Diterpenoids with Schistosomula-Killing and Anti-Fibrosis Activities In Vitro from the Leaves of Croton tiglium

The leaves of C. tiglium have been comprehensively researched for their structurally novel bioactive natural compounds, especially those with anti-schistosomiasis liver fibrosis activity, because ethyl acetate extract, which can be extracted from the leaves of C. tiglium, has good anti-schistosomiasis liver fibrosis effects. One new tigliane-type diterpene, 20-acetyl-13-O-(2-metyl)butyryl-phorbol (1), and nine known (2–10) analogues were isolated from the leaves of C. tiglium. Their structures were elucidated on the basis of spectroscopic analysis and ECD analysis. All diterpenoids had a stronger insecticidal effect on schistosomula, and compounds 2, 4, and 10 had good anti-liver-fibrosis effects. Furthermore, compared with the model group, compound 2 significantly downregulated the protein and mRNA expression of COL-I, COL-III, α-SMA, and TGF-β1 on TGF-β1-induced liver fibrosis in LX-2 cells. Meanwhile, compound 2 also regulated the expression of TGF-β/Smad-pathway-related proteins. The results suggest that diterpenoids from C. tiglium may serve as potential schistosomula-killing and anti-liver-fibrosis agents in the future.


Introduction
Schistosomiasis is a zoonosis caused by Schistosoma parasites that is widely prevalent in tropical and subtropical areas and has drawn limited research attention [1,2].There are multiple types of schistosomes, mainly including Schistosoma aegypti, S. mansoni, and S. japonicum [3].Schistosomiasis affects more than 70 countries or regions worldwide.According to statistics from the World Health Organization (WHO), at least 240 million people worldwide are infected with schistosomiasis, and over 800 million people live in high-risk areas [1,4].Schistosomiasis has a long history of prevalence in China.Typical eggs of S. japonicum were found in the Western Han female corpses in Mawangdui, Changsha, Hunan, and in the Western Han male corpses in Jiangling, Hubei.This confirms that as early as 2100 years ago, there was an epidemic of S. japonicum in China [5].As of the end of 2020, there were a total of 450 schistosomiasis-endemic counties (cities, districts) in China, 3352 schistosomiasis-endemic townships (towns), 28,376 endemic villages, and a total population of 71.3704 million in endemic villages [6].
Schistosomiasis is an immune disease that can cause damage to the human body during its infection process, including cercariae, schistosomula, adults, and eggs.The main reason for the damage is that antigens released at different stages of the worm's life can induce a series of immune pathological changes and some complications [7].The biggest damage is secondary fibrosis caused by Schistosoma cercariae eggs deposited in the liver [8,9].TGF-β 1 is currently recognized as a pro-fibrotic factor, mainly acting through the phosphorylation of smad2/3 in the smad protein family.It has the effects of activating hepatic stellate cells, promoting collagen synthesis, and ultimately leading to liver fibrosis [10].There have been multiple reports on the relationship between liver fibrosis in schistosomiasis and TGF in hepatic stellate cells-β 1 /Smads signal transduction [11,12].
At present, the preferred drug for treating schistosomiasis is praziquantel, which has good therapeutic effects.In addition to directly acting on schistosomiasis, praziquantel also has a certain degree of immune dependence and immune synergy, which can reduce the degree of liver fibrosis but still cannot change the fibrosis process or reverse liver fibrosis [13][14][15].In addition, praziquantel has some mild side effects such as headache, nausea, anorexia, and reports of drug resistance [16,17].The research on schistosome vaccines has been going on for over half a century, but vaccines obtained through traditional methods have unsatisfactory or unstable immune protection against human induction.Recombinant vaccines using genetic engineering technology are either in the laboratory research stage or in the clinical trial stage, and there is still a certain distance from practical application [18].Chinese herbs, which are essential components of global traditional medicine, have been widely used for the treatment of schistosomiasis, such as artemisinin and its derivatives, which have become drugs for the prevention and early treatment of schistosomiasis.More importantly, Chinese herbs have unique advantages in treating liver fibrosis caused by schistosomiasis [19,20].Therefore, the search for new anti-schistosomiasis liver fibrosis drugs in Chinese herbs is a research hotspot.
The seeds and leaves of Croton tiglium, which belongs to the Euphorbiaceae family, are known as traditional medicinal plants in China [21,22].In the early stage, we found a series of active diterpenoids against tumor from C. tiglium and that the ethyl acetate extract of the leaves of C. tiglium has good anti-schistosomiasis liver fibrosis effects [23][24][25][26].As part of our ongoing efforts to discover structurally novel bioactive natural compounds, especially those with anti-schistosomiasis liver fibrosis activity, in C. tiglium, we re-examined the chemical constituents of the leaves of C. tiglium.One new tigliane-type diterpene (1), together with nine known analogues were isolated from the ethyl acetate extract of C. tiglium.Herein, we report the in vitro isolation, structural elucidation, and schistosomula-killing and antifibrosis activities of these compounds.

