacteria in licorice has a cough stopping effect similar to that of the host decoction, total flavonoids, and total saponins. Our preliminary experimental research has confirmed that endophytic bacteria in licorice have various pharmacological effects such as antibacterial, anti-inflammatory, expectorant, and anti-tumor. They can metabolize ammonium glycyrrhizinate, glycyrrhizin, and glycyrrhizin, which are similar to the host licorice. Therefore, endophytic bacteria in licorice may have detoxification effects similar to licorice. Previous detoxification experiments have shown that licorice can effectively alleviate renal toxicity caused by CP. However, there has been no systematic mechanism study on the nephrotoxicity caused by CP by endophytic bacteria in licorice.

Therefore, this article aims to establish a CP induced HEK293 cell injury model through in vitro experiments, and explore the protective effect and mechanism of licorice endophytic bacteria on CP induced HEK293 cells. In order to provide a basis for the combined application of licorice endophytic bacteria and CP to enhance efficiency and reduce toxicity, and to provide theoretical reference for the detoxification effect of licorice endophytic bacteria, further expanding the pharmacological effects of licorice endophytic bacteria.

As the main excretory organ of the body, CP is mainly excreted through the kidneys and accumulates in the proximal tubules, forming a high concentration and long-term accumulation state, promoting the generation of ROS and inhibiting its metabolic enzyme degradation, resulting in a large accumulation of ROS in the body, which in turn induces cell apoptosis and causes kidney damage. Licorice has pharmacological effects of anti-tumor, anti-inflammatory, antioxidant, and free radical scavenging, which can reduce ROS generation and improve cell apoptosis status. The endophytic bacteria of licorice coexist with licorice and belong to medicinal microbial resources. They have pharmacological activities similar to the host licorice and can reduce kidney damage caused by horsehair. They also have the potential to improve cisplatin nephrotoxicity. This study used MTT assay to measure the absorbance of different drug intervention groups. The results showed that CP induced HEK293 cell damage was time-dependent and dose-dependent. Compared with the CP group, the licorice endophytic bacteria treatment group significantly improved cell damage, and the difference was statistically significant (P<0.01). Under an inverted microscope, it was observed that the shrinkage morphology of cells in the combination therapy group was significantly reduced, and the adhesion ability was restored, consistent with literature reports. Flow cytometry analysis revealed that compared with the Con group, the apoptosis rate of the CP group was significantly increased (P<0.01). Compared with the CP group, the apoptosis rate of the combination therapy group was reduced, and the CPJT-H group had the most significant effect (P<0.01). Consistent with the MTT assay results, it further suggests that licorice endophytic bacteria have a protective effect against CP induced HEK293 cell damage.
The process of cell apoptosis is very complex and can be divided into endogenous pathways, exogenous pathways, and endoplasmic reticulum stress pathways. The Bcl-2 family plays a key role in cell apoptosis, including anti apoptotic proteins such as Bcl-2 and pro apoptotic proteins such as Bax. Bcl-2 plays a major regulatory role in the mitochondrial apoptosis pathway and can cause cell apoptosis by activating downstream genes. Caspase-3, Caspase-8, and Caspase-9 are the main components of the Caspase family and are often used as important indicators to detect the degree of cell apoptosis. GRP78 and CHOP are marker proteins for endoplasmic reticulum stress and important components of endoplasmic reticulum stress-induced cell apoptosis. By examining the Bcl-2 family, Caspase family, and endoplasmic reticulum stress-induced apoptosis proteins, the protective mechanism of licorice endophytic bacteria against CP induced apoptosis in HEK293 cells was explored. The experimental results showed that compared with the Con group, CP intervention upregulated the relative expression levels of Bax, Caspase-3, Caspase-8, Caspase-9, GRP78, and CHOP mRNA and protein (P<0.05, P<0.01), while reducing the relative expression levels of Bcl-2 mRNA and protein (P<0.01). Compared with the CP group, the intervention of various therapeutic drugs for licorice endophytic bacteria showed a certain degree of improvement in CP induced HEK293 cell damage. The relative expression levels of Bax, Caspase-3, Caspase-8, Caspase-9, GRP78, and CHOP mRNA and protein in the CPJT-H group were significantly reduced (P<0.05, P<0.01), while the relative expression levels of Bcl-2 mRNA and protein increased (P<0.01).
In summary, endophytic bacteria of licorice can reduce CP induced apoptosis in HEK293 cells, and alleviate CP induced nephrotoxicity by inhibiting the expression of pro apoptotic proteins Bax, Caspase-3, Caspase-8, Caspase-9, GRP78, CHOP and promoting the expression of anti apoptotic protein Bcl-2. This provides a basis for the combined use of endophytic bacteria of licorice and CP for detoxification and expands the pharmacological effects of endophytic bacteria of licorice.