Bioactivity Evaluation Of Two Natural Products 1.Effect Of Kuwanon M On The Proliferation Of Lung Cancer Cells 2.Effect Of Liquiritigenin Against MPTP-Induced Parkinson’s Disease

Lung cancer is one of the tumor types with the most significant mortality rate.Although the mortality rate of lung cancer has been effectively controlled with the application of molecularly targeted drugs and immunotherapy,acquired drug resistance caused by long-term drug usage is still an obstacle to lung cancer treatment.Therefore,searching for novel and effective drugs is crucial for lung cancer treatment.Apoptosis is the most usual form of programmed cell death.Currently,the majority of available chemotherapeutic agents reach their anti-tumor purposes by causing apoptosis and apoptosis resistance has become the main cause of tumor chemotherapy resistance.Paraptosis is a non-apoptotic form of programmed death characterized by extensive cytoplasmic vacuolization caused by mitochondria and/or endoplasmic reticulum(ER)expansion,and is closely associated with activation of MAPKs signaling pathway.Discovering compounds that induce paraptosis may provide new options to improve apoptosis resistance.Mulberry(Morus alba L.)has been planted in China since thousands of years ago.Mulberry Diels-Alder-type adducts(MDAAs)are characteristic components of the moraceous plants.In terms of biosynthesis,most of these compounds are derived from chalcone and isoprenylphenols via[4+2]cycloaddition.The unique structure and diverse bioactivities of MDAAs have attracted the attention of researchers.Our laboratory previously isolated an MDAAs compound from the traditional Chinese medicine Morus alba L.,Kuwanon M(KWM),which inhibited the proliferation and migration while inducing extensive cytoplasmic vacuolization in human lung cancer A549 and NCI-H292 cells.Based on the previous study,this project carried out a further study about KWM.The results showed that KWM-induced proliferation inhibition of A549 and NCI-H292 cells was associated with both apoptosis and paraptosis.DAPI staining showed significant nuclear collapse and rupture;Annexin V-FITC/PI double staining revealed increased apoptotic cells;the cleavage of apoptosis-associated proteins such as Caspase 3/9 indicated that KWM induced apoptosis in lung cancer cells.The loss of mitochondrial membrane potential(MMP),the transfer of cytochrome c from mitochondria into the cytoplasm and the abnormal regulation of Bax/Bcl-2 suggested that KWM-induced apoptosis occurred through the endogenous pathway.KWM-treated cells showed significant cytoplasmic vacuolation,while the apoptosis inhibitor Z-VAD(OMe)-FMK partially antagonized KWM-induced cell death while having no effect on the production of cell vacuoles,so we speculated that KWM may cause another mode of cell death.The ER and mitochondrial fluorescent probe staining imaging revealed that the KWM-induced cytoplasmic vacuoles were derived from endoplasmic reticulum swelling and mitochondrial expansion,so we speculated that KWM induced paraptosis.Furthermore,KWM reduced the expression of Alix,a molecular marker of paraptosis,while activating the MAPK signaling pathway,suggesting the occurrence of paraptosis.The JNK inhibitor SP600125 and ERK inhibitor U0126 both antagonized KWM-induced cell death and cytoplasmic vacuolization,further confirming that KWM induced paraptosis in A549 and NCI-H292 cells.The onset of paraptosis is often related to persistent endoplasmic reticulum stress(ER stress).In cells,the ER is mainly responsible for protein synthesis,modification and folding.When ER stress happens,unfolded proteins will accumulate in the lumen of the ER and the cell activates the unfolded protein response(UPR)to restore its homeostasis.When UPR fails to correct the imbalance of ER homeostasis and will eventually cause cell death.In A549 and NCI-H292 cells,KWM increased the expression of ER chaperone protein GRP78/Bip,and three branches of UPR signaling pathway,IRE1α,PERK and ATF-6,were activated.Furthermore,cells were pretreated with protein synthesis inhibitor cycloheximide(CHX)and