Effect of Schistosomula Killing
Compounds have strong killing effects on schistosomula.Compared with the blank control group, the survival rate of schistosomula in different concentration groups at 24, 48, and 72 h decreased (p < 0.05), with the 34.00 µg/mL group having the best killing effect, and after 72 h, the mortality rate of schistosomula in different concentrations of each compound group reached 100%.The results also showed that the survival rate of the Schistosoma cercariae body in 34.00 µg/mL of all compounds was lower than that in the praziquantel group (p < 0.05, Table 2), indicating that the diterpenoid components in the leaves of C. tiglium had a stronger insecticidal effect on schistosomula compared to the praziquantel group.Compounds 2, 4, and 10 showed a certain inhibition effect on the proliferation of LX-2 cells with different concentrations.As the concentration increased, the inhibitory effect of the compounds on the viability of LX-2 cells was enhanced (Figure 4).Compounds 2, 4, and 10 showed a certain inhibition effect on the proliferation of LX-2 cells with different concentrations.As the concentration increased, the inhibitory effect of the compounds on the viability of LX-2 cells was enhanced (Figure 4).

Anti-Fibrosis Activities of
According to the calculation results of SPSS (version 26) statistical software, the IC50 values of compounds 2, 4, and 10 are 103.89,123.29, and 315.01 µM, respectively, while their TC0 values are 2.14, 5.17, and 11.80 µM, respectively (Table 3).Thus, for the low administration group, the concentrations of compounds 2, 4, and 10 with an anti-fibrosis effect are set as 0.50, 1.25, and 3.00 µM, respectively; the medium concentrations of compounds 2, 4, and 10 are set as 1.00, 2.50, and 6.00 µM; and the high-dose group of compounds 2, 4, and 10 are set as 2.00, 5.00, and 12.00 µM, respectively.According to the calculation results of SPSS (version 26) statistical software, the IC 50 values of compounds 2, 4, and 10 are 103.89,123.29, and 315.01 µM, respectively, while their TC 0 values are 2.14, 5.17, and 11.80 µM, respectively (Table 3).Thus, for the low administration group, the concentrations of compounds 2, 4, and 10 with an anti-fibrosis effect are set as 0.50, 1.25, and 3.00 µM, respectively; the medium concentrations of compounds 2, 4, and 10 are set as 1.00, 2.50, and 6.00 µM; and the high-dose group of compounds 2, 4, and 10 are set as 2.00, 5.00, and 12.00 µM, respectively.Compared with the blank group, the contents of COL-I, COL-III, α-SMA, and TGF-β1 in the TGF-β1-treated model group were significantly increased, which indicated that the TGF-β1 model was successfully established (Figures 5-8).Compared with the model group, compound 2 significantly decreased the contents of COL-I, COL-III α-SMA, and TGF-β1 (p < 0.01); compound 4 decreased the contents of them (p < 0.05); and compound 10 decreased the contents of α-SMA and TGF-β1 in each dose group (p < 0.05, Figures 5-8).Based on the above results, compound 2 is selected for the next research on inhibiting liver fibrosis.
the TGF-β1 model was successfully established (Figures 5-8).Compared with the group, compound 2 significantly decreased the contents of COL-I, COL-III α-SM TGF-β1 (p < 0.01); compound 4 decreased the contents of them (p < 0.05); and com 10 decreased the contents of α-SMA and TGF-β1 in each dose group (p < 0.05, Figu 8).Based on the above results, compound 2 is selected for the next research on inh liver fibrosis.Compared with the blank group, the contents of COL-I, COL-III, α-SMA, and TG β1 in the TGF-β1-treated model group were significantly increased, which indicated th the TGF-β1 model was successfully established (Figures 5-8).Compared with the mod group, compound 2 significantly decreased the contents of COL-I, COL-III α-SMA, a TGF-β1 (p < 0.01); compound 4 decreased the contents of them (p < 0.05); and compou 10 decreased the contents of α-SMA and TGF-β1 in each dose group (p < 0.05, Figures 8).Based on the above results, compound 2 is selected for the next research on inhibiti liver fibrosis.The results showed that compared with the blank group, the mRNA expression of COL-Ⅰ, COL-Ⅲ, α-SMA, and TGF-β1 was upregulated in the model group (p < 0.05).Compared with the model group, the different dosage groups of compound 2 significantly downregulated the mRNA expression levels of COL-I, COL-III, α-SMA, and TGF-β1 (p < 0.01).Compared with the colchicine group, the different dosage groups of compound 2 downregulated the mRNA expression levels of COL-III and α-SMA at a lower level (p < 0.05) (Figure 9).The results showed that compared with the blank group, the mRNA expression of COL-I, COL-III, α-SMA, and TGF-β1 was upregulated in the model group (p < 0.05).Compared with the model group, the different dosage groups of compound 2 significantly downregulated the mRNA expression levels of COL-I, COL-III, α-SMA, and TGF-β1 (p < 0.01).Compared with the colchicine group, the different dosage groups of compound 2 downregulated the mRNA expression levels of COL-III and α-SMA at a lower level (p < 0.05) (Figure 9).The immunohistochemical results showed that compared with the blank group, the expression levels of COL-I, COL-III, α-SMA, and TGF-β1 were significantly upregulated in the model group (p < 0.05).Compared with the model group, the expression level of COL-I was downregulated (p < 0.05), while those of COL-III, α-SMA, and TGF-β1 were significantly downregulated (p < 0.01) in different dosage groups of compound 2 (Figure 11).

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The Expression of COL-I, COL-III, α-SMA, and TGF-β1 in LX-2 Cells Were Evaluated Using Immunohistochemistry Staining The immunohistochemical results showed that compared with the blank group, the expression levels of COL-I, COL-III, α-SMA, and TGF-β1 were significantly upregulated in the model group (p < 0.05).Compared with the model group, the expression level of COL-I was downregulated (p < 0.05), while those of COL-III, α-SMA, and TGF-β1 were significantly downregulated (p < 0.01) in different dosage groups of compound 2 (Figure 11).The mRNA Expression of TGF-βRⅠ, TGF-βRⅡ, Smad2, and Smad3 Compared with the blank group, the mRNA expressions of TGF-βRⅠ, TGF-βRⅡ, Smad2, and Smad3 were increased in the model group that was established by stimulating LX-2 cells with TGF-β1 (p < 0.05).Compared with the model group, compound 2 at different doses and SB431542 significantly reduced the mRNA expressions of TGF-βRⅠ, TGF-βRⅡ, Smad2, and Smad3 (p < 0.01) (Figure 12).Compared with the blank group, the mRNA expressions of TGF-βRI, TGF-βRII, Smad2, and Smad3 were increased in the model group that was established by stimulating LX-2 cells with TGF-β1 (p < 0.05).Compared with the model group, compound 2 at different doses and SB431542 significantly reduced the mRNA expressions of TGF-βRI, TGF-βRII, Smad2, and Smad3 (p < 0.01) (Figure 12).The Protein Expression of TGF-βRⅠ, TGF-βRⅡ, Smad2, and Smad3 Compared with the blank group, the protein expressions of TGF-βRⅠ, TGF-βRⅡ, Smad2, and Smad3 were upregulated in the model group that was established by stimulating LX-2 cells with TGF-β1 (p < 0.05).Compared with the model group, compound 2 at different doses significantly decreased the protein expressions of TGF-βRⅠ and TGF-βRⅡ (p < 0.01) and reduced the protein levels of Smad2 and Smad3 (p < 0.05) (Figure 13).

The Protein Expression of TGF-βRI, TGF-βRII, Smad2, and Smad3
Compared with the blank group, the protein expressions of TGF-βRI, TGF-βRII, Smad2, and Smad3 were upregulated in the model group that was established by stimulating LX-2 cells with TGF-β1 (p < 0.05).Compared with the model group, compound 2 at different doses significantly decreased the protein expressions of TGF-βRI and TGF-βRII (p < 0.01) and reduced the protein levels of Smad2 and Smad3 (p < 0.05) (Figure 13).