ER stress inhibitor 4-phenylbutyric acid(4-PBA),both of which reversed KWM-induced cell death and cytoplasmic vacuolization,indicating that ER stress mediated KWM-induced paraptosis.The results also indicated that ER stress also mediated KWM-induced apoptosis in lung cancer cells.In conclusion,KWM induced both apoptosis and paraptosis in lung cancer A549 and NCI-H292 cells,and ER stress is a critical mediator in the process,indicating that KWM is expected to be a potential therapeutic agent for lung cancer.Parkinson’s disease(PD)is the second most common neurodegenerative disease around the world.As the population aging,the incidence of PD will keep increasing and tend to be younger,and the number of PD patients will increase to 14 million worldwide by 2040,so there is urgent to develop new effective PD therapeutics.The primary pathology of PD is the gradual absence of dopaminergic neurons in the compact part of substantia nigra(SNc)and striatum(Str),as well as the accumulation of misfolded α-synuclein(α-syn)in Lewy vesicles,leading to motor dysfunction.The molecular mechanisms of PD are complex,including abnormal α-syn aggregation.mitochondrial dysfunction and oxidative stress,neuroinflammation and genetic factors.Ferroptosis is an iron-dependent form of cell death.There is growing evidence that ferroptosis has become a crucial goal in the prevention and treatment of a wide range of chronic diseases,including neurodegenerative and cardiovascular diseases.As a member of transcription factors,Nrf2(NF-E2-related factor 2)is an essential mechanism defending cells against oxidative stress.Nrf2 regulates cellular redox homeostasis by encoding the expression of target genes with antioxidant response element(ARE)at the initiation end of the cells.GPX4,the most critical regulator of ferroptosis,has been proved to be a target gene of Nrf2,and targeting Nrf2 provides an option for treating ferroptosis-related diseases.Starting from the Nrf2 pathway to discover drugs to block ferroptosis is a promising approach for the treatment of neurodegenerative diseases such as PD.Additionally,studies have shown that activated microglia exist in the SNc and Str of PD patients as well as the neuroinflammation they cause.MPTP,LPS.and α-syn can induce microglia to polarize toward M1 type that can generate inflammatory cytokines and chemokines.leading to neuroinflammation and neuronal cell death.In contrast,M2-type microglia are able to release anti-inflammatory mediators and promote tissue repair.Therefore,balancing microglia M1/M2 polarization is also crucial for the treatment of neurodegenerative diseases.Liquiritigenin(LG)is a dihydroflavonoid isolated from Glycyrrhiza uralensis Fisch.,which can activate the Nrf2 signaling pathway.Firstly,we evaluated the effectiveness of LG on PD by constructing an acute PD mouse model using MPTP.The results showed that LG could alleviate the motor impairment,promote the increase of tyrosine hydroxylase(TH)expression in SNc and Str,as well as boost the production of nissl bodies,showing a protective effect on the neurological damage caused by MPTP.To further investigate the neuroprotective mechanism of LG,we conducted an in vitro study with dopaminergic neuronal cells SH-SY5Y and microglia HMC3 cells.The results showed that LG could active the Nrf2 signaling pathway in SH-SY5Y cells,prolong the half-life of Nrf2,antagonize MPP+-induced ferroptosis and apoptosis,as well as Erastin-induced ferroptosis and improve cell morphology.The results of Nrf2 silencing experiments indicated that the protective effect of LG on SH-SY5Y cells was Nrf2-dependent.Meanwhile,MPP+and LPS could induce HMC3 cells convert to M1 type,and LG pretreatment could promote HMC3 to transform from M1 to M2 type.The results of co-culture suggested that LG induced HMC3 self-protection while protecting SH-SY5Y cells against LPS-induced inflammatory injury.In summary,LG had neuroprotective effects,including direct protection of neuronal cells and inhibition of microglia activation,and may be a possible substance for the treatment of Parkinson’s disease.