Materials and Reagents
In August 2020, the leaves of C. tiglium were collected from Yibin County in Sichuan Province, China, identified by the corresponding author (X.J. Zhou).A voucher specimen (ZHXJ-0050) was deposited at our laboratory in Hunan University of Chinese Medicine.

ECD Calculation
Conformational analyses were carried out via random searching in Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 5 kcal/mol.The results revealed the nine lowest energy conformers.Subsequently, geometry optimizations and frequency analyses were implemented at the B3LYP-D3(BJ)/6-31G* level in CPCM methanol using ORCA5.0.1 [32].All conformers used for property calculations in this work were characterized to be at a stable point on a potential energy surface (PES) with no imaginary frequencies.The excitation energies, oscillator strengths, and rotational strengths (velocity) of the first 60 excited states were calculated using the TD-DFT methodology at the PBE0/def2-TZVP level in CPCM methanol using ORCA5.0.1 [32].The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma = 0.30 for all) [33].Gibbs free energies for conformers were determined using thermal correction at the B3LYP-D3(BJ)/6-31G* level and electronic energies evaluated at the wB97M-V/def2-TZVP level in CPCM methanol using ORCA5.0.1 [32].To obtain the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G).By comparing the experiment spectra with the calculated model molecules, the absolute configuration of the only chiral center was determined.
The ECD calculation results of compound 1 are available in the Supplementary Materials.

Killing of Schistosomula Test In Vitro
Oncomelania snails confirmed positive for schistosomiasis infection were provided by the Hubei Provincial Institute of Schistosomiasis Control.We left the snails in chlorinated water for 2 h at room temperature, and then collected the cercariae on the water surface into a 15 mL plastic centrifuge tube.Afterwards, the cercaria of Schistosoma collected were centrifuged and washed with pre-cooled medium at 4 • C twice.Every time of wash lasted for 5 min.Finally, the supernatant was discarded and the cercariae were collected.Schistosoma cercariae was added into a culture medium (containing 10% fetal bovine serum, 90% RPMI1640, 200 U/mL penicillin, 200 µg/mL streptomycin, 2.5 µg/mL amphotericin B and 10 mM 4-hydroxyethyl piperazine ethanesulfonic acid solution) and then submitted to inhaled syringe passes back and forth for certain times to shed its tail.After resuspension, it became schistosomula, and the tail breakage rate was more than 99%, as indicated by microscopic examination.After centrifuging, the severed tail was removed from the supernatant and schistosomula was collected.Schistosoma cercariae was divided into a blank control group, DMSO control group, praziquantel group and different concentrations of diterpenoid compounds group.During the experiment, samples 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were taken and dissolved with a little DMSO.Culture medium and distilled water were added for ultrasound suspension, and the concentrations of 8.50, 17.00, and 34.00 µg/mL in vitro test culture medium were prepared.Meanwhile, after dissolving praziquantel with a little DMSO, we added culture medium and distilled water for ultrasound suspension and prepared them into concentrations of 30.00 µg/mL in vitro test culture medium.
A measure of 1 mL culture medium was added to a centrifuge tube containing schistosomula, drawn well, and dripped onto a 6-well plate with 40 schistosomula per well.According to the above groups, 2 mL of drug-containing culture medium was added to each group, and culture medium was used for the blank control group.The 6-well plate was incubated in a 37 • C CO 2 incubator, and the changes and survival status of the worms were observed under a microscope at 24, 48, and 72 h.Finally, the survival rate of the worms was calculated.

Cell Culture
Human hepatic stellate cell LX2 is a non-parenchymal cell type, and LX2 cells usually serve as an in vitro model for liver fibrosis study, which helps to understand the occurrence and development of liver fibrosis.LX2 cells are a living cell line derived from the human liver, characterized by astrocytes, phenotype stability, expressing liver-specific markers, and so on.The features of LX2 cells make them an important tool for studying the mechanism of liver fibrosis, as well as screening for new compounds.LX-2 were provided by Abiowell Biotechnology Co., Ltd.(Changsha, China).The cell cryopreservation tubes in liquid nitrogen were transferred into a 37 • C water bath for thawing, and then the supernatant was centrifuged and discarded, adding 1 mL of complete medium (10% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin) to resuspend the cells.We transferred the cell suspension into a culture flask and added 4 mL of complete medium, gently stirred, and observed the cell morphology under a microscope.The cells were incubated at 37 • C in a humidified atmosphere containing 5% CO 2 .When the cells covered 70-80% of the culture dish, they could be passed on.The cells were at a ratio of 1:3 and gently blow-dried to evenly distribute them, and cells could be frozen and stored in a liquid nitrogen tank when the cell growth conditions were good.

Cytotoxicity Testing
The cells were divided into a blank control group, compound 2 treatment group (10.00, 20.00, 40.00, 80.00, 160.0 µM), compound 4 treatment group (20.00, 40.00, 80.00, 160.0, 320.0 µM), and compound 10 treatment group (10.00, 20.00, 40.00, 80.00, 160.0 µM).Each group had 6 wells, and the experiment was repeated 3 times.The CCK-8 method (https://www.abiowell.com/fuzhushiji/1271.htm, accessed on 9 January 2023) was used to detect cell proliferation.LX-2 cells were collected in the logarithmic growth stage, adjusting the cell concentration to 5 × 10 3 mL.Cells were seeded in 100 µL per well in 96-well plates.After overnight cell culture, the treatment group received specific drug stimulation, while the control group did not receive treatment.First, 100 µL of prepared cell culture medium was added to each well after washing twice with PBS, and then 10 µL of CCK-8 solution was added to each well.After 4 h of incubation in a cell incubator, the absorbance of each well at 450 nm was measured using a multifunctional enzyme-linked immunosorbent assay.Using SPSS26.0 statistical software, the cell growth inhibition rates of different concentrations of drugs on influenza viruses were calculated, and probit regression analysis was used to calculate the half inhibitory concentration (IC 50 ) of the drug.
Cell growth inhibition rate = (normal group OD value − sample group OD value/normal group OD value) × 100%

Establishing Cell Models
Normal LX-2 cells were seeded into a 96-well plate with 1 mL per well, incubated overnight to allow the cells to adhere to the wall, and changed to serum-free medium and starved for 12 h to synchronize the cell growth cycle.In addition to the blank control group, 5 ng/mL TGF-β1 was added to each treatment group to stimulate cells for 12 h to activate them.TGF-β1 is a recognized factor that promotes the formation of liver fibrosis.During the process of liver fibrosis, TGF-β1 induces stromal cell proliferation and transformation into fibroblasts, increases the synthesis of collagen and other matrix components, and aggravates the deposition of cellulose, collagen, and elastin, thereby causing liver fibrosis.

The Extraction of Total RNA and Protein of the Cells
The LX-2 cells were divided into a negative control group; TGF-β1 model group; the low-, middle-, and high-dose groups of compound 2 (0.50 µM, 1.00 µM, 2.00 µM); the low-, middle-, and high-dose groups of compound 4 (3.00 µM, 6.00 µM, 12.00 µM); the low-, middle-, and high-dose groups of compound 10 (1.25 µM, 2.50 µM, 5.00 µM); colchicine control group (2.5 µg/mL); and SB431542 group (10 µmol/L).In addition to the negative control group, all other groups were treated with TGF-β1.After 2 h of adsorption, the supernatant was discarded, and then cleaning was performed twice with PBS, followed by the addition of different concentrations of the compounds mentioned above.The total RNA and total protein of the cells were extracted after 48 h of exposure.The mRNA and protein expression of COL-I, COL-III, α-SMA, TGF-β1, TGFβRI, TGFβRII, Smad2, and Smad3 were detected by RT-qPCR and Western blot assay, respectively.

ELISA Detection
The contents of COL-I, COL-III, α-SMA, TGF-β1 were examined by ELISA assay.The reagent kit and sample to be tested were taken out of the refrigerator 20 min in advance to increase to room temperature, and then the diluted liquid of the sample was added to the well plate and incubated at 37 • C for 90 min.Next, we dried the liquid in the orifice plate and added 1× incubated biotinylated antibody working solution under the same conditions for 1 h, and then dried the liquid in the well plate and added 350 µL 1× washed liquid to each well, shaking and mixing well for 1 min, followed by shaking to dry and patting dry with thick absorbent paper.Next, 100 µL 1× enzyme conjugate working solution was added to each well, which was incubated in the dark at 37 • C for 30 min after coating with a sealing film.After washing the plate 3 times, 90% of the substrate was added to each well.The plate was incubated in the dark at 37 • C for 15 min; finally, 50 µL of the termination solution was added to each well to terminate the reaction, and an enzyme-linked immunosorbent assay was used to detect the optical density (OD value) at a wavelength of 450 nm for different pores.Then, the OD value of the blank control well was subtracted from the OD value of the detection well as the calibration value, and the standard curve was drawn with the concentration as the horizontal axis and the standard OD value as the vertical axis.The OD value of the test sample was substituted into the standard curve to obtain the detection concentration of the test sample.

RT-PCR Detection
The mRNA levels of COL-I, COL-III, α-SMA, TGF-β1, TGFβRI, TGFβRII, Smad2, and Smad3 were detected by RT-qPCR assay.Total RNA from cells were extracted using the Trizol method, and its concentration was measured using a UV spectrophotometer.Its absorbance values at 260 and 280 nm were calculated to determine the concentration and purity.Then, cell RNA agarose gel electrophoresis was performed, and the gel imaging system was used for observation and photography.Transcription of cDNA was reversed using cell total RNA as a template.The specific reaction system, operational steps, and reverse transcription conditions are outlined in Table 4.The reaction system was mixed well, and a vortex oscillator was used to centrifuge at 2000 rpm for 10 s, allowing the solution on the tube wall to be collected at the bottom of the tube.The tube was incubated in a 42 • C water bath for 60 min and then in an 85 • C water bath for 5 min.After the reaction was completed, centrifugation was performed at 3000 rpm for 10 s, and then the tube was placed on ice for cooling.Reverse transcripts can be directly used for fluorescence quantitative PCR reactions.The sequences of the target genes were searched on NCBI, and the primer sequences of COL-I, COL-III, α-SMA, TGF-β1, TGFβRI, TGFβRII, Smad2, and Smad3 were, respectively, designed using primer5 software; the primer sequences for each gene are shown in Table 5.Briefly, 30 µL of reaction volume contained 15 µL of SYBR Green PCR Master Mix, 1 µL of Primer R (10 µM), 1 µL of Primer F (10 µM), 11 µL of diethyl pyrocarbonate (DEPC)-treated water and 2 µL of template.The optimum conditions for the PCR amplification of the cDNA were established by following the manufacturer's instructions.The PCR cycle conditions were 95 • C, pre-denaturation for 10 min, then 95 • C for 15 s and 60 • C for 30 s, repeated for 40 cycles.The dissolution curves were determined at 60-95 • C. The data were analyzed using StepOne software (version 2.3) (Applied Biosystems, Waltham, MA, USA), and the cycle numbers at the linear amplification threshold values (Ct) for the endogenous dog GAPDH gene and the target genes were recorded.Relative gene expression (target gene expression normalized to the expression of the endogenous GAPDH gene) was calculated using the comparative Ct method (2 −∆∆Ct ).The experiments and analysis were conducted independently three times.

Western Blotting Detection
The total proteins of cells were extracted and lysed in each test group, and the concentration of sample protein in each group was measured with a BCA protein quantitative kit.We prepared sodium dodecyl sulfate polyacrylamide gel for electrophoresis, transferred the semi-dry electric membrane converter to NC membrane, and added the corresponding TGF-β1 primary antibody (diluted with 1:2000) after sealing skimmed milk.The membrane and primary antibody were incubated in a refrigerator at 4 • C overnight.The membrane and secondary antibody were incubated at room temperature for 90 min.After incubation, ECL chemiluminescence solution was used for color development, and X-ray film was exposed for several seconds to several minutes.The results were observed through development and fixation scanning.The grayscale of the target protein bands in the scanned image was analyzed using Image-ProPlus software (version 6.0).The gray ratio of each target band and β-actin is the relative expression of the target protein.The protein expressions of COL-I, COL-III, α-SMA, TGF-β1, TGFβRI, TGFβRII, Smad2, and Smad3 were detected by Western blot assay.

Immunohistochemical Detection
The expressions of COL-I, COL-III, α-SMA, TGF-β1, TGFβRI, TGFβRII, Smad2, and Smad3 were measured by immunohistochemical staining assay.The climbing film was fixed with 4% paraformaldehyde for 30 min and was then flushed with PBS three times for 5 min each time, and it was then added to 0.3% tramadol and allowed to penetrate for 30 min at 37 • C. The climbing film was added to 3% H 2 O 2 at room temperature for 10 min to inactivate endogenous enzymes.The climbing film was incubated with primary antibodies (COL-I, COL-III, α-SMA, TGF-β1, TGFβRI, TGFβRII, Smad2, Smad3) overnight at 4 • C. The climbing film was incubated with secondary antibody at 37 • C for 30 min and was rinsed with PBS three times for 5 min each time.The climbing film was incubated with DAB working solution for 1 min and was washed with distilled water.The climbing film was stained with hematoxylin for 5-10 min and was rinsed with distilled water and then returned to blue with PBS.The climbing film was dehydrated with all levels of alcohol (60-100%) for 5 min per level.After removal, the climbing film was placed in xylene for 10 min and was sealed with neutral gum for observation under a microscope.

Statistical Analysis
The data were processed using SPSS26.0.Measurement data (x ± s) were represented, significance comparison was performed using analysis of variance, inter-group comparison was performed using one-way ANOVE, and inter-group comparison was performed using LSD multiple analysis.The difference was statistically significant with p < 0.05.

Conclusions
At present, chemotherapy is the main means of treating schistosomiasis, and praziquantel is the preferred treatment drug [13][14][15].However, praziquantel has some mild side effects such as headache, nausea, anorexia, and reports of drug resistance [16,17].In this research, we found that all diterpenoids isolated from the leaves of C. tiglium have a stronger insecticidal effect on schistosomula, and that compounds 2, 4, and 10 have good anti-liver-fibrosis effects.As far as we know, this is the first report of the compounds from C. tiglium showing activities of schistosomula killing and anti-liver-fibrosis.These data suggest that diterpenoids from C. tiglium may serve as potential schistosomula-killing and anti-liver-fibrosis agents in the future.
In the pathogenesis of schistosomiasis, the biggest damage is secondary fibrosis caused by Schistosoma cercariae eggs deposited in the liver [8,9].TGF-β 1 is currently recognized as a pro-fibrotic factor and has the effects of activating hepatic stellate cells, promoting collagen synthesis, and ultimately leading to liver fibrosis [10].In the process of liver fibrosis disease, TGF-β1 promotes the production of extracellular matrix, reduces the degradation of COL-I and COL-III, and aggravates liver fibrosis.TGF-β ligands bind with TGFβRII on the cell surface, activate the serine/threonine kinase region of TGFβRI, forming heterotrimer, TGFβRI phosphorylates, and activate Smad2 and Smad3 to form an active transcription complex into the nucleus, and the signal is transferred from the cytoplasm to the nucleus to regulate gene transcription.The results in this research show that compounds 2, 4, and 10 have good anti-liver-fibrosis effects and that compound 2 can regulate the expression of TGF-β/Smad-pathway-related proteins.Our experimental results suggest that compound 2 plays an anti-liver-fibrosis role in regulating the TGF-β1/Smad signaling pathway.
In conclusion, one new tigliane-type diterpene and nine known analogues were isolated from the leaves of C. tiglium.We first demonstrated, in vitro, the potential schistosomulakilling and anti-liver-fibrosis effects of diterpenoids from C. tiglium.Our findings offer a potential new schistosomula-killing and anti-liver-fibrosis agent for medicinal applications.However, the safe usage and application of these diterpenoids should be further investigated in animal models.

Figure 9 .
Figure 9. COL-I (A), COL-III (B), α-SMA (C), and TGF-β 1 (D) mRNA expression levels (n = 3) (Con: blank control group; Mod: model group; Low: low-dose group; Med: middle-dose group; Hig: highdose group; Col: colchicine group).Compared with the blank control group, * p < 0.05; compared with model group, ## p < 0.01; compared with colchicine group, & p < 0.05.The Protein Expression of COL-I, COL-III, α-SMA, and TGF-β1 in LX-2 Cells As shown in Figure 10, compared with the blank group, the protein expression levels of COL-I, COL-III, α-SMA, and TGF-β1 were upregulated in the model group (p < 0.05).Compared with the model group, the different dosage groups of compound 2 significantly downregulated the protein expression of COL-I, COL-III, α-SMA, and TGF-β1 (p < 0.01).Compared with the colchicine group, the different dosage groups of compound 2 downregulated the protein expression levels of COL-III and α-SMA at a lower level (p < 0.05).

Table 2 .
The effect of compounds on schistosomula killing in vitro.